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Kindle Money Mastery

If you have ever wanted to be able to be an author for a living or as a side hobby, this is the online course for you! This course gives you access to all kinds of ebook and materials on how to make the most of the Amazon Kindle Store to make a huge amount of money! You don't need to be a creative genius, spend Hours on end writing, or even know how to use Kindle! All that you have to do is follow the instructions in this course by Stefan Pylarinos. Stefan built this course based on what he does for a living Every Single DAY. This is REAL information that has been perfected in a real business Why would this NOT work for you? This is how Stefan makes his money Why not you? Just think You can make living money writing Kindle books. And you can learn all about how to get started making money with K Money Mastery! Read more here...

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Modification of Receptor Binding Elements

However, the construction of receptor subtype-specific non-visual arrestins is not a trivial task. The main difficulty is in numbers even if only 10-15 amino acids determine receptor specificity of arrestin proteins (Vishnivetskiy et al. 2004), the number of possible combinations is enormous (> 1020). Obviously, screening so many mutants to identify receptor subtype-specific arrestins is impractical. Thus, relatively few residues that play the most important role must be identified first. Our recent data indicate that we can narrow down the list of prime suspects to four or five (Vishnivetskiy S.A. and Gurevich V.V., unpublished observations). Next, intelligently designed combination mutants that contain a residue of one of the four main types (positively charged, negatively charged, hydrophilic neutral, or hydrophobic) in each of these four to five positions should be constructed. This strategy brings the number of arrestin mutants that have to be tested to a manageable range (from...

Nterminal Domain And Copperbinding Properties Of Wndp

Recent studies by Sarkar and colleagues demonstrated that copper binding to N-WNDP is accompanied by conformational changes and by changes in the tertiary structure of the protein (53). These results are very interesting, but it remains uncertain how closely the reported structural rearrangements resemble the changes taking place under more physiological conditions. Our studies using limited proteolytic digestion of soluble copper-free and copper-bound N-WNDP maltose-binding fusion (N-WNDP-MBP) demonstrate that, although some protein regions slightly change their exposure to protease as a result of copper binding, the overall proteolytic pattern of copper-free and copper-bound N-WNDP remain the same. This result suggests that there is no dramatic alterations in the overall fold of these proteins upon in vivo copper binding (unpublished observation).

Agonist Concentration Effect Curves

Agonist concentration-effect curve data. An isoprenaline E A curve obtained in the guinea pig isolated, spontaneously beating right atrium preparation. The data was fitted to Eq. (1) yielding the Hill function parameters shown. Note the curve is a rectangular hyperbola (m 1) p A 50 (-log10 A 50). (Unpublished data.) Agonist concentration-effect curve data. An isoprenaline E A curve obtained in the guinea pig isolated, spontaneously beating right atrium preparation. The data was fitted to Eq. (1) yielding the Hill function parameters shown. Note the curve is a rectangular hyperbola (m 1) p A 50 (-log10 A 50). (Unpublished data.)

And Biochemical Analysis Of The Wndp Functional Domains

Most of the biochemical studies described earlier utilized the heterologous expression, purification, and analysis of isolated functional domains of WNDP. Although, in general, this approach has certain limitations, it proved to be very fruitful and informative for WNDP. The key to successful biochemical characterization of isolated domains is the ability to obtain them in a soluble and well-folded form. The N-terminal domains of WNDP and MNKP contain over 600 amino acid resides and a large number of cysteines, which makes their expression in a soluble form a challenging task. In fact, in our first experiments the expression of N-MNKP and N-WNDP as fusions with maltose-binding protein (N-MNKP-MBP and N-WNDP-MBP, respectively) led to largely insoluble proteins deposited in inclusion bodies. Using the fluorescent Cys-directed probe (see Section 6.2. for details), we determined that most of the Cys residues in these proteins were unavailable for modification without prior reduction with...

G3139 in Melanoma Clinical and Preclinical Results

Injected at 80mgkg_1 intraperitoneally (i.p.) from days 12 to 16. Apoptosis, as assessed by terminal uridine deoxynucleotidyl transferase nick end labeling (TUNEL) assay, increased from 0.7 in the control, saline or mismatched ON group to 3.3 in the antisense group. Levels of Bcl-2 protein expression were stated as being reduced by 66-72 in the G3139-treated group, but only a single Western blot example was shown. Furthermore, the reverse control oligo altered the flanking sequences of the CpG motifs relative to G3139, while the mismatched oligo eliminated them entirely. The authors of this work were aware of these problems, and in a follow-up22 the issue of immunostimulation by the two CpG motifs of G3139 was re-examined. SCID mice bearing 518A2 melanoma xenotransplants were treated with G3139 or with G4232, a G3139 variant that contains C5-methylcytosine at each CpG motif. This substitution is purported to abolish immune stimulation by CpG phosphorothioates, but the extent to which...

Indirect Indicators Of Irs Activation In Major Depression

IRS activation is accompanied by a fall in serum Zn (Mussalo-Rauhamaa, Konttinen, Lehto, & Honkanen, 1988 Solomons, 1988 Srinivas, Braconier, Jeppsson, Abdulla, Akesson, & Ockerman, 1988). Zn is a trace element, which, in the plasma, is firmly bound to a2-macroglobulin (40 ), while the remaining Zn is loosely bound to Alb (55 ) or amino acids (5 ).The loosely bound Zn fraction provides the Zn delivery to the tissues (Solomons, 1988). There is now evidence that depression is accompanied by lower serum Zn (Little, Castellanos, Humphries, & Austin, 1989 McLoughlin & Hodge, 1990 Maes, Scharpe, D'Haese, De Broe, & Cosyns, 1994d Maes, Vandoolaeghe, Neels, Demedts, Wauters, Meltzer, Altamura, & Desnyder, 1997d).There are two factors which can explain lower serum Zn in depression. First, because IRS activation results in decreased serum Alb concentrations and Alb is the major Zn binding protein, there is potentially less Zn binding protein available, which could in part...

Role of Regulation of Rubisco Activity

Possibility is that it decreases the turnover of the Rubisco protein. This idea is supported by data showing that deactivation and association with CA1P or RuBP can protect Rubisco against protease activity (Khan et al., 1999). Since Rubisco is so abundant in ( plants, the turnover of Rubisco protein represents a significant energetic cost which would be expected to be manifested as increased maintenance respiration. Thus regulation of Rubisco activity may serve to increase NUE by decreasing maintenance respiration (Mitchell, Andralojc and Parry, unpublished).

Clinical Trials in Advanced Melanoma

New, unpublished data have revealed the dramatic extent to which LDH stratification, even within the 'normal' range, is predictive of prognosis in advanced melanoma. As shown in Figure 2.7, which is a retrospective examination of a recently completed large study in advanced melanoma (EORTC 18951, N 330), there is an almost linear progression to improved prognosis as the value of LDH descends. Moreover, this improved prognosis with diminishing LDH could also be observed in the current trial. As shown in Figure 2.8 for patients (N 108) whose LDH is < 0.8 x ULN, the median survival (at 24 months, Genasense-dacarbazine vs. dacarbazine alone) was 12.3 months vs. 9.9 months (p 0.0009, HR 0.64). LDH seems clearly destined to emerge as an important parameter for the identification of patients most likely to benefit from Genasense-dacarbazine treatment in the future. Figure 2.7 Depicted are Kaplan-Meier survival curves analyzed by LDH decile in melanoma patients receiving chemotherapy...

Comparisons of Steroid Hormonal Factors in Nested Case Control Studies

From the studies discussed above it must again be concluded that, with few exceptions, no clear or convincing patterns are apparent about associations between circulating hormone concentrations and prostate cancer risk at the individual level. In most studies an association was found between increased risk and increased ratios of testosterone to DHT, but this was statistically significant in only three of six studies. Although these findings suggest a relation between reduced 5a-reductase activity and prostate cancer risk, no associations were found between risk and the levels of 5a-reduced androgen metabolites that are indicators of 5a-reductase activity. Significant associations were found between prostate cancer risk and elevated levels of testosterone and androstenedione and decreased levels of SHBG and 17b-estradiol,but they each occurred in only a single study 160,164 and were not found in other studies. It is possible that relevant associations may have been missed in most...

Natural Occurrence of Oleamide

First to quantitatively report on oleamide levels in rat plasma (10 ng mL) and cerebral spinal fluid (44 ng mL) (Hanus et al., 1999). Oleamide has also been reported to be present in three human breast cancer lines (Bisogno et al., 1998) and in human tear film, although there is disagreement over the relative contribution of oleamide to the total content of nonpolar lipids in this secretion (Butovich et al., 2007 Nichols et al., 2007). Reports on the natural occurrence of oleamide must be considered with caution, due to its widespread use as a slip agent in the production of polyethylene products commonly used in laboratory research. This complication is avoided with a biosynthetic labeling approach using either radioactive or stable mass isotopes. Biosynthetic labeling has been used to demonstrate the biosynthesis of oleamide by different cell types. Initial studies were carried out in the mouse neuroblastoma N18TG2 cell line (Bisogno et al., 1997 Merkler et al., 2004). As will be...

Chemokine Glycosaminoglycan Interactions

Three-dimensional structures of chemokine-GAG complexes are difficult to determine because of the propensity of GAGs to induce the precipitation of chemokines at the high concentrations required for structural work. Nuclear magnetic resonance studies have been used to study the affinity, oligomeriza-tion, and or GAG binding site of CXCL4 (68), CXCL8 (69), CXCL12 (Murphy et al., unpublished results), and CCL5 (65,70). The crystal structures of CCL5 and two heparin-derived disaccharides have been determined by x-ray crystallography (Figure 5A see color plate) (71). The interaction between chemokines and the disaccharides are largely electrostatic, yet specific. However, these studies may reveal a potential template for structure-based drug optimization that increases both the affinity and selectivity for disaccharide-based derivatives (67). Interestingly, there is variation in the GAG interaction site within a chemokine family. Nuclear magnetic resonance studies of CXCL4 (68) indicate...

Properties Of The Yeast Hexose Transporters

Hxt5 is expressed only in glucose-deprived cells or under a variety of starvation and stress conditions (Diderich et al., 1999 J. Becker and E. Boles, unpublished). It has an intermediate affinity for glucose and seems to be used to ensure that the yeast cells can utilize glucose rapidly as soon as it becomes available. What is the reason for such an extreme redundancy of hexose transporter genes in a unicellular organism S. cerevisiae possesses glucose transporters with high, intermediate, low, and very low affinity. It could be argued that constitutive expression of one high-affinity, high-capacity glucose transporter should be enough to fulfill all the requirements of glucose uptake under any conditions. However, such an assumption seems not to be true. It has been shown that in yeast cells exhibiting only high-affinity glucose transport, at high extracellular glucose concentrations, the intracellular free glucose causes a significant inhibition of glucose uptake, which thereby...

