Structural insights into the design of nonpeptidic isothiazolidinone-containing inhibitors of protein-tyrosine phosphatase 1B

Structural analyses of the protein-tyrosine phosphatase 1B (PTP1B) active site and inhibitor complexes have aided in optimization of a peptide inhibitor containing the novel (S)-isothiazolidinone (IZD) phosphonate mimetic. Potency and permeability were simultaneously improved by replacing the polar peptidic backbone of the inhibitor with nonpeptidic moieties. The C-terminal primary amide was replaced with a benzimidazole ring, which hydrogen bonds to the carboxylate of Asp(48), and the N terminus of the peptide was replaced with an aryl sulfonamide, which hydrogen bonds to Asp(48) and the backbone NH of Arg(47) via a water molecule. Although both substituents retain the favorable hydrogen bonding network of the peptide scaffold, their aryl rings interact weakly with the protein. The aryl ring of benzimidazole is partially solvent exposed and only participates in van der Waals interactions with Phe(182) of the flap. The aryl ring of aryl sulfonamide adopts an unexpected conformation and only participates in intramolecular pi-stacking interactions with the benzimidazole ring. These results explain the flat SAR for substitutions on both rings and the reason why unsubstituted moieties were selected as candidates. Finally, substituents ortho to the IZD heterocycle on the aryl ring of the IZD-phenyl moiety bind in a small narrow site adjacent to the primary phosphate binding pocket. The crystal structure of an o-chloro derivative reveals that chlorine interacts extensively with residues in the small site. The structural insights that have led to the discovery of potent benzimidazole aryl sulfonamide o-substituted derivatives are discussed in detail.     

Genotyping of recombinant Pichia pastoris strains

A simplified amplified-fragment length polymorphism (AFLP) method was used to genotype Pichia pastoris strains obtained by transformation of P. pastoris strain GS115 with a single integration vector. A total of 14 transformants and 3 control strains were analyzed, which generated 16 different band patterns. A clonal variation was obtained after the transformation process due to genetic differences generated during the transformation event of the host strain. Furthermore, the cluster analysis showed that the transformants with lesser genetic differences with respect to the P. pastoris host strain are the recombinant strains with the highest level of recombinant protein production.     

Tuberin: a stimulus-regulated tumor suppressor protein controlled by a diverse array of receptor tyrosine kinases and G protein-coupled receptors

Tuberin, a tumor suppressor protein, is involved in various cellular functions including survival, proliferation, and growth. It has emerged as an important effector regulated by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Regulation of tuberin by RTKs and GPCRs is highly complex and dependent on the type of receptors and their associated signaling molecules. Apart from Akt, the first kinase recognized to phosphorylate and inactivate tuberin upon growth factor stimulation, an increasing number of kinases upstream of tuberin have been identified. Furthermore, recruitment of different scaffolding adaptor components to the activated receptors appears to play an important role in the regulation of tuberin activity. More recently, the differential regulation of tuberin by various G protein family members have also been intensively studied, it appears that G proteins can both facilitate (e.g., G(i/o)) as well as inhibit (e.g., G(q)) tuberin phosphorylation. In the present review, we attempt to summarize our emerging understandings of the roles of RTKs, GPCRs, and their cross-talk on the regulation of tuberin.     

Normal T-cell development and immune functions in TRIM-deficient mice

The transmembrane adaptor molecule TRIM is strongly expressed within thymus and in peripheral CD4(+) T cells. Previous studies suggested that TRIM is an integral component of the T-cell receptor (TCR)/CD3 complex and might be involved in regulating TCR cycling. To elucidate the in vivo function of TRIM, we generated TRIM-deficient mice by homologous recombination. TRIM(-/-) mice develop normally and are healthy and fertile. However, the animals show a mild reduction in body weight that appears to be due to a decrease in the size and/or cellularity of many organs. The morphology and anatomy of nonlymphoid as well as primary and secondary lymphoid organs is normal. The frequency of thymocyte and peripheral T-cell subsets does not differ from control littermates. In addition, a detailed analysis of lymphocyte development revealed that TRIM is not required for either positive or negative selection. Although TRIM(-/-) CD4(+) T cells showed an augmented phosphorylation of the serine/threonine kinase Akt, the in vitro characterization of peripheral T cells indicated that proliferation, survival, activation-induced cell death, migration, adhesion, TCR internalization and recycling, TCR-mediated calcium fluxes, tyrosine phosphorylation, and mitogen-activated protein family kinase activation are not affected in the absence of TRIM. Similarly, the in vivo immune response to T-dependent and T-independent antigens as well as the clinical course of experimental autoimmune encephalomyelitis, a complex Th1-mediated autoimmune model, is comparable to that of wild-type animals. Collectively, these results demonstrate that TRIM is dispensable for T-cell development and peripheral immune functions. The lack of an evident phenotype could indicate that TRIM shares redundant functions with other transmembrane adaptors involved in regulating the immune response.     

