Aquaporin-1 in the peritoneal membrane: Implications for water transport across capillaries and peritoneal dialysis

Peritoneal dialysis (PD) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced in the peritoneal cavity. The dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores (radius < 3 Å) responsible for the transport of solute-free water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water perfectly fit with the characteristics of the ultrasmall pore. Treatment with corticosteroids induces the expression of AQP1 in peritoneal capillaries and increases water permeability and ultrafiltration in rats, without affecting the osmotic gradient and the permeability for small solutes. Studies in knockout mice provided further evidence that osmotically-driven water transport across the peritoneal membrane is mediated by AQP1. AQP1 and endothelial NO synthase (eNOS) show a distinct regulation within the endothelium lining peritoneal capillaries. In acute peritonitis, the upregulation of eNOS and increased release of NO dissipate the osmotic gradient and result in ultrafiltration failure, despite the unchanged expression of AQP1. These data illustrate the potential of the peritoneal membrane to investigate the role and regulation of AQP1 in the endothelium. They also emphasize the critical role of AQP1 during peritoneal dialysis and suggest that manipulating AQP1 expression may be used to increase water permeability across the peritoneal membrane.     

Aquaporin-1 in the peritoneal membrane: Implications for water transport across capillaries and peritoneal dialysis

Peritoneal dialysis (PD) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced in the peritoneal cavity. The dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores (radius < 3 A) responsible for the transport of solute-free water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water perfectly fit with the characteristics of the ultrasmall pore. Treatment with corticosteroids induces the expression of AQP1 in peritoneal capillaries and increases water permeability and ultrafiltration in rats, without affecting the osmotic gradient and the permeability for small solutes. Studies in knockout mice provided further evidence that osmotically-driven water transport across the peritoneal membrane is mediated by AQP1. AQP1 and endothelial NO synthase (eNOS) show a distinct regulation within the endothelium lining peritoneal capillaries. In acute peritonitis, the upregulation of eNOS and increased release of NO dissipate the osmotic gradient and result in ultrafiltration failure, despite the unchanged expression of AQP1. These data illustrate the potential of the peritoneal membrane to investigate the role and regulation of AQP1 in the endothelium. They also emphasize the critical role of AQP1 during peritoneal dialysis and suggest that manipulating AQP1 expression may be used to increase water permeability across the peritoneal membrane.     

Increased survival of mesothelial cells from the peritoneum in peritoneal dialysis fluid

Peritonitis remains the most important factor in patient morbidity and technical failure associated with continuous ambulatory peritoneal dialysis (CAPD). In vitro examination of bacterial infection of cultured human peritoneal mesothelial cells (HPMC) is an attractive approach to the study of peritonitis in CAPD, yet there are few reports on this subject. Previous studies have shown two limitations: (i) cell cultures of HPMC lasted for days only when incubated in culture medium and (ii) short-term studies of <30 min were done in HPMC when incubated with peritoneal dialysis fluid (PDF). Human peritoneal mesothelial cells, maintained in a conventional single chamber culture system with PDF alone, were unable to survive more than 40 min. The present study was designed to prolong the viability of HPMC cultured in PDF, with the object of using cells under different conditions, such as that of simulating CAPD. HPMC were cultured using plastic microtiter plates, where they were grown to confluence and growth was arrested. PDF containing different concentrations of NaHCO3and human serum albumin was added. Cell viability after exposure for up to 24 h was measured by trypan blue, Cell Death Detection ELISA and Annex-V flow cytometry. The data confirmed the 'toxic' effect of PDF, with cell viability being <40% after 2 h incubation in 4.25% glucose in PDF. However, the survival time of HPMC increased significantly in 4.25% glucose PDF at a physiological pH and even further after the addition of human albumin. These experimental conditions simulating CAPD may allow future in vitro studies of mesothelial physiology and peritonitis related to CAPD treatment.     

Sulfotransferases of two specificities function in the reconstitution of high endothelial cell ligands for L-selectin.