Beck Depression Inventory

In an unpublished study, the author has found that 25 percent of 821 chronic pain patients score in the moderate to severe range of depression on the BDI. The BDI is a 21-item questionnaire requiring the patient to endorse various symptoms of depression that produces

EpoxyalkaneCoM Transferase A Zinc Dependent Epoxidase

Extended X-ray absorption fine structure analysis of recombinant EaCoMT supports a mechanism of thiol group activation similar to that seen in MetE (8 K. Peariso, S.A. Ensign, and J.E. Penner-Hahn, unpublished data). Specifically, the addition of CoM to EaCoMT resulted in a change of the zinc environment from 2S + 2N O to 3S + 1N O, consistent with CoM replacing a dissociable water (or other N O ligand). The addition of epoxypropane resulted in a return to a 2S + 2N O environment, consistent with the mechanism shown in Figure 5b. R-HPCDH has been cloned and expressed in a fully active form, but unfortunately, S-HPCDH has not been successfully expressed due to its tendency to form inclusion bodies in heterologous expression systems (55). Therefore, studies of S-HPCDH are lagging behind those of R-HPCDH. Studies of native and mutant forms of R-HPCDH showed that it follows a mechanistic pattern consistent with other members of the SDR family. The enzyme binds and releases substrates in...

Biosynthesis of NADA and other endocannabinoids more questions than answers

Partly due to the fact that their roles as endocannabinoids have not yet been conclusively confirmed, little information still exists on the biosynthetic and catabolic pathways of NADA, virodhamine and noladin ether. Regarding the latter compound, after the initial work of Hanus et al. (2001), two more studies have reported contrasting results on its actual occurrence in brain tissue (Fezza et al. 2002 Oka et al. 2003), and this may have somehow hampered the performance of further studies on noladin ether's metabolic pathways. However, it was clearly established that this putative endocannabinoid is not produced in intact mouse neuroblastoma cells from arachidonic acid incorporated into phospholipids and after ionomycin stimulation, i.e. using conditions leading to the biosynthesis of large amounts of 2-AG (Fezza et al. 2002). As to NADA, strong evidence has been reported against this compound being produced from the metabolism of N-arachidonoyl-tyrosine and using the same enzymes...

The Yeast Expression System

The requirements to the optimal host are that the transformation frequency is high, that most enzymes can be made in reasonable amounts, i.e. in the range of 10-50mg l, and that no post translational modification that may hamper the enzyme activity takes place. In order to identify other yeasts that might be more suitable as hosts Yarrowia lipolytica, Schizosaccharomyces pombe, Kluyveromyces lactis and Hansenula polymorpha expression systems were developed and compared to S. cerevisiae (Table 1) (Sandal et al. 1995 unpublished results). Five enzyme genes, ( eel , gal I, pga I, from Aspergillus aculeatus, encoding an endoglucanase, galactanas and polygalacturonase, respectively, eel II from Humicola insolens, encodeing an endoglucanase and lip I from Thermomyces lanuginosus were selected for the expression studies. Each gene was for convenience of cloning and in order to maintain identical 5' and 3' sequences adapted with an Sfi I and an Not I site in the 5' and 3' ends respectively....

Overall Structure Of Complex Ii

And FrdAB) to bind to the membrane and thus be active in quinone reduction (34, 60-62). The QFR structures show a close interaction between the 3Fe-4S iron-sulfur cluster and the membrane anchor subunit(s). The E. coli QFR membrane domain is composed of two subunits (FrdC and FrdD), each of which contains three membrane-spanning segments with helical secondary structures. These helices have been termed helices I-VI (I-III in FrdC, and IV-VI in FrdD). The single subunit FrdC of Wolinella QFR contains five transmembrane helices it lacks the one corresponding to helix III in E. coli FrdC. This may indicate that a gene fusion between a corresponding frdD gene and the 5' end of an ancestral frdC gene is responsible for the five transmembrane helices and single FrdC subunit forms of QFR and SQR. As mentioned, the structures of the hydrophobic anchor subunits from the two organisms can be aligned to a significant degree however, this requires a rotation around the membrane normal and...

Chronic lymphocytic leukemia CLL

In addition to CTCL, there remain several variant forms of T-cell lymphocytic malignancies that might potentially benefit from treatment with DAB389IL-2. Theoretically, as long as the neoplastic cells express the IL-2 receptor, they should be sensitive to intoxication with the fusion protein toxin. A phase II clinical trial to evaluate DAB389IL-2 for the treatment of CLL has recently been launched at Wake Forest School of Medicine (Frankel, A.E. unpublished). As described by Bannerji and Byrd (2000), CLL is characterized by lesions of small mature lymphocytes with low proliferation rates, and commonly affects the elderly. Although 98 of cases are of B cell in origin, the 2 of cases which are of T cell in origin are significantly more aggressive and are characterized by significantly increased proliferation rates. These patients usually present with splenomegaly and symptoms related to anemia due to marked lymphocytosis. Examination of the bone marrow reveals either focal or diffuse...

Catalysis In Complex Ii

From the N5 position of the flavin ring. Hydride transfer from the N5 of the flavin to C2 is followed by protonation at the C3 position by FrdA Arg287 (96, 101). Reprotonation of FrdA Arg287 is accomplished by a proton pathway involving FrdA Glu245 and FrdA Arg248 (96). The product of this reaction succinate is then released by movement of the capping domain, which also would allow entrance of another molecule of the substrate. In principle, the reverse reaction (succinate oxidation), which is catalyzed efficiently by membrane-bound QFRs containing covalently bound flavin, would proceed by the reverse of the mechanism described above. Mutation of the amino acid residues indicated in Figure 4 has been done in E. coli QFR, and results are consistent with the mechanism proposed using the S. frigidimarina enzyme (47, 90, 96-99) I. Schroder, E. Maklashina, & G. Cecchini, unpublished data .

Cytokine gene delivery using HSV1derived vectors

We also used another non-replicative HSV-1-derived viral vector created in an HSV-1 triple deletion mutant backbone lacking the two copies of the ICP4 gene, the ICP27 gene, and the ICP22 gene and containing the mouse IL-1 receptor antagonist (IL-1ra) gene.23 The deletion of the ICP4, ICP22 and ICP27 genes, which are essential for viral in vitro and in vivo replication and viral cytotoxicity, reduces considerably the intrinsic cytotoxicity of this HSV-1-derived mutant.23 After assessing the proper in vivo distribution of the vector into naive mice, we intracerebrally injected the IL-1ra-containing HSV-1-derived vector into C57 BL6 mice immunized with MOG35-55. The treatment was performed either before or after the onset of EAE. The results (R. Furlan, unpublished) indicate that the IL-1ra vector was able to significantly delay the onset and reduce the severity of EAE when preventively injected, but not when the vector was injected after EAE onset. No interference with the proper...

Future Directions and Concluding Remarks

Strategies for defining the biosynthetic pathway for oleamide will include both pharmacologic- and genetic-based strategies although substantial challenges face both approaches. For example, while several drugs target PAM in vitro (Driscoll et al., 2000 Mains et al., 1986) these agents lack specificity and high efficacy in vivo (Mueller et al., 1993, 1999). Moreover, pharmacologic agents against cytochrome c have not been developed, although, immune-neutralization with monoclonal antibodies may prove useful for this purpose. It appears, therefore, that genetic strategies offer the most effective means for defining the mechanism(s) for oleamide biosynthesis. These strategies are particularly powerful in cell culture where targeted gene disruption and RNA inhibition (RNAi) can be used to selectively eliminate the expression of PAM or cytochrome c in specific cell types. In this regard, we have observed in preliminary studies that cells lacking both somatic and testicular forms of...

Materials and Methods

Two studies examined the relation between vitamin C intake and broad definitions of CVD in representative samples of U.S. adults (Table 7.2). The first study, using data from NHANES I with 13-16 y of follow-up, found that vitamin C intakes > 50 mg d in combination with vitamin C supplement use had a standardized mortality ratio (SMR) of 0.7 (17). However, SMR, unlike RR estimates, are estimated using an external comparison group and do not permit adjustment for potential confounding factors. In NHANES II, vitamin C intakes of 32-73 mg d among men were associated with an increased (60 ) risk of CVD mortality after adjustment for CVD risk factors, supplement use, and total energy intake, but there was no increased risk in the middle two dietary intake quartiles (Loria, unpublished data). There was no association between vitamin C intake and CVD mortality among women in NHANES II. This study also used a narrower definition of CVD than the NHANES I study, excluding rheumatic,...

Cytokines In Major Depression And

With responders-to-pharmacological treatment. These results correspond with those obtained from 33 TRD hospitalized patients in our Department during the last 20 months (Sluzewska et al., unpublished data) in whom we have found elevated levels of IL-6, sIL-6R, product term, sIL-2R, and sTfR. There was a significant correlation between AGP and IL-6 in TRD patients (Sluzewska et al., 1995b) as well as between AGP, ACT, and IL-6 in 33 TRD patients, hospitalized in Department of Adult Psychiatry in Poznan during the year of 1995 (Sluzewska et al., 1997b). One of the indicators of activation of T lymphocytic arm of the CMI is the presence of soluble CD8 in plasma. In 33 TRD patients studied, increased plasma concentration of sCD8 was found (Sluzewska et al., unpublished). The sCD8 molecule or suppressor cytotoxic T cell antigen is secreted by activated T lymphocytes such as CD8+T cells (Tomkinson et al., 1989). A positive relationship between increased levels of sIL-2R and sCD8 is...

Estrogen Receptor Mediated Mechanisms

The prostate contains estrogen receptors, and both the estrogen receptor-a and -b are present in the rat prostate 247 . Thus, involvement of direct receptor-mediated effects of estrogens in prostate carcinogenesis is plausible, but rodent studies using anti-estrogen treatments (such as tamoxifen and ICI-182,780) have yielded contradictory results. The prostate tumor-promoting effects of testosterone may involve estrogen generated by aromatization. However, simultaneous administration of testosterone and tamoxifen did not alter the prostate carcinogenesis enhancing effect of the androgen in experiments in rats injected with prostatic carcinogens prior to the hormone treatment 248 McCormick and Bosland, unpublished data . On the other hand, ICI-182,780 blocked the induction of epithelial dysplasia in the prostatic periphery in NBL rats treated with testosterone and 17b-estradiol 249 the effects of this antiestrogen on induction of periurethral prostate carcinomas are not known.