Evidence for intracellular spatial separation of hexokinases and fructokinases in tomato plants

Four hexokinase (LeHXK1–4) and four fructokinase (LeFRK1–4) genes were identified in tomato plants. Previous GFP fusion studies indicate that the gene product of LeHXK3 is associated with the mitochondria while that of LeHXK4 is located within plastids. In this study we found that the enzyme encoded by the fructokinase gene LeFRK3 is also located within plastids. The presence of LeFrk3 enzyme in plastids raises the question of the origin of fructose in these organelles. The other three FRKs enzymes, LeFrk1&2&4, are located in the cytosol. Unlike LeFrk1&2&4, the two additional HXKs, LeHxk1&2, share a common membrane anchor domain and are associated with the mitochondria similar to LeHxk3. The difference in the locations of the cytoplasmic FRK and HXK isozymes suggests that glucose phosphorylation is confined to defined special intracellular localizations while fructose phosphorylation is less confined.Contribution from the Agriculture Research Organization, The Volcani Center, Bet Dagan, Israel, No. 126/2006 series.     

Alteration of the menstrual cycle in baboons placed on tethering devices and moved to individual housing--a stress model for a follicular phase defect

BACKGROUND: During an attempt to identify endocrine characteristics in the baboon that would more precisely predict ovulatory status for assisted reproductive techniques, we observed severe alterations in the menstrual cycle length upon introducing an environmental stress. This environmental stress involved moving animals from their baseline gang cage environment to individual indoor caging and placing them on a tethering apparatus. METHODS: Five adult female baboons were followed for changes in sex skin indicative of menstrual cycle timing and move from outdoor gang gages to individual indoor cages during the early follicular phase of their cycle. A tether device including a surgically implanted cannula was then installed to facilitate daily blood draws without sedation. Radioimmuonoassays were performed to monitor serum estradiol levels and lapraroscopic surveillance was used to confirm time of ovulation. RESULTS: Complete data sets were collected from four of the female baboons. In each case, a prolongation of the menstrual cycle was noted either during the cycle during which the females were moved to indoor caging or during the cycle immediately following the move. This prolongation was isolated to the follicular phase of the affected cycle. CONCLUSIONS: We conclude that otherwise normal handling procedures, including movement to new caging, and/or installation of a tether device, can impart a stress effect on reproductively cycling adult female baboons, such that folliculogenesis is delayed.     

Pyridazines. Part XXIX: synthesis and platelet aggregation inhibition activity of 5-substituted-6-phenyl-3(2H)-pyridazinones. Novel aspects of their biological actions

A series of 6-phenyl-3(2H)-pyridazinones with a diverse range of substituents in the 5-position have been prepared and evaluated in the search for new antiplatelet agents. A significant dependence of the substituent on the inhibitory effect has been observed. The pharmacological study of these compounds confirms that modification of the chemical group at position 5 of the 6-phenyl-3(2H)-pyridazinone system influences both variations in the antiplatelet activity and the mechanism of action.     

Crystal structure of a human rhinovirus that displays part of the HIV-1 V3 loop and induces neutralizing antibodies against HIV-1

We report the 2.7 A resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I beta turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.     

Developmental endocrinology of the reproductive axis in the chicken embryo

In mammals, the phenotype of the homogametic sex develops in the (relative) absence of steroids and the phenotype of the heterogametic sex is imposed by the early action of steroids. In contrast, the heterogametic sex in avian species is the female and the presence of estrogens and their receptors plays a crucial role in female sexual differentiation. The time- and sex-dependent expression of enzymes involved in steroidogenesis which determine the ratio of androgens/estrogens produced by the gonads has been extensively investigated during the last 5-6 years. These results all show that the lack of estrogen synthesis in the male appears to be due to the extremely low levels of 17beta-hydroxysteroid dehydrogenase and P450aromatase expression. In females, extensive expression of the aromatase gene (around day 5-6 of incubation), leading to estrogen synthesis, and specific expression of the estrogen receptor-mRNA in the left gonad results in the development of a functional left ovary. Other sex differences can be found in the expression of the inhibin subunit genes in gonads of chicken embryos and in circulating concentrations of inhibin, follicle stimulating hormone (FSH) and steroids. Sex reversal attempts have been made by varying incubation temperatures, by using anti-estrogens, androgens, aromatase inhibitors and synthetic steroids. In ovo administration of a sex steroid hormone or an inhibitor of endogenous sex steroid synthesis can cause phenotypical sex reversal. All these experiments show that the development of gonads in birds is very sensitive to changes in the embryonic hormonal environment, sometimes resulting in changes of postnatal reproduction and even growth.