L-selectin, a lectin-like receptor, mediates rolling of lymphocytes on high endothelial venules (HEVs) in secondary lymphoid organs by interacting with HEV ligands. These ligands consist of a complex of sialomucins, candidates for which are glycosylation- dependent cell adhesion molecule 1 (GlyCAM-1), CD34, and podocalyxin. The ligands must be sialylated, fucosylated, and sulfated for optimal recognition by L-selectin. Our previous structural characterization of GlyCAM-1 has demonstrated two sulfation modifications, Gal-6-sulfate and GlcNAc-6-sulfate in the context of sialyl Lewis x. We now report the cloning of a Gal-6-sulfotransferase and a GlcNAc-6-sulfotransferase, which can modify GlyCAM-1 and CD34. The Gal-6-sulfotransferase shows a wide tissue distribution. In contrast, the GlcNAc-6-sulfotransferase is highly restricted to HEVs, as revealed by Northern analysis and in situ hybridization. Expression of either enzyme in Chinese hamster ovary cells, along with CD34 and fucosyltransferase VII, results in ligand activity, as detected by binding of an L-selectin/IgM chimera. When coexpressed, the two sulfotransferases synergize to produce strongly enhanced chimera binding.     

SK HEP-1: A human cell line of endothelial origin

Summary SK-HEP-1 is an immortal, human cell line derived from the ascitic fluid of a patient with adenocarcinoma of the liver. We have determined that these cells are of endothelial origin. Despite the location of the tumor from which SK HEP-1 was derived, the cell line does not have properties of hepatocytes. Northern blot analysis of total cellular RNA shows no messenger RNA for the hepatic-specific proteins albumin, alpha-fibrinogen, or gamma-fibrinogen. Endothelial characteristics are seen by transmission electron microscopy. These features include numerous pinocytotic vesicles, electron dense granules consistent with Weibel-Palade bodies, and abundant intermediate filaments, identified immunocytochemically as vimentin. Cultures grown on plastic dishes grow in bundles of polygonal to spindle-shaped cells. Proteins characteristic for endothelial cells are identified by immunocytochemistry. Addition of basement membrane material (Matrigel) or type I collagen to the cultures induces these cells to organize into a tubular network.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.     

Platelet endothelial cell adhesion molecule‐1 (PECAM1) plays a critical role in the maintenance of human vascular endothelial barrier function

Cardiovascular endothelial barrier dysfunction is associated with a number of cardiovascular diseases. This study aims to investigate the role of platelet endothelial cell adhesion molecule‐1 (PECAM1) in the maintenance of the vascular endothelial barrier integrate. Human umbilical vein endothelial cells (HUVECs) were cultured into monolayers using as an in vitro model to assess the endothelial barrier function. Knockdown of the gene of PECAM1 markedly reduced the transendothelial resistance and increased the permeability of the HUVEC monolayers. From the wild HUVECs, we detected a complex of PECAM1, claudin1, occluding and endothelial cell selective adhesion molecule (ESAM); such a complex was not detected in the PECAM1‐deficient HUVECs. Knockdown of either claudin1, or occludin, or ESAM, did not affect the formation of the tight junction (TJ) complex. Exposure to recombinant interleukin (IL)‐13 inhibited the expression of PECAM1 and down‐regulated the HUVEC monolayer barrier function. PECAM1 plays an important role in the formation of TJ complex. Copyright © 2015 John Wiley & Sons, Ltd.     

The product of the gene GEF1 of Saccharomyces cerevisiae transports Cl- across the plasma membrane

Expression of GEF1 in Xenopus laevis oocytes and HEK-293 cells gave rise to a Cl- channel that remained permanently open and was blocked by nitro-2-(3-phenyl-propylamino) benzoic acid and niflumic acid. NPPB induced petite-like colonies, resembling the GEF1 knock-out. The fluorescent halide indicator SPQ was quenched in a wild-type strain, in contrast to both a GEF1 knock-out strain and yeast grown in the presence of NPPB. Immunogold and electron microscopy located Gef1p in the plasma membrane, vacuole, endoplasmic reticulum and Golgi apparatus. Eleven substitutions in five residues forming the ion channel of GEF1 were introduced; some of them (S186A, I188N, Y459D, Y459F, Y459V, I467A, I467N and F468N) did not rescue the pet phenotype, whereas F468A, A558F and A558Y formed normal colonies. All the pet mutants showed reduced O2 consumption, small mitochondria and mostly disrupted organelles. Finally, electron microscopy revealed that the plasma membrane of the mutants develop multiple foldings and highly ordered cylindrical protein-membrane complexes. All the experiments above suggest that Gef1p transports Cl- through the plasma membrane and reveal the importance of critical amino acids for the proper function of the protein as suggested by structural models. However, the mechanism of activation of the channel has yet to be defined.     