Non Receptor Mechanisms

Estrogens are capable of producing DNA damage in the target tissues for estrogen carcinogenicity, independent of their interaction with the estrogen receptor 252 . Gladek and Liehr 253 have found a direct DES-DNA adduct in the kidney of male hamsters treated with DES, as well as indirect estrogen-generated DNA adducts (perhaps of endogenous origin and of undetermined structure) detectable by 32P-postlabeling 254 . Both observations are thought to be related with the formation of catechol estrogens that undergo redox cycling during which reactive intermediates and reactive oxygen species are generated and lipid peroxidation can be initiated 252 . In the NBL rat, treatment for 16 weeks with testosterone plus 17 -estradiol enhanced the formation of a chromato-graphically unique endogenous adduct selectively in the periurethral region of the rat dorsolateral prostate, which is the site of the carcinogenic effect of this treatment 255, Bosland, unpublished data . Ho and Roy 256 found...

C Nitrite Reductase Genes and Mutants

It has proved much more difficult to produce mutants deficient in NiR (nii mutants) than in NR, probably because accumulation of nitrite caused by NiR deficiency would be more detrimental than accumulation of nitrate. In addition, there are no direct selection methods available for isolating nii mutants, like chlorate resistance for NR-deficient mutants. Nevertheless, one nii mutant has been isolated in barley (Duncanson et al., 1993) by screening a M3 population of mutagenized barley seeds for nitrite accumulation. NiR activity was strongly reduced in this mutant line. Since the mutation segregated with RFLP markers associated with the NiR apoenzyme gene, the mutant is probably affected in this gene (Ward et al., 1995). Recently, nii mutants were also obtained in the unicellular algae Chlorella soro-kiniana (Burhenne and Tischner, 2000) and Chlamydomonas reinhardtii (Navarro et al., 2000). These mutants were unable to grow with nitrate or nitrite as nitrogen sources and excreted...

Xray Structures Of The Redox Active Metal Sites

The electron density of the CuA site is spherical at 2.8 A resolution, as shown in Fig. 4A (6). However, location of amino acid side chains clearly shows that the CuA site includes two copper atoms. The arrangement of these amino acids suggests that two cysteine sulfur atoms bridge the two copper atoms and form a rhombic square planar coordination structure. Each copper atom has two other amino acid ligands arranged perpendicularly against the rhombic plane in tetrahedral arrangement. The two tetrahedral copper sites are symmetrically opposed at 2.8 A resolution. However, at 2.3 A resolution, some asymmetry is detectable (unpublished observation). In any case, the structure is fully consistent with the multifrequency EPR data (34).

Historical introduction

Pope (1945) reported that using Staphylococcus aureus and a papain digest medium produced a turbidity-penicillin concentration curve that was considerably steeper than with most other media (unpublished data reported in Garrod and Heatley, 1944). The curve, which was sigmoid in shape, had an unusually long and straight central portion, and by extrapolating this back to the axis representing penicillin concentration, a very high degree of accuracy was obtainable.

Solubilization Of Substrates

We found that the new fluorogenic lipase substrates are readily dispersed in aqueous buffer, when fatty acid-free albumin is present (4, 5). Under these conditions, activities of various lipases can be determined in a highly reproducible manner within minutes. This unique form of lipid substrate was identified as a bimolecular complex of lipid and albumin. Data obtained by dynamic light scattering showed the respective particles have a diameter of 8 nm and are, therefore, similar in size to the lipid-free protein (M. Duque, N. Maurer, O. Glatter, and A. Hermetter, unpublished).

Choosing a Region of Study

Are most likely to originate from the hippocampus (see Engel and Cahan86), a statistic consistent with the low AD thresholds found with hippocampal kindling in rats,25-34-38-41 it is not surprising that this region has received much of the investigative energy. However, it is important that the results of experiments involving hippo-campal kindling, or those that make use of hippocampal tissue to identify kindling-induced changes, are interpreted in a manner that is consistent with current theories of kindled seizure networks. If the experimental objective is to identify kindling-induced changes that are critical to kindled seizure development, it is important to assay for such changes in tissue that is known to be modified to the extent that it is kindled. Based on what is known of the circuits involved in kindled seizure propagation (see Section III. A), it makes little theoretical sense to kindle the amygdala and look to the hippocampus to identify the modifications critical to...

Dose versus Treatment Chronicity

Subcutaneous injection of cytokines is generally considered to be a benign route. However, the intraventricular route for the treatment of leptomeningeal metastases (LMD) has proved to be particularly toxic. In one study, 78 of patients developed a wakeful vegetative state after receiving intraventricular IFN-a at a wide range of doses (cumulative dose of 15 to 54MIU) (Meyers, Obbens, Scheibel, & Moser, 1991). It took an average of 3 weeks off of therapy for the patients to recover to their baseline performance status. The toxicity of intraventricular IL-2 for leptomeningeal melanoma appears to be more sporadic, with some individuals tolerating treatment without mood or cognitive side effects, and others developing severe reactions including delirium, obtundation, and death (unpublished data). Delayed effects are also problematic. Intraventricular IL-2 has been reported to cause a progressive dementia in a patient who appeared to be otherwise cured of LMD (Meyers & Yung, 1993).

Clinical Relevance of Kindling

A common criticism of kindling is that there is little documented evidence that humans kindle. For obvious reasons this concern is difficult to address. Although there exist at least two reports of kindled epileptogenesis occurring in the human brain following repeated direct stimulation with implanted intracranial elec-trodes,9697 and several instances of recurrent spontaneous convulsions in schizophrenic patients receiving repeated electroconvulsive therapy,54 a definitive answer to this criticism is impossible. A question that can be addressed, however, is whether the phenomenon of clinical epilepsy has kindling-like properties. A second clinical phenomenon that many have postulated to reflect kindling of the human brain is posttraumatic epilepsy.6,54,86,98 Patients with this condition often present with CPS, months to years after a serious brain insult. During the intervening silent period between the initial trauma and the appearance of clinical seizures, injured brain tissue, or...

Privileged Scaffolds Look Where the Light is Brightest

Recurrent in drugs, and such privileged scaffolds were originally defined15 as a single molecular framework able to provide ligands for diverse receptors Benzodiazepines are the quintessential privileged scaffold, and were the target of the first16 parallel solid-phase synthesis of a small-molecule non-oligomer library. Although only eight examples were reported, this first publication by Ellman was instrumental in alerting the pharmaceutical industry to the potential of combinatorial chemistry, and numerous benzodiazepine libraries have since appeared17 (Figure 4). These are undoubtedly the tip of the iceberg, as many others remain unpublished in corporate collections. In the same manner, many other privileged scaffolds are popular targets for discovery libraries. Our group, for example, has prepared libraries based on well-known structural motifs such as P-amino alcohols,18 hydantoins and thiohydantoins,19 quinolinones,20 biaryls,21 and acyclic guanidines.22 While it is easy to...

Kinetic Evaluation Of Fet3p Structure And Function

The yeast-expression system has allowed for the production of the 100-mg quantities of Fet3p required for detailed structure-function studies. These studies have been of two general types kinetic and spectroscopic biophysical. Most recently, diffraction-quality crystals of Fet3p have been produced. A sample of these are shown in Fig. 18. These particular crystals are approx 300 m in length and diffract to approx 3 A, indicating that crystallographic structure determination of Fet3p will be accomplished in the near future (Taylor et al., unpublished results). Fig. 15. Computer modeling of the ferroxidase site in Fet3p. Model proposed by Buonaccorsi di Patti et al. (21) based on the structure of ascorbate suggested that Glu185 was part of the Fet3p ferroxidase site. A model of Fet3p based on the more similar laccase structure (9) or a de novo model generated in InSightII places this residue > 20 A away from the type 1 Cu(II) (panel A). Instead, this model places residue D278 within 7...

Body condition effect

The residence time of subcutaneously administered ML compounds is influenced by the body condition of the animal. The volume of distribution and elimination half-lives of ML compounds, particularly moxidectin, are generally larger in animals with high body fat content such as pigs and sheep, compared with cattle. Examining the disposition of ivermectin and moxidectin, Craven et al. (2001) observed that despite having less body fat than fat pigs, lean animals exhibited similar apparent volumes of distribution (5.1 versus 5.3 l kg-1, respectively) and while mean residence time in tissue was shorter in lean pigs, body condition had no other effect on ivermectin pharmacokinetics. Similarly, the quantity of fat binding sites had no effect on the volume of distribution of the more lipophilic moxidectin (17.9-18.7 l kg-1) even though it was much greater than for ivermectin. However, the mean transit time of moxidectin through the central and tissue compartments was shorter in lean than fat...

Dopamine Transporter and Drugs of Abuse

We have previously constructed 3D models of DAT 127-129,136 based on various low-resolution structural data and transporters with low homol-ogy with DAT. The A. aeolicus LeuTAa X-ray crystal structure 64 provides the possibility of updating the previous DAT models. Figure 11 shows a putative binding site of cocaine in DAT (unpublished). Site-directed mutagenesis studies and docking studies of cocaine binding to DAT indicated that cocaine

Genomics of G Protein CoupLed Cannabinoid Receptors

The relative lack of homology between the human CB1 and CB2 receptors has made it difficult to identify other possible cannabinoid receptors in the human genome using sequence analysis searching. A series of searches (using Ensembl W.L. Davies and C.R. Hiley, unpublished results) against the transmembrane domains (TMDs) of the CB1 receptor reveals homology primarily only with the CB2 receptor, and that occurred in TMDs 1, 2, 3, 4, and 7. Focus on orphan receptors reveals that GPR3 (TMD2, TMD4, TMD7), GPR6 (TMD4, TMD7), and GPR12 (TMD2, TMD4, TMD7) share identity over multiple TMDs and it is noteworthy that the binding sites for rimonabant (TMD3 6 McAllister et al., 2003) and anandamide (TMD2, TMD3, TMD7 Barnett-Norris et al, 2002) show a limited overlap with these TMDs. However, the few studies that have described effects of ligands on GPR3, GPR6, and GPR12 suggest sphingosine-1-phosphate and related lipids are agonists, not endocannabinoids (Uhlenbrock ). This

WNTFrizzled Interactions

A more quantitative analysis of dose-effect relationships 30, 31 . This is important because the broad expression of FZDs in various cell types renders a pharmacological and cell-based analysis of single WNT-FZD pairs difficult. In fact, we have not been able to identify a suitable mammalian cellular system without endogenous FZD expression. On the contrary, many cell types express multiple isoforms as well as co-receptors (unpublished observations).

