Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation

Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-kappaB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.     

Cell-specific localization of insulin-like growth factor binding protein mRNAs in rat liver

The liver is a major source of circulating insulin-like growth factor I (IGF-I), and it also synthesizes several classes of IGF binding proteins (IGFBPs). Synthesis of IGF-I and IGFBPs is regulated by hormones, growth factors, and cytokines. They are nutritionally regulated and expressed in developmentally specific patterns. To gain insight into cellular regulatory mechanisms that determine hepatic synthesis of IGF-I and IGFBPs and to identify potential target cells for IGF-I within the liver, we studied the cellular sites of synthesis of IGF-I, IGF receptor, growth hormone (GH) receptor, and IGFBPs in freshly isolated rat hepatocytes, endothelial cells, and Kupffer cells. We also localized cellular sites of IGFBP synthesis by in situ hybridization histochemistry. Western ligand and immunoblot analyses were used to determine IGFBP secretion by isolated cells. Two IGF-I mRNA subtypes with different 5' ends (class 1 and class 2) were detected in all isolated liver cell preparations. Type 1 IGF receptor mRNA was detected in endothelial cells, indicating that these cells are a local target for IGF actions in liver. GH receptor was expressed in all cell preparations, consistent with GH regulation of IGF-I and IGFBP synthesis in multiple liver cell types. The IGFBPs expressed striking cell-specific expression. IGFBP-1 was synthesized only in hepatocytes, and IGFBP-3 was expressed in Kupffer and endothelial cells. IGFBP-4 was expressed at high levels in hepatocytes and at low levels in Kupffer and endothelial cells. Cell-specific expression of distinct IGFBPs in the liver provides the potential for cell-specific regulation of hepatic and endocrine actions of IGF-I.     

Deuterium-proton exchange on the native wild-type transthyretin tetramer identifies the stable core of the individual subunits and indicates mobility at the subunit interface

Transthyretin is a human protein capable of amyloid formation that is believed to cause several types of amyloid disease, depending on the sequence deposited. Previous studies have demonstrated that wild-type transthyretin (TTR), although quite stable, forms amyloid upon dissociation from its native tetrameric form into monomers with an altered conformation. Many naturally occurring single-site variants of TTR display decreased stability in vitro, manifested by the early onset familial amyloid diseases in vivo. Only subtle structural changes were observed in X-ray crystallographic structures of these disease associated variants. In this study, the stability of the wild-type TTR tetramer was investigated at the residue-resolution level by monitoring (2)H-H exchange via NMR spectroscopy. The measured protection factors for slowly-exchanging amide hydrogen atoms reveal a stable core consisting of strands A, B, E, F, and interestingly, the loop between strands A and B. In addition, the faster exchange of amide groups from residues at the subunit interfaces suggests unexpected mobility in these regions. This information is crucial for future comparisons between disease-associated and wild-type tetramers. Such studies can directly address the regions of TTR that become destabilized as a consequence of single amino acid substitutions, providing clues to aspects of TTR amyloidogenesis.     

Serological estimation of prey-protein gut-residence time and quantification of meal size for grass shrimp consuming meiofaunal copepods

A series of experiments using serological reagents was conducted to examine predation, ingestion and digestion in a model predator-prey system. The harpacticoid copepod Amphiascoides atopus, obtained from mass culture, was used as prey and the grass shrimp, Palaemonetes pugio, as predator. Bulk-gut passage time in P. pugio was measured by visualization of latex beads and ranged from 0.5 to 4 h in starved and continuously-fed grass shrimp. A polyclonal antibody was prepared from crude extracts of A. atopus; cross reactions with P. pugio and three other crustaceans were either negligible or not detected using slide agar-gel-double-immunodiffusion (AGID) and Western blot preparations. The presence of A. atopus antigens was detected with great sensitivity (e.g., seven copepods, 35 μg dry weight, gave positive results) in grass shrimp gut contents even when proteins of other crustacean prey were present. Prey-proteins could be detected for as long as 4 h with AGID and 8 h with Western blot techniques. Individual grass shrimp that were fed A. atopus and consumed from 0 to 98 copepods h(-1) were subjected to Western-blot preparation with chemiluminescence detection and densitometric evaluation. There was a significant curvilinear relationship between protein content and the number of copepod prey ingested. Results suggest that serological techniques can be modified to estimate the mass or abundance of standard-sized prey ingested by field-collected predators.     