Pathway and kinetics of vitellogenin-gold internalization in the Xenopus oocyte

After in vitro incubation of Xenopus oocytes with vitellogenin (VTG)-gold conjugate, the gold particles are distributed on the whole plasma membrane. Their concentration in coated pits still occurs at 0 degrees C. At +20 degrees C the label quickly (30 sec) appears in multi-vesicular endosomes (MVE) which segregate together with primary endocytic vesicles into distinct clusters below the plasma membrane. From this step up to crystallization of the yolk platelets, the gold particles stay in the same compartment. During 5.5 h the label progressively increases along the MVE membrane, first (1.5 h) by fusion of primary endocytic vesicles with consecutively enlarging endosomes, then (4 h) by decreasing of the MVE membrane. As concerns the yolk platelet formation, concentration of primordial yolk platelets (PYP) occurs at 5.5 h from the incubation onset, the labeling of preexisting yolk platelets starts at 7 h, while crystallization of PYP begins only after 12-13 h. Our results indicate that VTG receptors are not preclustered in coated pits and their lateral translation is not inhibited at 0 degrees C. The yolk protein processing takes place within one compartment only. The VTG condensation begins with a long concentration phase of receptor-VTG complexes still integrated in the endosome membrane. It occurs in MVE by: i) a repeated fusion of primary endocytic vesicles; ii) removing part of the endosome membrane by internal vesiculation. Fusion between endosomes occurs only after VTG has dissociated from its receptors and VTG dissociates only when when the density of the VTG-receptor complexes in the endosome membrane is sufficient. Crystallization begins after a 7-8 h delay. The endosome migration into the oocyte is also controlled by the binding of VTG to its receptors. Our results also demonstrate that binding of VTG colloidal gold modifies neither the vitellogenic pathway nor the duration of the vitellogenin internalization. However when vitellogenin is bound to colloidal gold, dissociation of ligand-receptor complexes is delayed because the amount of ligand in the incubation medium is necessarily low.     

Identification of minimum carbohydrate moiety in N-glycosylation sites of brain endothelial cell glycoprotein 96 for interaction with Escherichia coli K1 outer membrane protein A

Bacterial meningitis is a serious central nervous system infection and Escherichia coli K1 (E. coli K1) is one of the leading etiological agents that cause meningitis in neonates. Outer membrane protein A (OmpA) of E. coli K1 is a major virulence factor in the pathogenesis of meningitis, and interacts with human brain microvascular endothelial cells (HBMEC) to cross the blood-brain barrier. Using site-directed mutagenesis, we demonstrate that two N-glycosylation sites (NG1 and NG2) in the extracellular domain of OmpA receptor, Ecgp96 are critical for bacterial binding to HBMEC. E. coli K1 invasion assays using CHO-Lec1 cells that express truncated N-glycans, and sequential digestion of HBMEC surface N-glycans using specific glycosidases showed that GlcNAc1-4GlcNAc epitopes are sufficient for OmpA interaction with HBMEC. Lack of NG1 and NG2 sites in Ecgp96 inhibits E. coli K1 OmpA induced F-actin polymerization, phosphorylation of protein kinase C-α, and disruption of transendothelial electrical resistance required for efficient invasion of E. coli K1 in HBMEC. Furthermore, the microvessels of cortex and hippocampus of the brain sections of E. coli K1 infected mice showed increased expression of glycosylated Ecgp96. Therefore, the interface of OmpA and GlcNAc1-4GlcNAc epitope interaction would be a target for preventative strategies against E. coli K1 meningitis.     