Glutamate Dehydrogenase

Taking into account the two reactions catalysed by GDH and the obvious regulation of gene expression and enzyme activity, its is clear that GDH must play an important role at the interface of C and N metabolism. Despite the availability of mutants of maize and A. thaliana lacking one of the GDH subunits (Pryor, 1990 Melo-Oliveira et al., 1996), this role is still not clear. Ameziane et al. (2000) transformed tobacco with the gdhA gene of E. coli encoding a high affinity assimilatory NADPH-dependent GDH, targeted to the cytosol. Over a three year period the gdhA transgenic tobacco produced significantly more dry weight than the control plants, particularly during water shortage. Increases in soluble amino acids (in particular proline) and carbohydrates were also detected. In addition, the transgenic plants were less sensitive to PPT. Other studies on transformed plants expressing the assimilatory GDH from E. coli or Chlorella sorokiniana have also demonstrated increased growth and...

Dominant Negative Approach to Inhibition by Displacement

Residues that were predicted to play critical roles in catalysis (Xu et al. 2000), and that were also absolutely conserved were selected for individual mutagenesis to alanine. The original test mutations were performed in PDE4D3 rather than PDE4B2 so the cognate sites were Tyr 325, His 326, His 330, His 366, His 370, Asp 484 and Glu 505. Lysates from cells transfected with these PDE4D3 point mutants were found to have PDE activity between 96 and 98 lower than lysates from cells transfected with wild type PDE4D3, indicating that all the mutated species are effectively catalytically inactive (M. Magiera, unpublished data).

Mechanisms of Estrogen Receptora Action

Gene expression microarray study carried out on 12,000 genes reported that more than 400 genes showed estrogen regulation (Frasor et al. 2003 Frasor et al. 2004), whilst our own data using recently available microarray platforms providing total genome coverage, show that the expression of 1,128 genes are altered within 24 hours following estrogen addition (Buluwela and Ali, unpublished). These studies are in good agreement with ChlP-chip analyses, which identified 3,665 ERa binding regions in MCF-7 cells (Carroll et al. 2006). Hence, the estrogen-stimulated growth of breast cancer cells appears to require the concerted action of a large number of genes. It is also important to note that these studies have shown that in addition to stimulating gene expression, ERa represses the expression of many genes in breast cancer cells, for example the expression of several pro-apoptotic and growth inhibitory genes is repressed by estrogen.

Allosteric effects and biased agonism

Alternatively, the apparent agonist effects in GTPgS-binding may be due to allosteric modulation. If, for example, low concentrations of LPI were present in the membrane preparations used by Ryberg et al. (2007) and Johns et al. (2007) cannabinoids such as anandamide might act at as modulators, potentiating effects at an orthosteric LPI-binding site but without having being pure agonists in their own right. This seems unlikely, because CP55,940 would be predicted to behave as an antagonist, but in fact it activates GTPgS binding. Furthermore, 0-1602 and abnormal cannabidiol have not been observed to potentiate GPR55 agonists in the yeast assay (A. J. Brown, unpublished observations). A third property of GPCRs that can underlie differing patterns ofligand specificity is biased agonism. This is not fully satisfactory for GPR55, however, primarily because most examples of biased agonism involve changes in orders of efficacy among panels of ligands when comparing different signaling...

Concerted Recruitment of mDCs and pDCs The Role of Chemokines

CCL3 and induces mobilization of both mDC and pDC precursors (both are positive for CCR1 and CCR5) into the circulation, possibly from bone marrow. (iv) Serum TNF-a also upregulates CXCL9 in systemic LN HEVs. (v) Naive mDC precursors are recruited to sites of invasion, capture antigens to become primed mDCs, and consequently migrate into the draining LNs through afferent lymphatics. Local TNF-a accelerates influx of naive mDC precursors via CCL3 as well as efflux of primed mDCs via CCL21. (vi) In contrast, naive pDC precursors transmigrate across TNF-a stimulated HEVs in a CXCL9-dependent manner to become primed pDCs. Primed mDCs in the LNs attract and form clusters with primed pDCs. (vii) Primed pDCs further differentiate into effector pDCs possibly through interaction with antigen-derived factors. Therefore, naive mDC precursors are peripheral tissue-seeking DCs and (viii) finally act as effector APCs to prime tissue-derived antigen-specific T cells. In contrast, naive pDC...

Heterodimers 5HT2C and 5HT2A

To date, there are no published studies reporting the heterodimerization of 5-HT2C receptors with other members of the serotonin receptor family. Of the 13 different serotonin receptors identified in humans, the 5-HT2A receptor is structurally the most similar to the 5-HT2C receptor, and they are coexpressed in regions such as the hippocampus and cortex. Do 5-HT2C receptors form heterodimers with 5-HT2A receptors Given the large body of evidence suggesting that heterodimers can have unique signaling properties, this is an appropriate question to ask. In preliminary studies, we have observed cotrafficking of CFP- and YFP-tagged 5-HT2C and 5-HT2A receptors in transfected HEK 293 cells. In addition, we have observed co-staining of 5-HT2C and 5-HT2A receptors within individual primary hippocampal neurons (unpublished observations). During these studies, we identified commercially available antibodies with good specificity for 5-HT2C (Santa Cruz, C-20) and 5-HT2A (Calbiochem, N22-41)...

Isocitrate Dehydrogenase Provides 2Oxoglutarate for Ammonia Assimilation

Lacking the 2-OG dehydrogenase enzyme complex (Stanier and Cohen-Bazire, 1977). Therefore, in cyanobacteria, since 2-OG produced in the ICDH reaction cannot be further oxidized, it directly enters the GS-GOGAT cycle. Indeed, decrease of ICDH activity leads to a depletion of the intracellular Glu pool (Vega-Palas and Florencio, unpublished). This fact connects the ICDH reaction to biosynthetic N metabolism and rules out a role of ICDH in energy production as in other organisms. Although NADP-dependent and NAD-dependent ICDHs have been described in prokaryotes, most bacteria have only the NADP-linked enzyme. Cyanobacterial ICDH is strictly dependent on NADP and no NAD-ICDH activity has been reported so far (Friga and Farkas, 1981 Muro-Pastor and Florencio, 1992 Muro-Pastor and Florencio, 1994). NADP-ICDH has been purified from the unicellular cyanobacteria Synechocystis sp. PCC 6803 andAnacystis nidulans (Friga and Farkas, 1981 Muro-Pastor and Florencio, 1992) and from the filamentous...

Other Antiepileptic Drugs

Used in various chronic pain conditions, usually neuropathic pain and or headache. The results are mostly disappointing and the few that claim efficacy are far from convincing. Some clearly negative results from large trials involving antiepileptic drugs remain unpublished. The clinician should therefore resist the temptation to try just another drug from this class when others have failed many old and new AEDs require good understanding of their pharmacological properties and attention to detail if they are to be used safely.

Activation of NFkB by the Kinase Cascade and Trx Mediated Redox Regulation

There are at least two independent steps in the NF-kB activation cascade kinase pathways and redox-signaling pathways. These two distinct pathways are involved in the NF-kB activation cascade in a coordinate fashion, which may contribute to fine tune, as well as fail-safe, regulation of NF-kB activity. In most of the cases, NF-kB dissociation by the kinase cascade is a primary step of NF-kB activation. At least two distinct types of kinase pathways are known to be involved in NF-kB activation NF-kB kinase and IkB kinase (Fig. 2). The IKK complex is considered to be responsible for IkB phosphorylation in a wide variety of signal transduction pathway (Zandi et al., 1997 Woronicz et al., 1997). However, NF-kB was shown to be phosphorylated in some cell lines in response to stimulation with TNF or IL-1 followed by NF-kb-dependent gene expression (Ostrowski et al., 1991 Naumann and Scheidereit, 1994 Li et al., 1994). We found a 43-kD serine kinase, called NF-kB kinase, that is associated...

Absorption and Bioavailability

Information about the absorption of phenolic acids in humans is very limited. Our own recent research data suggest that plasma levels of caffeic acid are significantly increased within 1-4 h of drinking a glass of red wine (Abu-Amsha-Caccetta, unpublished results). The increase in plasma caffeic acid occurred using red wine with or without the alcohol removed (Fig. 9.5).

Specificity Of The Involvement Of Peripheral And Central Cytokines In Different Components Of Sickness Behaviour

The previously described experiments show that IL-ip is important both at the periphery and in the brain for social activities but not for food-motivated behavior. In this last case, IL-ip appears to be more important in the brain than at the periphery. To test further this possibility, the depressing effects of LPS were examined on the food intake of mice pretreated with IP or ICV IL-lra. As expected, IP pretreatment with IL-lra had no effect. However, ICV administration of IL-lra attenuated the decrease in cumulative food intake that was induced by LPS (Gheusi, unpublished observations). The importance of brain IL-lp in the depression of social behavior that occurs in sick individuals needs to be qualified. As described earlier, brain IL-lp is involved in the depression of social exploration that occurs in response to peripheral administration of IL-ip, and this has been observed both in rats and in mice (Bluthe et al., 1997 Kent et al., 1992a). This effect appears to be mediated by...

Presence of epicatechin and oxidative stress in vivo

Chocolate confectionary for 4 weeks, when blood was collected after an overnight fast (Actis-Goretta et al., unpublished). This is consistant with reports from acute feeding studies that the majority of absorbed epicatechin is cleared from the blood by 8 hours 18,19,25 , Wan et al. 26 also reported a rapid clearance of epicatechin in subjects fed 22 g of cocoa powder and 16 g of dark chocolate. Consistent with the findings by Osakabe et al. 24 , these investigators observed an 8 increase in LDL-oxidation lag time after subjects consumed the chocolate products for a period of 4 weeks. The observation by Osakabe et al. 24 that there was a significant increase in lag time to LDL-oxidation after 1 and 2 weeks of cocoa consumption, independent of the concurrent presence of epicatechin in the plasma, suggests that the protective effects of flavanols on LDL-oxidation may be due to their effect on the amount of vitamins C and or E. or other antioxidants, associated with the LDL particle. Such...

