Congenital malformations among newborns and developmental abnormalities among human embryos in Belarus after Chernobyl accident.

Evaluation of the effects of radioactive contamination on human populations is important for an understanding of the present and future risk for human health, including the genetic risk. This review centers on the results of population monitoring of developmental anomalies among human embryos and congenital malformations among newborn in the Republic of Belarus before and after Chemobyl accident. The data revealed that the incidences of developmental anomalies and congenital malformation from the mostly radionuclide-contaminated rural regions of Belarus reliably exceed the indices in control areas.     

Structural consensus in ligand-protein docking identifies recognition peptide motifs that bind streptavidin

Computational structure prediction of streptavidin-peptide complexes for known recognition sequences and a number of random di-, tri-, and tetrapeptides has been conducted, and mechanisms of peptide recognition with streptavidin have been investigated by a new computational protocol. The structural consensus criterion, which is computed from multiple docking simulations and measures the accessibility of the dominant binding mode, identifies recognition motifs from a set of random peptide sequences, whereas energetic analysis is less discriminatory. The predicted conformations of recognition tripeptide and tetrapeptide sequences are also in structural harmony and composed of peptide fragments that are individually unfrustrated in their bound conformation, resulting in a minimally frustrated energy landscape for recognition peptides. Proteins 28:421–433, 1997. © 1997 Wiley-Liss, Inc.     

Synthesis and biological evaluation of novel 5-aryl-4-(5-nitrofuran-2-yl)-pyrimidines as potential anti-bacterial agents

A facile two-step synthetic approach to fluorinated and non-fluorinated 5-aryl-4-(5-nitrofuran-2-yl)-pyrimidines from readily available 5-bromo-4-(furan-2-yl)pyrimidine has been developed. All synthesized compounds were screened in vitro for their antibacterial activities against twelve various bacterial strains. It is demonstrated that some of these compounds exhibited significant antibacterial activities against strains Neisseria gonorrhoeae and Staphylococcus aureus, comparable and even higher with that commercial drug Spectinomycin.     

Tumor suppressor Tsc1 is a new Hsp90 co‐chaperone that facilitates folding of kinase and non‐kinase clients

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat‐shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co‐chaperone for Hsp90 that inhibits its ATPase activity. The C‐terminal domain of Tsc1 (998–1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co‐chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1‐Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co‐chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90‐mediated folding of kinase and non‐kinase clients—including Tsc2—thereby preventing their ubiquitination and proteasomal degradation.     

Interfering with Wnt signalling alters the periodicity of the segmentation clock

Somites are embryonic precursors of the ribs, vertebrae and certain dermis tissue. Somite formation is a periodic process regulated by a molecular clock which drives cyclic expression of a number of clock genes in the presomitic mesoderm. To date the mechanism regulating the period of clock gene oscillations is unknown. Here we show that chick homologues of the Wnt pathway genes that oscillate in mouse do not cycle across the chick presomitic mesoderm. Strikingly we find that modifying Wnt signalling changes the period of Notch driven oscillations in both mouse and chick but these oscillations continue. We propose that the Wnt pathway is a conserved mechanism that is involved in regulating the period of cyclic gene oscillations in the presomitic mesoderm.     

Identification of a New Functional Domain in Angiopoietin-like 3 (ANGPTL3) and Angiopoietin-like 4 (ANGPTL4) Involved in Binding and Inhibition of Lipoprotein Lipase (LPL)

Angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4) are secreted proteins that regulate triglyceride (TG) metabolism in part by inhibiting lipoprotein lipase (LPL). Recently, we showed that treatment of wild-type mice with monoclonal antibody (mAb) 14D12, specific for ANGPTL4, recapitulated the Angptl4 knock-out (-/-) mouse phenotype of reduced serum TG levels. In the present study, we mapped the region of mouse ANGPTL4 recognized by mAb 14D12 to amino acids Gln²⁹–His⁵³, which we designate as specific epitope 1 (SE1). The 14D12 mAb prevented binding of ANGPTL4 with LPL, consistent with its ability to neutralize the LPL-inhibitory activity of ANGPTL4. Alignment of all angiopoietin family members revealed that a sequence similar to ANGPTL4 SE1 was present only in ANGPTL3, corresponding to amino acids Glu³²–His⁵⁵. We produced a mouse mAb against this SE1-like region in ANGPTL3. This mAb, designated 5.50.3, inhibited the binding of ANGPTL3 to LPL and neutralized ANGPTL3-mediated inhibition of LPL activity in vitro. Treatment of wild-type as well as hyperlipidemic mice with mAb 5.50.3 resulted in reduced serum TG levels, recapitulating the lipid phenotype found in Angptl3^-/- mice. These results show that the SE1 region of ANGPTL3 and ANGPTL4 functions as a domain important for binding LPL and inhibiting its activity in vitro and in vivo. Moreover, these results demonstrate that therapeutic antibodies that neutralize ANGPTL4 and ANGPTL3 may be useful for treatment of some forms of hyperlipidemia.Lipoprotein lipase (LPL)⁵ plays a pivotal role in lipid metabolism by catalyzing the hydrolysis of plasma triglycerides (TGs). LPL is likely to be regulated by mechanisms that depend on nutritional status and on the tissue in which it is expressed (1–3). Two secreted proteins, angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), play important roles in the regulation of LPL activity (4, 5). ANGPTL3 and ANGPTL4 consist of a signal peptide, an N-terminal segment containing coiled-coil domains, and a C-terminal fibrinogen-like domain. The N-terminal segment as well as full-length ANGPTL3 and ANGPTL4 have been shown to inhibit LPL activity, and deletion of the N-terminal segment of ANGPTL3 and ANGPTL4 resulted in total loss of LPL-inhibiting activity (6, 7). These observations clearly indicate that the N-terminal region of ANGPTL4 contains the functional domain that inhibits LPL and affects plasma lipid levels. The coiled-coil domains have been proposed to be responsible for oligomerization (8); however, it is not known whether the coiled-coil domains directly mediate the inhibition of LPL activity.To define the physiological role of ANGPTL4 more clearly, we characterized the pharmacological consequences of ANGPTL4 inhibition in mice treated with the ANGPTL4-neutralizing monoclonal antibody (mAb) 14D12 (9). Injection of mAb 14D12 significantly lowered fasting TG levels in C57BL/6J mice relative to levels in C57BL/6J mice treated with an isotype-matched anti-KLH control (KLH) mAb (9). These reduced TG values were similar to decreases in fasting plasma TG levels measured in Angptl4 knock-out (-/-) mice. This study demonstrated that mAb 14D12 is a potent ANGPTL4-neutralizing antibody that is able to inhibit systemic ANGPTL4 activity and thereby recapitulate the reduced lipid phenotype found in Angptl4^-/- mice. The readily apparent pharmacological effect of mAb 14D12 prompted new questions about the epitope recognized by mAb 14D12 and how this antibody-antigen binding event affected ANGPTL4 function as an LPL inhibitor.Although ANGPTL4 is able to interact directly with LPL (10), it is not clear which amino acids within ANGPTL4 mediate this interaction. Here we show that amino acids Gln²⁹–His⁵³ of mANGPTL4 contain the epitope for mAb 14D12. This region, hereby designated specific epitope 1 (SE1), also defines a domain that mediates the interaction between ANGPTL4 and LPL and the subsequent inactivation of LPL. With this information we present evidence that ANGPTL3 also contains an SE1 region, and with antibodies specifically reactive with ANGPTL3 SE1 we examine whether the ANGPTL3 SE1 region is involved in LPL binding and inhibition. We also determined whether treatment of C57BL/6 mice with an anti-ANGPTL3 SE1 mAb can recapitulate the phenotype of lower serum TG and cholesterol levels found in Angptl3^-/- mice. Finally we tested the therapeutic potential of an anti-ANGPTL3 SE1 mAb for treatment of hyperlipidemia in apolipoprotein E^-/- (ApoE^-/-) or low density lipoprotein receptor^-/- (LDLr^-/-) mice.     

Enzymes involved in metabolism of extracellular nucleotides and nucleosides: Functional implications and measurement of activities

Abstract

Extracellular nucleotides and nucleosides mediate diverse signaling effects in virtually all organs and tissues. Most models of purinergic signaling depend on functional interactions between distinct processes, including (i) the release of endogenous ATP and other nucleotides, (ii) triggering of signaling events via a series of nucleotide-selective ligand-gated P2X and metabotropic P2Y receptors as well as adenosine receptors and (iii) ectoenzymatic interconversion of purinergic agonists. The duration and magnitude of purinergic signaling is governed by a network of ectoenzymes, including the enzymes of the nucleoside triphosphate diphosphohydrolase (NTPDase) family, the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, ecto-5′-nucleotidase/CD73, tissue-nonspecific alkaline phosphatase (TNAP), prostatic acid phosphatase (PAP) and other alkaline and acid phosphatases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). Along with “classical” inactivating ectoenzymes, recent data provide evidence for the co-existence of a counteracting ATP-regenerating pathway comprising the enzymes of the adenylate kinase (AK) and nucleoside diphosphate kinase (NDPK/NME/NM23) families and ATP synthase. This review describes recent advances in this field, with special emphasis on purine-converting ectoenzymes as a complex and integrated network regulating purinergic signaling in such (patho)physiological states as immunomodulation, inflammation, tumorigenesis, arterial calcification and other diseases. The second part of this review provides a comprehensive overview and basic principles of major approaches employed for studying purinergic activities, including spectrophotometric P_i-liberating assays, high-performance liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analyses of purine substrates and metabolites, capillary electrophoresis, bioluminescent, fluorometric and electrochemical enzyme-coupled assays, histochemical staining, and further emphasizes their advantages, drawbacks and suitability for assaying a particular catalytic reaction.