Human microvascular endothelial cell prostaglandin E1 synthesis during in vitro ischemia-reperfusion

Ischemia-reperfusion injury is a microvascular event documented in numerous in vivo animal models. In animal models, prostaglandin and prostaglandin analogues have been found to ameliorate reperfusion injury. These studies were undertaken to evaluate human microvascular endothelial PGE(1) synthesis during in vitro ischemia followed by reperfusion. Human (neonatal) microvascular endothelial cell (MEC) cultures (n = 6) were subjected to sequential 2 h periods of normoxia (20% O(2)), ischemia (1.5% O(2)), and reperfusion (20% O(2)). Prostaglandin E(2) synthesis in conditioned media was determined by ELISA. Steady state levels of MEC prostaglandin H synthase (PGHS)-1 and -2 mRNA were assessed at the end of each 2-h period using RT-PCR and a quantitative mRNA ELISA. MEC PGHS protein levels were analyzed using an ELISA. PGE(1) release increased significantly during the initial 30 min of ischemia, but rapidly fell below normoxic levels by 90 and 120 min. During reperfusion, PGE(1) release returned to normoxic levels at 30, 60, and 90 min, and exceeded normoxic levels at 120 min. PGHS-1 mRNA levels were undetectable during all experimental conditions. PGHS-2 mRNA levels were unchanged by ischemia, but were decreased by reperfusion. In contrast, PGHS-2 protein levels increased 3-fold during ischemia, and remained elevated during reperfusion. Human MEC do not express PGHS-1 mRNA in vitro. Prolonged ischemia decreases MEC PGE(1) synthesis, and stimulates increased PGHS-2 protein levels without altering the steady state levels of COX-2 mRNA. During reperfusion, increased PGHS-2 protein levels persist and are associated with stimulated PGE(2) secretion, despite relative decreases in PGHS-2 mRNA.     

Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine.     

Ion channel involvement in the temperature-sensitive response of the rabbit corneal endothelial cell resting membrane potential

Previous studies have shown that the resting potential (E _m) of the corneal endothelium hyperpolarizes following an increase in temperature above 24°C. Whole-cell studies using the perforated-patch technique were used to compare currents and E _mvalues from isolated corneal endothelial cells at 24 and 32°C. These studies revealed a small, outwardly rectifying, slowly activating, weakly voltage-dependent current with a reversal potential showing K⁺ selectivity (E _rev = –80 mV). This current had features similar to the whole-cell current seen following addition of HCO₃ ^– to these cells. E _mmeasurements found an average 24 mV hyperpolarization following temperature elevation in NaCl Ringer. Single channel studies found the only change in channel activity following an elevation in temperature to be an increase in the open probability (P _o) of a K⁺ channel previously reported in this cell type to be activated by external anions. P _o(–30 mV) at 24 and 32°C equaled 0.003 and 0.06, respectively. Increases in P _owere found at all voltages examined. This increased P _ocan account for the magnitude of the hyperpolarization seen in these cells following temperature elevation. Addition of HCO₃ ^– along with elevated temperature produced a synergistic effect on the increase in P _oalong with an increased hyperpolarization of the cell, pointing to separate mechanisms of activation from these two stimuli.The authors would like to thank Ms. Helen Hendrickson for her technical support and Drs. Gianrico Farrugia and Adam Rich for their helpful comments. This work was supported by NIH grants EY09673, EY03282, EY06005, and an unrestricted award from Research to Prevent Blindness.     