An Hypothesis For Atypical Antipsychotic Drug Action

Our hypothesis suggests that direct dopamine D1 receptor agonists may be particularly effective in modulating the cognitive dysfunction in schizophrenia. A recent study in PCP-treated rats suggests that, in fact, dopamine D1 agonists are highly effective in alleviating cognitive dysfunction associated with reduced cortical dopamine transmission (our unpublished observations). Further investigations of the cognitive effects of dopamine D1 receptor agonists in normal and schizophrenic subjects are clearly warranted. The concept of directly or indirectly modulating cortical dopamine D1 receptors as a treatment for cognitive dysfunction in schizophrenia must, however, be viewed in the perspective of recent studies that demonstrate that working memory can be impaired by both insufficient and excess stimulation of the dopamine D1 receptor33,73 (see Chapter 7) and that these effects are due to D1 receptor-mediated modulation of the memory-related firing of prefrontal neurons.74

Conclusions and Perspectives for the Future

Much less potent than ToxB with regard to glucosylation potency (Chaves Olarte et a ., unpublished). However, the toxins obviously also have different cell surface receptors, and this could be part of the explanation. Despite the high homology at the primary sequence level, the native 3D-structures certainly differ. This is evident from the different epitope patterns, and also from the finding thatToxA is highly resistant to trypsin whereas ToxB is inactivated by this enzyme (Lyerly et a ., 1989). The uneven epitope distribution on ToxA may imply that the native toxin is more or less covered by its C-terminal repeat structure, possibly by being dimerized in a manner that exposes predominantly the repeat structure. In contrast, ToxB is likely to have a 3D-configuration that also exposes some N-terminal parts of the molecule, as its epitopes appear to be distributed over the entire primary sequence of the molecule. Establishment of the 3D-structures for both toxins will be helpful in...

Glutamate Receptors in Peripheral Tissues

A recent surge of publications (and this book) supports the presence, importance, and functionality of GluRs outside the CNS with unique distributions within various tissues and species. These tissues include adrenal medulla (Yoneda and Ogita, 1986 Watanabe et al., 1994 Hinoi et al., 2002a), peripheral nerves both myelinated and unmyelinated (Aas et al., 1989 Coggeshall and Carlton, 1998), bone (Chenu et al., 1997 Bhanga et al., 2001 Itzstein et al., 2001 Chenu, 2002 Gu et al., 2002 Hinoi et al., 2002), bone marrow (Genever et al., 1999), bronchial smooth muscle, endocrine pancreas (Betrand et al., 1992,1993 Gonoi et al., 1994 Inagaki et al., 1995 Molnar et al., 1995 Weaver et al., 1996 Liu et al., 1997 Gill et al., 2000 Morley, chapter 8, this volume), gut (Moroni et al., 1986 Shannon and Sawyer,1989 Burns etal., 1994 Tsai etal., 1994,1999), esophagus (Burns etal., 1994), duodenum, ileum, and descending colon (Burns et al., 1994), hepatocytes (Sureda et al., 1997 Storto et al., 2000...

PsaA Subunit Ia and PsaB Subunit Ib

Which showed that the N terminal sequence of PsaB is just as predicted from the gene except for the N terminal methionine 35 . A similar fragment with the predicted N-terminal sequence of PsaA without the N-terminal methionine has been isolated by using HPLC from a Staphylococcus V8 protease digest of a spinach PsaA PsaB preparation (A. Ohinata, H. Hir-ata, H. Hiraiwa, and T. Hiyama, unpublished results). These results suggest that the N terminal residues of the mature PsaA and PsaB are possibly unprocessed formylmethionine. From these sequences, the molecular weights of these two polypeptides would be calculated as 82,000 to 83,000 with 750 to 800 amino acid residues. These two have some 40 homologies to each other.

Aialglhttt Awdafylavf Dylwlnssql Qelietlawa Aafliastsg

The separation of PsaA and PsaB has been achieved only by using SDS-PAGE with urea containing gel 35 . It should be noted that an apparent separation achieved with SDS-PAGE without urea in an earlier pioneering report 71 was wrong. N terminal sequencing and immunoblotting of the two separated bands revealed that the lower band obtained by that method was a mixture of degraded PsaA and PsaB, while the upper band was the unresolved mixture of PsaA and PsaB (H. Hiraiwa, H. Hirata, and T. Hiyama, unpublished results).

Renal Delivery of Captopril Lysozyme

Accumulation of a radiolabelled LMWP in the lysosomes of the proximal tubular cell. Electron microscope autoradiography of renal proximal tubular cells from a rat injected i.v. with 125I -tyramine-cellobiose-labelled cytochrome-c, 4 h prior to fixation through the abdominal aorta. An intense lysosomal accumulation of the protein is observed in three dark electron-dense lysosomes . A few grains are seen over the apical endocytic apparatus. Part of the luminal brush border is found in the upper right hand corner. Magnification, x 25 000. Unpublished data from E. I. Christensen, Arhus, Denmark, and M. Haas, Groningen, Netherlands. Figure 5.10. Accumulation of a radiolabelled LMWP in the lysosomes of the proximal tubular cell. Electron microscope autoradiography of renal proximal tubular cells from a rat injected i.v. with 125I -tyramine-cellobiose-labelled cytochrome-c, 4 h prior to fixation through the abdominal aorta. An intense lysosomal accumulation of the protein is...

Pharmacological Aspects of Locked Nucleic Acid Oligonucleotides Targeting Messenger RNA and MicroRNA

The pharmacological aspects of LNA ONs targeting mRNA are extensively reported in the literature and have recently been reviewed.33 In Section 5.4.2 dealing with mRNA inhibition we focus on recent data from our own, mainly unpublished, work to illustrate new aspects of mRNA targeting. In Section 5.4.3 on miRNAs we take a slightly different approach and present this novel class of targets more broadly and then, finally, focus on the pharmacological work that we have generated.

The Activated Enzyme

What are the functional consequences of activation under different conditions of toxic insult The conclusions that can be drawn from steady-state kinetic studies involving a series of increasingly reactive electrophilic substrate analogues and glutathione at low (R. Morgenstern, unpublished observation) or physiological concentrations (Morgenstern et al., 1988) are as follows. At physiological glutathione concentrations and at high concentrations of reactive electrophiles, activation increases conjugation dramatically. On the other hand, if the reactive electrophile is present in low concentrations, activation does not increase the enzymatic rate appreciably. Of course, activation by covalent modification is less likely in this case. The activity toward less reactive electrophilic substrates does not change regardless of the activation state of the enzyme when the glutathione concentration is high. When the glutathione concentration is low ( 0.1 mM), reactive electrophiles are...

Renal Catabolism of Captopril Lysozyme

After renal uptake, captopril was rapidly released from the conjugate as indicated by the rapid decrease in renal captopril levels with time (Figure 5.9b).The difference in renal t1 2 of naproxen and captopril after delivery with lysozyme is likely to be due to an unequal rate of release from the lysozyme conjugates. Whereas naproxen-lysozyme requires a peptidase for cleavage, captopril is released from the conjugate enzymatically by -lyase and or non-enzy-maticaly by thiol-disulfide exchange with endogenous thiols. To reduce the rate of capto-pril-lysozyme breakdown, two different cross-linking reagents, SPDP and SMPT, were tested. Although an SMPT link between two proteins is in principle less susceptible to disulfide reduction 83 , no difference in degradation rate was found between the SPDP and the SMPT captopril-lysozyme conjugates (Kok et al., unpublished data).

Biodistribution of Locked Nucleic Acid Oligonucleotides

Tritium labelling was also used to image the biodistribution of the LNA compounds SPC2968 and SPC2996 in mice. The LNA ONs were 16-mer gapmers and designed to target Hypoxia-inducible Factor 1 Alpha (Hif-1a) and Bcl-2, respectively. The biodistribution was measured as whole-body auto-radiography in sagittal sections between five minutes and 18 days post dosing. The highest uptake of radioactivity was registered in the kidney cortex, uterus, uvea of the eye, skin, liver, bone marrow, lymph nodes and spleen (Figure 5.7 unpublished data). The tissue half-lives were estimated on the basis of disappearance of tritium-label over 18 days and were found to be remarkably similar across tissues, estimated at approximately 8 days. This is much longer than reported for first-generation PS and can be related to the improved nuclease resistance of LNA.65 Both ONs showed a very high blood clearance of radioactivity with elimination half-lives of about one hour.

Checking the Dessert Menu First

If one wishes to use a biomarker to differentiate between normal healthy persons and those with a disease, for example, one of the many questions to be considered prior to deciding which indeed is the most informative biomarker for the job concerns the potential degrees of measurement between them. In many cases the biomarker of interest is present in normal healthy persons as well as in those with disease, but with different typical levels in the two populations. In this case it is very important to establish a priori the descriptive characteristics of the two groups in order to define what a clinically relevant treatment effect might be and how one might judge that such an effect occurred. Published literature, unpublished previous data, and pilot studies all provide potential resource material for defining these descriptive characteristics and should be evaluated carefully in advance. Since both populations will present with a range of values, relevant descriptive characteristics...

Renal and Systemic Effects of Captopril Lysozyme

With regard to the pharmacological effects of the captopril-lysozyme conjugate, the following observations were made (Kok et al., unpublished data). The extent of ACE-inhibition in the plasma and kidney tissue was measured after i.v. administration of captopril-lysozyme and an equimolar dose of free captopril. It was shown that conjugation to lysozyme caused a similar though more sustained inhibition of renal ACE-activity by captopril. The inhibition of plasma ACE-activity was clearly reduced but not entirely prevented by conjugation of cap-topril to lysozyme. Possibly, the S-S linked drug conjugate is partly degraded in the circulation. It is also possible that after degradation of the conjugate in the kidney, captopril was transported back into the bloodstream. The rapid intracellular release may provide a sufficient driving force for transport across the basolateral membranes.

Regulation of prostaglandin synthesis in mesangial cells

As in other cell types, induction of COX-2 mRNA and or protein in mesangial cells is strongly suppressed by glucocorticoids 32 , Partial inhibition of COX-2 is also obtained by the immunosuppressive drug cyclosporine A, which is widely used to prevent transplant rejection ( 24 and unpublished results). It may be linked to cyclosporine A-related hypertension, a common adverse effect of this drug.