Current status of antisense DNA methods in behavioral studies

The antisense DNA method has been used successfully to block the expression of specific genes in vivo in neuronal systems. An increasing number of studies in the last few years have shown that antisense DNA administered directly into the brain can modify various kinds of behaviors. These findings strongly suggest that the antisense DNA method can be used as a powerful tool to study causal relationships between molecular processes in the brain and behavior. In this article we review the current status of the antisense method in behavioral studies and discuss its potentials and problems by focusing on the following four aspects; (i) optimal application paradigms of antisense DNA methods in behavioral studies; (ii) efficiencies of different administration methods of antisense DNA used in behavioral studies; (iii) determination of specificity of behavioral effects of antisense DNA; and (iv) discrepancies between antisense DNA effects on behaviors and those on protein levels of the targeted gene.      

Src-mediated Post-translational Regulation of Endoglin Stability and Function Is Critical for Angiogenesis

Endoglin is a transforming growth factor β (TGF-β) co-receptor essential for angiogenesis and tumor vascularization. Endoglin modulates the crucial balance between pro- and anti-angiogenic signaling by activin receptor-like kinase (ALK) 1, 5, and TGF-β type II (TβRII) receptors. Despite its established role in physiology and disease, the mechanism of endoglin down-regulation remains unknown. Here we report that the conserved juxtamembrane cytoplasmic tyrosine motif (⁶¹²YIY⁶¹⁴) is a critical determinant of angiogenesis. Src directly phosphorylates this motif to induce endoglin internalization and degradation via the lysosome. We identified epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as Src-activators that induce endoglin turnover following ⁶¹²YIY⁶¹⁴ phosphorylation. Interestingly, Src phosphorylation of endoglin-⁶¹²YIY⁶¹⁴ was also an important process for receptor down-regulation by TRACON105 (TRC105), an endoglin-targeting antibody currently in clinical trials. The regulation of ⁶¹²YIY⁶¹⁴ phosphorylation was critical for angiogenesis, as both the phosphomimetic and unphosphorylatable mutants impaired endothelial functions including proliferation, migration, and capillary tube formation. Collectively, these findings establish Src and pro-angiogenic mitogens as critical mediators of endoglin stability and function.     

Drag reduction in polysaccharide solutions

Dilute solutions of many polysaccharides have lower frictional resistance in pipe flow than pure water. As yet unexplained, this effect may have important industrial ramifications.     

Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+) regulatory T cells in human autoimmune diseases

A lack of regulatory T (T(Reg)) cells that express CD4, CD25 and forkhead box P3 (FOXP3) results in severe autoimmunity in both mice and humans. Since the discovery of T(Reg) cells, there has been intense investigation aimed at determining how they protect an organism from autoimmunity and whether defects in their number or function contribute to the development of autoimmunity in model systems. The next phase of investigation - that is, to define the role that defects in T(Reg) cells have in human autoimmunity - is now underway. This Review summarizes our progress so far towards understanding the role of CD4(+)CD25(+)FOXP3(+) T(Reg) cells in human autoimmune diseases and the impact that this knowledge might have on the diagnosis and treatment of these diseases.     

The size heterogeneity of rat insulin-like growth factor-I mRNAs is due primarily to differences in the length of 3'-untranslated sequence

Previous studies have revealed multiple size classes of rat insulin-like growth factor-I (IGF-I) of estimated size 7.5-7.0, 1.9-1.5, and 1.2-0.9 kilobases (kb). Available sequence information accounts for only 2.1 kb of the 7.5-7.0 kb IGF-I mRNAs. We used oligomer directed ribonuclease H (RNase H) mapping to define the extent to which the unknown sequence in the large molecular weight mRNAs lies 5' or 3' to known sequence. Rat liver polyadenylated RNAs were incubated with oligomer probes complementary to internal rat IGF-I precursor (E domain) coding sequences. RNase H was used to hydrolyze IGF-I mRNAs at the point of annealment with the oligomers. Resultant 5' and 3'-IGF-I mRNA fragments were analyzed on Northern blots. A probe specific for type 1 (class C) 5'-sequences (the most predominant of multiple 5'-sequence types found on rat IGF-I mRNAs) identifies intact IGF-I mRNAs of 7.5-7.0, 1.9-1.5 and 1.2-0.9 kb but, after oligomer directed RNase cleavage of these mRNAs, identified only a single IGF-I mRNA 5'-fragment. Major differences in the length of sequence 5' to the IGF-I coding sequence therefore, do not account for the multiple size classes of type 1 (class C) IGF-I mRNAs. The size of the 5'-fragment suggests that the extent of sequence 5' to the IGF-I coding sequence is 0.4-0.7 kb in type 1 (class C) IGF-I mRNAs. Identification of multiple 3'-fragments of IGF-I mRNAs demonstrated heterogeneity in the 3'-ends of rat IGF-I mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)