Dietary fats and membrane function: implications for metabolism and disease

Lipids play varied and critical roles in metabolism, with function dramatically modulated by the individual fatty acid moities in complex lipid entities. In particular, the fatty acid composition of membrane lipids greatly influences membrane function. Here we consider the role of dietary fatty acid profile on membrane composition and, in turn, its impact on prevalent disease clusters of the metabolic syndrome and mental illness. Applying the classical physiological conformer-regulator paradigm to quantify the influence of dietary fats on membrane lipid composition (i.e. where the membrane variable is plotted against the same variable in the environment--in this case dietary fats), membrane lipid composition appears as a predominantly regulated parameter. Membranes remain relatively constant in their saturated (SFA) and monounsaturated (MUFA) fatty acid levels over a wide range of dietary variation for these fatty acids. Membrane composition was found to be more responsive to n-6 and n-3 polyunsaturated fatty acid (PUFA) levels in the diet and most sensitive to n-3 PUFA and to the n-3/n-6 ratio. These differential responses are probably due to the fact that both n-6 and n-3 PUFA classes cannot be synthesised de novo by higher animals. Diet-induced modifications in membrane lipid composition are associated with changes in the rates of membrane-linked cellular processes that are major contributors to energy metabolism. For example, in the intrinsic activity of fundamental processes such as the Na+/K+ pump and proton pump-leak cycle. Equally, dietary lipid profile impacts substantially on diseases of the metabolic syndrome with evidence accruing for changes in metabolic rate and neuropeptide regulation (thus influencing both sides of the energy balance equation), in second messenger generation and in gene expression influencing a range of glucose and lipid handling pathways. Finally, there is a growing literature relating changes in dietary fatty acid profile to many aspects of mental health. The understanding of dietary lipid profile and its influence on membrane function in relation to metabolic dysregulation has exciting potential for the prevention and treatment of a range of prevalent disease states.     

An Accurate Model for the Expression of the Antisense RNA Block Gene:the Firefly Luciferase Gene-Xenopus Oocyte System

The plasmid p~(SV-Luc20)or the mRNA of luciferase gene transcribed from p~(SP64-Luc12)was introduced intothe nucleus or cytoplasm of Xenopus oocytes at stages 5-6 by microinjection.Then the injectedoocytes were incubated in MB medium at 18℃ for definite periods,and the crude enzyme ofluciferase was prepared.Results indicated that the luciferase gent and its mRNA could be transcribedand translated into the enzymatic protein of luciferase with high biological activity,and could alsocatalyze the substrates to emit light.If different ratios of firefly lucifcrasc gene and its antisense RNA were introduced together into thenucleus or cytoplasm of Xenopus oocytes,then the expression of firefly luciferase gone was severelyblocked.Since the lucifcrasc activity can be measured rapidly and quantitatively and the Xenopusoocytes obtained easily,the firefly luciferase gene-Xenopus oocyte system is an excellent model forrevealing quantitatively how the antisense RNA can block gene expression.     

A requirement for NF-protocadherin and TAF1/Set in cell adhesion and neural tube formation

Neurulation in vertebrates is an intricate process requiring extensive alterations in cell contacts and cellular morphologies as the cells in the neural ectoderm shape and form the neural folds and neural tube. Despite these complex interactions, little is known concerning the molecules that mediate cell adhesion within the embryonic neural plate and neural folds. Here, we demonstrate the requirement for NF-protocadherin (NFPC) and its cytosolic partner TAF1/Set for proper neurulation in Xenopus. Both NFPC and TAF1 function in cell-cell adhesion in the neural ectoderm, and disruptions in either NFPC or TAF1 result in a failure of the neural tube to close. This neural tube defect can be attributed to a lack of proper organization of the cells in the dorsal neural folds, manifested by a loss in the columnar epithelial morphology and apical localization of F-actin. However, the epidermal ectoderm is still able to migrate and cover the open neural tube, indicating that the fusions of the neural tube and epidermis are separate events. These studies demonstrate that NFPC and TAF1 function to maintain proper cell-cell interactions within the neural folds and suggest that NFPC and TAF1 participate in novel adhesive mechanisms that contribute to the final events of vertebrate neurulation.     