Stress can Both Promote Temporary Motor Improvement and Lead a Worsening of Behavior

One of the paradoxes of PD is that when severely affected patients are exposed to a sudden stress they can respond with a temporary improvement in their motor behavior. This is exemplified by a number of anecdotes about patients responding to emergencies by catching a ball, running out of a burning building, or swimming out into the ocean to save a child 62 . Comparable findings were made in the 6-OHDA-treated rat. For example, Ed Stricker and I observed that if an akinetic rat were to be placed in a pail of water, it would swim with apparent ease to the edge to the pail and then climb out. However, as in the patient anecdotes, seconds later the rat would again freeze unpublished observation but see also 63,64 . The neurobiological basis of such phenomena is obscure. Although we found that stress increased DA release, even after 6-OHDA lesions 65 , the DA receptor antagonist haloperidol failed to block stress-induced kinesia 63 . Equally mysterious is the common clinical observation...

Renal Delivery of High Doses of LMWPs

Cojocel et al. demonstrated clear adverse effects after relatively high doses of lysozyme 96 . We studied these aspects in more detail and concluded that lysozyme should be given in a dose of less than 100 mg-1 kg-1 over 6 h to minimize the negative effects on systemic blood pressure, glomerular filtration and renal blood flow. From these data, it emerged that LMWPs are suitable to serve as drug carriers to the proximal tubular cell of the kidney. However, the conjugate should preferably be administered in low-dose by constant infusion to limit the systemic and renal toxicity and to reduce the urinary loss of the intact conjugate (unpublished data).

Cyclooxygenase expression in renal inflammation

Large amounts of prostanoids have been described to be synthesized in experimental models of glomerulonephritis. The data obtained from in vitro studies with mesangial cells in culture suggested COX-2 to be the major COX isoform involved in glomerular inflammation. Data on COX isoform expression in animal models are still limited but suggest a role for both isoforms. In a rat model of glomerulonephritis induced by anti-thymocyte antiserum, COX-2 expression was very transiently upregulated during the first hours of the inflammation, whereas there was a prolonged upregulation of COX-1 over several days 33 , A similar increase in COX-1 was also observed in other rat models of glomerulonephritis and in biopsies obtained from patients with immunoglobulin A (IgA) nephritis (A. Hartner and M. Goppelt-Struebe, unpublished results). In a very recent study, COX-2 was found to be upregulated in kidney biopsies obtained from patients with active lupus nephritis 34 , Enhanced COX-2...

Indirect Evidence Using the Nonselective 5HT2C Compounds Clozapine and Mesulergine

Similarly, mesulergine displays inverse agonist property at 5-HT2C receptors in vitro (Barker et al. 1994). However, mesulergine has been classically defined and used as a 5-HT2C antagonist in vivo (Pazos et al. 1984 Kennett and Curzon 1988 Prisco et al. 1994). In line with this, mesulergine, at the optimal dose of 0.1 mg kg (Fig. 10.3), blocks the decrease in subcortical DA release induced by Ro 60-0175. Interestingly, the selective 5-HT2C antagonist SB 242084 prevents a further increase in subcortical DA release induced by mesulergine (Navailles et al. unpublished data), confirming that mesulergine may affect DA neuron function independently of its DA-D2 antagonist properties (Di Matteo et al. 1998 Prisco et al. 1994 Van Wijngaarden et al. 1990). Although requiring further investigation, these data suggest that mesulergine may also behave as a 5-HT2C inverse agonist in vivo.

Of Calcium Homeostasis In Acute

Studies from our laboratory indicate that depletion of GSH and resulting oxidative stress also results in perturbation of calcium homeostasis within dopa-minergic cells via decreased activity of the Na+ K+ ATPase possibly as a result of decreased mitochondrial function and ATP synthesis (94, unpublished results). It is interesting to note in this light that levels of the calcium-binding protein calbindin have been found to be decreased in the autopsied brains of PD patients and those neurons that are spared in the disease appear to express elevated levels of the protein (99-101).

FerredoxinNADP Oxidoreductase

Membranes in higher plants, but readily solubilized by acetone treatment 167 . Once solubilized, this enzyme is soluble in water without any detergent and readily purified 167 . The amino acid sequence of the spinach enzyme is shown in Figure 9.21. The prosthetic group is flavin adenine dinucleotide (FAD). A three-dimensional structure has been proposed from x-ray crystallography with 2.6 A resolution 168 . For more about structures and functions, refer to a review 169 . The gene, petH, is nucleus encoded in eukaryotes 170 . Although this protein is believed to be peripheral and is located on the stromal side, it has been reported to be com-plexed with some other thylakoid constituents with the b6 f complex 179,182 , with a 17.5 kDa protein 180,181 , and with a 33 kDa protein (H. Yamazaki, T. Hiyama, unpublished result). More work has to be done on these matters, since FNR has often been implicated as a part of the cyclic electron transport 183 .

Concluding Remarks

The present review is admittedly biased and not well balanced, reflecting the author's long indulgence in this field since the 1960s. The emphasis is sometimes on the historical side rather than on hot news items, which appeared often too hot to handle for the present author. An old Chinese proverb says, Digging into classic literature provides useful hints and often leads to a new discovery.'' It may not be a waste of time to look back at the past once in a while. It may also be true that ''there's many a good tune played on an old fiddle.'' Some unpublished results in the author's hand have also been included here to back up the author's views. The readers might as well refer to excellent reviews for more details, for subjects not covered here, and for sometimes different and perhaps more ''balanced'' views in this field 16,31,38, 74,87,140,175,176 .

Factors Modifying Nicotinic Analgesia 541 Gender Studies

Heterogeneous, outbred populations were used. More recently, Flores et al.,129 have investigated the antinociceptive effect of epibatidine in eight inbred mouse strains using the tail-immersion withdrawal assay. They found that these strains differed significantly in their sensitivity to epibatidine, with DBA 2, BALB c, and A strains showing much greater sensitivity than all others. In particular, the A strain exhibited 20-fold higher antinociceptive potency compared with the C3H He strain. Importantly, the effect of epibatidine was blocked by mecamylamine in both strains, indicating the involvement of neuronal nAChRs in mediating the observed antinociception. Based on these data, Flores et al.,129 suggested that such pharmacogenetic differences most probably derive from pharmacodynamic rather than pharmacoki-netic mechanisms, and this view is supported by findings that similar strain differences in antinociceptive sensitivity using the mouse tail-flick and hot-plate tests were...

Bcrabl Induces Myeloid and Lymphoid Leukemias

In Ph+ ALL patients, imatinib therapy often induces relatively brief remissions, and tragically these patients have poor prognoses despite conventional combination chemotherapy and myeloablative bone marrow transplantation. Like most other cases of pediatric ALLs (Pui et al. 2004), these Ph+ leukemias often have sustained deletions at chromosome 9p21 that encompasses the INK4A ARF locus. In contrast, myeloblasts from CML patients in chronic phase (where imatinib is most effective), and in the clinically aggressive accelerated and blastic phases of the disease (where imatinib is less efficacious), display neither genomic loss at the INK4A ARF locus nor ARF promoter methylation (our unpublished observations). We therefore wondered whether INK4a ARF loss might contribute to imatinib resistance in Ph+ ALL.

Acquired Resistance to Egfr Tkis

It is unclear why this would be the case from a teleological point of view, although the oncogenic shock model could again be evoked. An EGFR double mutant that contains an activating mutation and the T790M mutation is significantly more active than either single mutant and displays a degree of ligand-independent activation (unpublished data). Therefore, an overly hyperactive double EGFR mutant could potentially trigger pro-death signaling and cells over-expressing the double mutant may be at a selective disadvantage in a tumor. Recent genetic studies have also identified familial single T790M EGFR mutations that confer a predisposition to lung cancer, lending credence to the idea that these mutants are hyperactivated (80).

Mice Carrying a Null Allele of the GR GRnulnuN Show Complete Penetrance of Perinatal Lethality

A finding that remained unexplained for a long time was the fact that about 20 of the homozygous GRhypo hypo mutants survived to adulthood without any gross abnormalities. The discovery of two unusual GR-mRNA species in homozygous mutants provides a potential explanation for this observation (unpublished data). In one of these transcripts exon 1 is directly spliced onto exon 3 of the GR gene, thereby omitting exon 2, which codes for the entire NH2-terminus of the protein. From studies on apoptosis it is known that a methionine in exon 3 can serve as a potential start site, giving rise to a GR protein of about half the normal size consisting only of the unmature at birth (unpublished data). In summary, from the analysis of GRnull null mice we conclude that the observations made in GRhypo hypo mice are indeed characteristic for an absence of the GR during development. However, interpretation of effects observed in adult GRhypo hypo mice requires caution, as it must be considered that a...

D A Novel Import Pathway for CFo subunit II

As if the complexities of thylakoidal protein insertion translocation were not bewildering enough, recent studies on the biogenesis of CFo subunit II (CFoII) suggest that this protein is imported into the thylakoids by yet another pathway. Structurally, CFoII is unique in that it is an integral membrane protein (containing a single membrane-spanning section near the N-terminus) that is synthesized with a bipartite presequence apparently similar to those of lumenal proteins (Herrmann et al., 1993). This presequence can act as a lumen-targeting signal, having been shown capable of targeting plastocyanin into the lumen (unpublished data) and it was at first thought likely that CFoII was transported by the thylakoid protein translocation machinery, with the membrane-spanning section acting as a 'stop-transfer' signal to abort the translocation process. Recent studies on the integration of CFoII into isolated thylakoids, however, strongly suggest that this protein is in fact integrated...

Laboratory techniquebased models and search strategies

Where Lpart is the concentration of RNA type i that is partitioned, LBT is the concentration of RNA type i that bound target in the equilibrium and Lfee is the concentration of RNA type i that remains unbound. (Note The notations of the various papers are converted into a single notation used throughout this review (Table 1).) For optimal partitioning, one would like CP 1 and BG 0. From unpublished experiments, they observed CP 0.8 and BG 0.001. This partitioning model makes two assumptions. It assumes that the retention of RNAs bound to nitrocellulose is independent of the randomized component, an unpublished observation made both by Irvine et al. 20 and Mandecki et al. 21 . Sequence-independent background binding may indicate that flow or mechanical issues are responsible for the background component more than actual binding. It also assumes that the retention of RNAs bound to nitrocellulose-bound proteins is independent of the randomized component. This is far less likely, as the...

Neurochemical Responses to Interleukin1 IL1

Tophan concentrations, as well as increases in 5-HIAA (Dunn, 1988b). Thus IL-1 mimicked quite closely the response to influenza virus infection, except that the IL-1-induced responses dissipated within a few hours. These results were subsequently replicated in rats (Kabiersch, del Rey, Honegger, & Besedovsky, 1988). It was also shown in rats that decreases in hypothalamic NA occurred after ip IL-1 (Fleshner, Goehler, Hermann, Relton, Maier, & Watkins, 1995). Depletions of NA are more readily obtained in rats than in mice (Stone, 1975 and Dunn, unpublished observations). Zalcman et al. also observed IL-1-induced increases in MHPGrNA and 5-HIAA 5-HT ratios in the prefrontal cortex and hippocampus, but also found increases in mouse prefrontal cortex DOPAC (Zalcman, Green-Johnson, Murray, Nance, Dyck, Anisman, & Green berg, 1994). In more than 100 experiments with ip IL-1 administration to mice, we have observed statistically significant increases of DOPAC in only a very few...

Arf Inactivation in a Mouse Bone Marrow Transplantation Model of Ph ALL

Utilizing a conventional bone marrow transplantation (BMT) approach to model BCR-ABL-induced ALL (Hu et al. 2004), unconditioned donor bone marrow, transduced with retroviruses coexpressing Bcr-Abl and GFP, was transplanted without intervening in vitro culture into lethally irradiated recipient mice. Mice that received transduced Arf+ + (WT) donor cells all succumbed to a lethal lymphoblastic leukemia with a median survival of 5-6 weeks. Analysis of recipients of transduced WT donor cells confirmed Bcr-Abl-dependent expression of p19Arf in circulating leukocytes at three weeks post-transplantation. Robust Bcr-Abl-dependent p19Arf and p53 induction was also detected in isolated bone marrow and splenic B-lineage cells recovered from clinically well mice as early as two weeks after transplantation of transduced WT donor cells. In contrast, all recipients of transduced Arfbone marrow cells developed a more aggressive leukemia and survived less than three weeks post-transplant (Williams et...

Specific Uptake into Cells

Figure 5 Pharmacological effects of liposome and liposome-free incubations for amikacin (A 132 ) and for triamcinolone acetonide phosphate (B, unpublished observation) in cell culture. A represents situation A in figure 3. B, represents situation B in Figure 3. Note the right shift for the liposomal preparation, indicating a less favorable uptake of liposomal encapsulated drug. Such formulations are only working as slow-release depot formulations of the drug. Figure 5 Pharmacological effects of liposome and liposome-free incubations for amikacin (A 132 ) and for triamcinolone acetonide phosphate (B, unpublished observation) in cell culture. A represents situation A in figure 3. B, represents situation B in Figure 3. Note the right shift for the liposomal preparation, indicating a less favorable uptake of liposomal encapsulated drug. Such formulations are only working as slow-release depot formulations of the drug.

Arf Inactivation Contributes to Aggressive Disease in Immunocompetent Mice

A more refined approach entails intravenous (IV) injection of serial log dilutions of p210+ or p185+ donor cells into recipient mice. While 2 x 105 Arf+ + p210+ cells failed to establish leukemias in recipient animals and 2 x 106 Arf+ + p185+ cells produced disease with a 4-5 week latency, as few as 200 Arf- p210+ cells or 20 Arf-- p185+ cells (the lowest numbers yet evaluated) were capable of producing an aggressive leukemia within four weeks of injection (our unpublished data). These experiments argue that short-term Arf-null, p185+ and Arf-null p210+ pre-B cell cultures are highly enriched in leukemia-initiating cell activity, and that there may be little requirement (or opportunity) for in vivo selection of other collaborating genetic events. Furthermore, as five logs fewer Arf-null p185+ cells than p185+ WT cells were required to initiate leukemia in recipient animals, these experiments underscore the exceptional potency of Arf as a tumor suppressor in vivo.

Temporal Trends and Geographic Variability in Adverse Male Endpoints

Rates of cryptorchidism are difficult to compare geographically or temporally because of their sensitivity to such factors as age at diagnosis, diagnostic practice, and age at orchidopexy. Nevertheless, it is notable that substantial increases have been reported in many countries in Northern Europe and the US since the mid-1950s. These trends are consistent with those for testicular cancer, for which cryptorchidism is a significant risk factor. In 1984 Chilvers et al. 59 reported an increase in the incidence of cryptorchidism in England and Wales, most marked among boys under ten years. Using hospital discharge data, the authors estimated that the cumulative incidence of crypt-orchidism by age 15 had risen from 1.4 to 2.9 between 1952 and 1977. These authors suggested that a trend towards earlier (and possibly unnecessary) orchidopexy might have contributed to this increase. The incidence of cryptorchidism was assessed at three months of age among boys born in the Oxford area, using...

Analytical Methods for Protein Oxidation

Glycogen metabolism utilizes several enzymes that contain reactive sulfhydryls, i.e. glycogen synthase, glycogen phosphorylase, protein phosphatases and kinases. Enzymes involved in glycogen metabolism were some of the first in which oxidative mechanisms were thought to represent important regulatory mechanisms.63'64 Phosphoryase b (dephosphorylated) has been used as a model protein for study of protein sulfhydryl oxidation because commercially available protein of high purity is suitable for definitive studies on the mechanism of both S-thiolation and dethiolation.13 It has two reactive cysteines per subunit and oxidative modification by either S-thiolation or S-nitrosylation of these cysteines does not cause any apparent activity change, although the protein may have less affinity for the glycogen particle in the oxidized state (unpublished observations). The protein is easily studied by gel electro-focusing and by other molecular techniques.30 It has been used as a substrate...

Pharmacophores as Full Molecular Descriptors

One application of pharmacophore fingerprints is as a ranking descriptor in database searches. Pharmacophore fingerprints can be pre-calculated for database compounds, with conformational sampling, and stored in an efficient format. An example of this are pre-stored four-center pharmacophore fingerprints, where one line of encoded information uses -11 kilobytes of space for 1000 pharmacophores, (S. J. Cho and J. S. Mason, unpublished results). Comparisons of such databases against probe fingerprints can be accomplished at speeds of more than 700000 compounds per hour on a single Silicon Graphics R10000,250Mhz CPU. The results of these comparisons provide a powerful 3-D virtual screening method, both directly and as an adjunct to other methods. Similarity is measured using potential pharmacophore counts (per compound and in common) and similarity indices such as the modified Tanimoto index 15,99 shown in Eq. (7.1)

Interacting Proteins That Selectively Modulate Or Signaling Pathways

In addition to in vitro analysis of qOR-mPKCI interactions, some behavioral tests were performed. We observed an enhanced morphine-induced analgesia in mPKCI knockout mice and noted a faster development of tolerance to morphine-induced analgesia in knockout mice than in wild-type controls. Therefore, studies from our laboratory revealed some roles for mPKCI in fine-tuning the signaling of the stimulated qOR. The sensitization and phosphorylation states of qOR appeared to depend on qOR-mPKCI interactions in vitro. In experiments that quantified morphine-induced antinociception, our results indicate that qOR-mPKCI interactions have potential to alter pain perceptions in a living organism. Our results in experiments that measured morphine-induced dependence and tolerance also indicate that qOR-mPKCI interactions participate in manifestations of tolerance and dependence to an opiate drug (data unpublished but reported in the 2002 INRC meeting). Taken together, these data indicate that...

Breath Acetone as a Measure of Systemic Ketosis

Whereas AcAc, P-HBA, and ACET can all be measured in urine and plasma, ACET is distinct in that it is volatile and can be measured in the breath. Dietary manipulations such as fasting and KD consumption cause an increase in breath ACET (2,11-16). Because blood ACET equilibrates readily with alveolar air (7), we have been exploring breath ACET as a noninvasive measure of systemic ketosis in children with refractory seizures on a KD. In a recent study, we showed that children on a classic KD (based on dairy fat) had about 115 times more ACET in their breath than healthy or epilepsy controls (Fig. 1). In that study, we also documented that breath ACET levels in KD children ranged from 555 to 6671 nmol L, although more recent studies we have conducted in KD children have revealed that the range is actually much larger (approx 100-20,000 nmol L unpublished). We have also found that in chronically ketotic rats and mildly ketotic adults, breath ACET is a significant predictor of plasma...

Structurebased Crosslinking Of Proteindna Complexes

A recent development combines the use of protein-engineering and site-specific DNA-modification techniques to introduce disulfide cross-links into protein-DNA interfaces (79) P. Zhou & G.L.V., unpublished observations . The resulting disulfide-trapped complexes have proven amenable to structure determination by X ray and NMR, whereas the corresponding uncross-linked complexes had proven refractory to structure determination by standard methods. In disulfide trapping, a Cys residue is introduced by site-directed mutagenesis into the DNA-binding surface of a protein, and an alkanethiol tether is incorporated at a nearby site in the protein-binding surface of the DNA through synthetic chemistry. Application of this technology obviously requires some knowledge about likely contact points in the protein and DNA. Several convenient methods exist to tether thiol functionality to various positions in DNA. Convertible nucleosides (80 - 82), several of which are commercially available (Glen...

Separation and Determination of Chl a Esterified with Alcohols other than Phytol

In dark-grown barley and corn seedlings, we have consistently observed the presence of trace amounts of Chl a esteri-fied with long chain fatty alcohols (LCFA) other than phytol (Figure 1, pathway 14). Experimental difficulties caused by the low concentration of these tetrapyrroles have so far hindered determination of the nature of the esterifying LCFA at position 7 of the macrocycle. Because of interference by the sizable Pchlide a E pool, the dark-formed Chlide a E pool constituents can only be observed after separation of the fully ester-ified tetrapyrroles by HPLC. In the HPLC system described in section 10.2, the dark-formed Chlide a Es exhibit retention times of about 10.4 to 11.1 minutes (Rebeiz, unpublished). ing. As evidenced by on-line emission spectra of eluting peaks, the formation of 4 different Chlide a E can be detected during the first 500 milliseconds, following a 2.5-millisecond light treatment of etiolated barley and corn (Figure 1, pathway 13). The various Chlide...