Quaternary structure and apical membrane sorting of the mammalian NaSi-1 sulfate transporter in renal cell lines

NaSi-1 encodes a Na⁺-sulfate cotransporter expressed on the apical membrane of renal proximal tubular cells, which is responsible for body sulfate homeostasis. Limited information is available on NaSi-1 protein structure and the mechanisms controlling its apical membrane sorting. The aims of this study were to biochemically determine the quaternary structure of the rat NaSi-1 protein and to characterize its expression in renal epithelial cell lines. Hexahistidyl-tagged NaSi-1 (NaSi-1-His) proteins expressed in Xenopus oocytes, appeared as two bands of about 60 and 75 kDa. PNGase F treatment shifted both bands to 57 kDa while endoglycosidase H treatment led to a downward shift of the lower molecular mass band only. Mutagenesis of a putative N-glycosylation site (N591S) produced a single band that was not shifted by endoglycosidase H or PNGase F, confirming a single glycosylation site at residue 591. Blue native-PAGE and cross-linking experiments revealed dimeric complexes, suggesting the native form of NaSi-1 to be a dimer. Transient transfection of EGFP/NaSi-1 in renal epithelial cells (OK, LLC-PK1 and MDCK) demonstrated apical membrane sorting, which was insensitive to tunicamycin. Transfection of the EGFP/NaSi-1 N591S glycosylation mutant also showed apical expression, suggesting N591 is not essential for apical sorting. Treatment with cholesterol depleting compounds did not disrupt apical sorting, but brefeldin A led to misrouting to the basolateral membrane, suggesting that NaSi-1 sorting is through the ER to Golgi pathway. Our data demonstrates that NaSi-1 forms a dimeric protein which is glycosylated at N591, whose sorting to the apical membrane in renal epithelial cells is brefeldin A-sensitive and independent of lipid rafts or glycosylation.     

The effects of diabetes and aminoguanidine treatment on endothelial function in a primate model of type 1 diabetes

Abnormalities of endothelial function have been demonstrated in diabetes and are thought to play a role in the pathogenesis of diabetic complications. The aims of this study were to determine whether aminoguanidine, an inhibitor of glycation, can prevent endothelial and microcirculation abnormalities in a primate model of type 1 diabetes. Male baboons (Papio hamadryas) were assigned to one of the four groups: control, diabetes, control treated with aminoguanidine or diabetes treated with aminoguanidine. Diabetes was induced by streptozocin (60 mg/kg) and treated with once daily injection of insulin. Aminoguanidine was given subcutaneously (10 mg/kg), once a day. Diabetic animals had a mean duration of diabetes of 8.9 +/- 3.4 years and HbA1c of 8.9 +/- 1.1%. Microvascular function was measured by laser Doppler velocimetry, with examination of endothelium-dependent increase in skin blood flow (SkBF) following iontophoresis of acetylcholine (ACh) and endothelium-independent increase in SkBF in response to the nitric oxide (NO) donor sodium nitroprusside (SNP). Multiple regression analysis identified diabetes (P = 0.049) and aminioguanidine treatment (P = 0.026) as significant determinants of ACh response. The diabetic baboons treated with aminoguanidine had less Ach-mediated SkBF response compared with controls (1.39 +/- 0.32 vs. 2.26 +/- 0.61, F = 3.3, P = 0.04), but there was no difference between groups in SkBF response to SNP. We conclude that endothelial dysfunction can be demonstrated in this primate model of type 1 diabetes at a stage when overt diabetic complications are not present. This occurred in the absence of insulin resistance or significant hypercholesterolemia. Administration of aminoguanidine from the onset of diabetes was not able to prevent this abnormality and in fact aggravated the endothelial response. Effects of aminoguanidine on NO synthase may contribute to this phenomenon.     

The DEAD box RNA helicase VBH-1 is required for germ cell function in C. elegans

Vasa and Belle are conserved DEAD box RNA helicases required for germ cell function. Homologs of this group of proteins in several species, including mammals, are able to complement a mutation in yeast (DED1) suggesting that their function is highly conserved. It has been proposed that these proteins are required for mRNA translation regulation, but their specific mechanism of action is still unknown. Here we describe functions of VBH-1, a C. elegans protein closely related to Belle and Vasa. VBH-1 is expressed specifically in the C. elegans germline, where it is associated with P granules, the C. elegans germ plasm counterpart. vbh-1(RNAi) animals produce fewer offspring than wild type because of defects in oocyte and sperm production, and embryonic lethality. We also find that VBH-1 participates in the sperm/oocyte switch in the hermaphrodite gonad. We conclude that VBH-1 and its orthologs may perform conserved roles in fertility and development.