Correction For The Dmso Effect By The Ashift Method

The ''A shift'' Avdeef, unpublished . It is thought to be caused in some cases by DMSO binding to the drugs. Just as the equilibrium model in Section 6.1.3 was expanded to allow for the salt solubility equilibrium, Eq. (6.4), the same can be done with a binding equation based on DMSO (e.g., in 0.5 v v)

Pharmacological Inhibition Of Cytokines Is Protective In Brain Injury

TNF-a production is regulated at both transcriptional and translational levels, so a TNF-a mRNA inhibitor such as rolipram (a phosphodiesterase inhibitor) or tyrphostins, which inhibit protein tyrosine kinases, could be used to treat TNF-a-mediated diseases.21,22 CNI-1493, a tetravalent guanylhydrazone compound that inhibits phosphorylation of p38 mitogen-activated protein (MAP) kinase, has been shown to selectively inhibit TNF-a synthesis.23,24 This drug, used in cerebral ischemia25 and trauma (unpublished observations from our laboratory), gave significant cerebroprotection. In our model of closed head injury, we inhibited TNF-a translation (dexanabi-nol),18 expression (pentoxyphylline), and activity (TNF-a-binding protein)26 and demonstrated neuroprotection. Namely, less edema formation and blood-brain barrier disruption were found, along with faster and greater clinical recovery. In addition, the anti-inflammatory cytokine interleukin-10 (IL-10) was used in a model of traumatic...

Additional Methodological Notes

The use of the anionic detergent sarkosyl to solubilize the overexpressed CTP from the inclusion body fraction is particularly critical. We have tested many nonanionic detergents that proved ineffective at solubilizing the CTP. Fiermonte et al. (1993) first demonstrated the effectiveness of sarkosyl in solubilizing mitochondrial transporters. We subsequently showed that 20 different mitochondrial transporters, which differ substantially in amino acid sequence, are effectively solubilized by this detergent (Kaplan et al., 1995 Mayor et al., 1997). It should be pointed out that, during the solubilization step, patience is required, as the complete extraction of transporter from inclusion bodies can take > 1 h of careful, repetitive pipetting and scraping. In addition, we have shown based on CD analysis that the detergent solubilized Cys-less CTP contains a substantial amount of a-helical content, which approaches that observed with transporter that has been reconstituted into...

Possible Effects Of Compartmentalized Electrical Signaling On Nmda Receptors

To fully reveal the effects of spine depolarization of synaptically evoked Ca transients, quantitative models of the action of Ca buffers61-65 on Ca handling may be used to calculate the Ca current into the spine. This can be accomplished by deconvolving the Ca transient with the impulse response of the spine to a brief, small increase in Ca.4,59 In regimes of linear Ca handling, this approach reveals the time course of the Ca current into the spine. In preliminary studies using this approach, the Ca current that underlies synaptically evoked Ca transients in spines of mouse CA1 pyramidal neurons is comprised of fast and slow components (Figure 9.1D through F authors' unpublished data).

Degeneration In The Cns Involves Common Mediators Of Toxicity

Memory.16 When its concentration increases, however, it can become cytotoxic.17-20 Extracellular glutamate is normally buffered via uptake by astro-cytes, and is recycled after being converted to glu-tamine.21-23 Under abnormal conditions, caused for example by acute or chronic CNS insults, the local buffering capacity in the CNS is apparently unable to control the inevitable increase in glutamate. Studies in our laboratory have shown that glutamate, when injected intravitreally at different concentrations into rats, exerts a dose-dependent effect on the recruitment of the immune system to counteract the cytotoxicity. This was demonstrated by comparing retinal ganglion cell (RGC) death in normal mice and mice devoid of T-cells (nude mice) from a strain capable of producing a protective T-cell-mediated response (Kipnis et al, unpublished results Schori et al, unpublished results Yoles et al, unpublished results). Interestingly, absence of T-cells was correlated with greater neuronal...

Role of ROS in the Regulation of Insulin Receptor Tyrosine Kinase Activity

Signaling by insulin involves autophosphorylation of the insulin receptor kinase domain at Tyr1158, Tyr1162, and Tyr1163. In line with the oxidative inhibition of tyrosine phosphatases by (approximately millimolar concentrations of) hydrogen peroxide or other strong oxidants (see previous section), it was found that similar strongly oxidative conditions enhance the tyrosine phosphorylation of the insulin receptor h-chain in an insulin-independent fashion. Lower and physiologically relevant concentrations (< 100 M) of hydrogen peroxide, in contrast, do not induce phosphorylation in the absence of insulin but enhance the response to insulin in a synergistic fashion 146 . More recent experiments have shown that the stimulatory effect of hydrogen peroxide can be demonstrated on highly purified preparations of the insulin receptor kinase domain, indicating that the insulin receptor kinase domain serves by itself as a redox-sensitive signaling compound (W. Droge, unpublished observation)....

Rhodopsin Phosphorylation A Rheostat Control for the Sensitivity of Phototransduction

It appears that similar complex patterns of receptor phosphorylation might be a common occurrence among GPCRs. For example, recent studies from our laboratory have determined that the M1-muscarinic is phosphorylated at least 11 times within the third intracellular loop and once within the C-tail (unpublished observation). Reconstitution of GRK-2 and casein kinase 1a with the partially purified M1-muscarinic receptor results in agonist-dependent phosphorylation (Haga et al. 1996 Waugh et al. 1999) indicating that like the M3-muscarinic receptor this receptor subtype might be a substrate for multiple protein kinases. Furthermore, the multi-site nature of GPCR phosphorylation has also been demonstrated from tryptic phosphosite mapping studies on the bradykinin B2 receptor (Blaukat et al. 2001), mass spectrometry analysis on the b2-adrenergic receptor and CXCR4 receptor (Busillo et al. 2010 Trester-Zedlitz et al. 2005) and the use of anti-phospho site antibodies on the somatostatin A2,...

Regulation of Catecholamine Synthesis in the Adrenal Medulla Is Achieved on Several Levels by the GR

Also unaltered, which was unexpected in the light of the description of a GRE in the rat promoter. However, when we analyzed hormone levels, we found a marked reduction of adrenalin and normal noradrenalin levels (unpublished data). Obviously, there must be a step in the control of adrenalin systhesis other than mRNA transcription that is dependent on DNA-binding of the receptor. This regulation could be at the level of PNMT enzyme activity, as it was reported that glucocorticoids are indeed involved in the control of this step (Wong et al., 1995). Our results suggest that DNA-binding-dependent mechanisms account for regulation of adrenalin synthesis by the GR, although not by influencing PNMT mRNA expression. Additionally, in contrast to previous findings (Ross et al., 1990), GRE binding to the PNMT promoter seems not to be crucial for basal expression of this gene.

Industrial Chemicals Toxication and Detoxication

Methane (Meyer et al. 1991a Hallier etal., 1993), ethylene oxide (Hallier el al., 1993), and probably (+ )-benzo(iv)pyrene-7,8-diol-9,10-oxide, which is a substrate for the rat homologue GST5-5 (B. Jernstrom, D. J. Meyer, and B. Ketterer, unpublished data). The GSTT1 null phenotype should reduce susceptibility to the toxicity of dichloromethane but may increase susceptibility to polycyclic aromatic hydrocarbon diol epoxides. In the etiology of tobacco-smoke-induced lung cancer, GSTT1 and GSTM1 poly-mophisms may be additive.

E GRdim Mice Offer the Opportunity to Further Analyze Molecular Mechanisms of Glucocorticoid Physiology

One of the major advantages of GRdim mice is the possibility of studying DNA-binding-independent regulation by the GR in adult mice. Among the most important functions which are considered to be mediated by crosstalk of the GR with various transcription factors such as AP-1 and NF-kB are all types of immunological processes (Gaillard, 1994). Therefore, we are presently analyzing various aspects of this type of control. First results suggest that repression of cytokines is widely intact in GRdim dim mice and also that the immunosuppressive action of the GR is largely retained in in vivo models for inflammatory reactions (unpublished data). Therefore, some of these paradigms may allow use of GRdim mice in the seach for pharmacological compounds acting preferentially through the DNA-binding-independent function of the GR.

Spanning a Topological Pharmacophore Space for Similarity Searching

As soon as some few lead structures have been identified, a very appealing approach is to derive a pharmacophore model from the known active structure (the seed or query structure) and perform a computer-based similarity search to speed up the process of lead-identification. It is known that ligands often bind to a receptor in a conformation other than their lowest energy conformations, where the degree of deformation can be correlated with the number of freely rotatable bonds in the molecule 64 , Thus it appears reasonable to develop pharmacophore models that are independent of 3-D structure, or accurately account for conformational flexibility. Several such approaches have been developed and successfully applied to similarity searching, database profiling, and design 65 , A simplistic alternative is to focus on special 2-D representations of molecules only. Topological correlation of generalized atom types builds on this ground. It is a special molecular descriptor that leads to a...

A cartoon representation of landscapes that map sequence to function

To test the relationships between sequence spaces of different sizes more directly, two different sequence spaces can be explored for function in parallel. The smaller space is a subset of the larger space (Fig. 9), but the topographies of different regions are unknown. We used libraries that span either 30 or 71 random sequence positions as the starting points for selections. The lengths of the random sequence tracts were arbitrary, but may be instructive 30-mers can form numerous minimal structural elements, such as stem-loops and pseudoknots, and many aptamers have been culled from 30-mer pools, while 71-mers can form more complex structural elements, such as helical junctions, and have the potential to generate architectures that are buttressed by tertiary structural interactions (e.g., tRNA at ca. 75 residues). RNA molecules that could bind to the tyrosine phos-phatase from Yersiniapestis (Yop51*A 162) were selected from these pools (S. Bell and A.D. Ellington, unpublished...

Acute and Chronic Transplant Rejection

For a brief period of warm ischaemia so that the decoy ODNs can be taken up by the endothelial cells of the donor organ (see Section 7.3). Moreover, these cells also appear to be the most appropriate target for the decoy ODN-mediated inhibition of pro-inflammatory transcription factors, since not only acute, but also chronic, transplant rejection is closely associated with endothelial dysfunction and microcirculatory perfusion failure. Consequently, decoy ODNs directed against NF-kB, which has been implicated in endothelial cell adhesion molecule and chemokine expression, at least in rodents, were the first to be tested in animal models of allograft rejection and vasculopathy. Pressure-mediated decoy ODN administration to rat cardiac allografts42 as well as perfusion of rat renal allografts with naked DNA43 or by using a contrast agent-ultrasound procedure44 resulted in a significantly increased survival of the transplants in the absence of immunosuppression or reduced graft...

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