Sperm binding to oviductal epithelium probably serves to form the isthmic sperm reservoir. This interaction of sperm and oviductal epithelium may involve species-specific carbohydrate recognition. We tested a series of carbohydrates and glycoproteins for inhibition of bovine sperm binding to oviductal epithelium in vitro. Explants of isthmic and ampullar epithelium were obtained from oviducts that had been surgically removed from preovulatory heifers. The explants were incubated (39 degrees C, 5% CO2) with fetuin, asialofetuin, ovalbumin, fucoidan, fucose, N-acetyl glucosamine, or N-acetyl glucosamine sulfate dissolved in a modified Tyrode's balanced salt solution, termed sperm-TALP (pH 7.4, 295 mOsm) for 10 min before frozen-thawed motile sperm obtained by swim-up were added. After 15 min, the explants were rinsed, and sperm binding density was evaluated. Oviductal explants treated with fucoidan (3 mg/ml; p < 0.001, n = 5) or fucose (31 mM, p < 0.01, n = 6) had reduced densities of bound sperm compared to the controls. Incubation of explants in increasing concentrations of fucose (4-62 mM) resulted in increased inhibition of sperm binding. Pretreating explants with fucosidase also reduced sperm binding (p < 0.001, n = 3) compared to that in controls containing the fucosidase inhibitor deoxyfuconojirimycin. The presence of fucosylated molecules on the surface of the oviductal epithelium was confirmed by labeling with fucose lectins from Ulex europeus and Lotus tetragonolobus. We conclude that fucose is involved in a specific interaction between bovine sperm and oviductal epithelium.

OBJECTIVENonalcoholic steatohepatitis (NASH) is increasingly common in obese patients. However, its metabolic consequences in patients with type 2 diabetes mellitus (T2DM) are unknown.RESEARCH DESIGN AND METHODSWe studied 154 obese patients divided in four groups: 1) control (no T2DM or NAFLD), 2) T2DM without NAFLD, 3) T2DM with isolated steatosis, and 4) T2DM with NASH. We evaluated intrahepatic triglycerides by proton MRS (1H-MRS) and assessed insulin secretion/resistance during an oral glucose tolerance test and a euglycemic-hyperinsulinemic clamp with glucose turnover measurements.RESULTSNo significant differences among groups were observed in sex, BMI, or total body fat. Metabolic parameters worsened progressively with the presence of T2DM and the development of hepatic steatosis, with worse hyperinsulinemia, insulin resistance, and dyslipidemia (hypertriglyceridemia and low HDL cholesterol) in those with NASH (P < 0.001). Compared with isolated steatosis, NASH was associated with more dysfunctional and insulin-resistant adipose tissue (either as insulin suppression of plasma FFA [33 ± 3 vs. 48 ± 6%] or adipose tissue insulin resistance index [9.8 ± 1.0 vs. 5.9 ± 0.8 mmol/L ⋅ µIU/mL]; both P < 0.03). Furthermore, insulin suppression of plasma FFA correlated well with hepatic steatosis (r = –0.62; P < 0.001) and severity of steatohepatitis (rs = −0.52; P < 0.001). Hepatic insulin sensitivity was also more significantly impaired among patients with T2DM and NASH, both fasting and with increasing insulin levels within the physiological range (10 to 140 µIU/mL), compared with other groups.CONCLUSIONSIn obese patients with T2DM, the presence of NAFLD is associated with more severe hyperinsulinemia, dyslipidemia, and adipose tissue/hepatic insulin resistance compared with patients without NAFLD. The unfavorable metabolic profile linked to NAFLD should prompt strategies to identify and treat this population early on.

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Oviductal sperm reservoirs have been found in cattle, mice, hamsters, pigs, and horses. In cattle (Bos taurus), the reservoir is evidently formed when sperm bind to fucosylated ligands resembling Le(a) trisaccharide on the surface of oviductal epithelium. The aim of this study was to characterize the fucose-binding protein on bull sperm. Fresh ejaculated sperm were extracted with 0.5 M KCl in Hepes-balanced salts. Extracts were subjected to affinity chromatography using immobilized Le(a) trisaccharide (alpha-L-Fuc[1,4]-beta-D-Gal[1,3]-D-GlcNAc). Two-dimensional PAGE of the affinity chromatography eluates revealed a prominent protein of approximately 16.5 kDa and a pI of 5.8. This protein inhibited binding of sperm to oviductal explants. A similar analysis of proteins extracted from capacitated sperm (which do not bind to oviductal epithelium) showed a reduction in the amount of the 16.5-kDa protein. When examined by epifluorescence microscopy, live uncapacitated sperm labeled over the acrosome with a fucose-BSA-fluorescein isothiocyanate (FITC) conjugate, while capacitated sperm did not. When capacitated sperm were treated with 16.5-kDa protein, labeling with fucose-BSA-FITC was partially restored. The comparative ease with which the protein was removed from sperm and its apparent reassociation with sperm suggested that it could be a peripheral protein derived from epididymal or accessory gland fluids. Blots of SDS-PAGE gels of seminal plasma proteins revealed the presence of a Le(a)-binding protein with an apparent mass of 16.5 kDA: Amino acid sequencing of two tryptic fragments of the protein purified from sperm extracts identified it as PDC-109 (BSP-A1/A2), a product of the seminal vesicles.

SummaryAcute pulmonary injury is known as acute chest syndrome (ACS) in patients with sickle cell disease (SCD). Secretory phospholipase A 2 (sPLA 2 ) was found to predict those at risk for ACS and a trial was designed to determine if red blood cell transfusion can be used to prevent ACS. Patients with an elevated sPLA 2 were randomised to either receive a single transfusion or standard care. Five of the eight patients (63%) randomised to standard care developed ACS versus none of the seven patients randomised to the transfusion arm (P ¼ 0AE026, Odds ratio ¼ 23AE6, 95% confidence interval 1, 557). This study suggests that transfusion may prevent ACS.

Sperm binding to oviductal epithelium produces a reservoir in vivo that may serve to maintain sperm fertility and provide sperm for fertilization when ovulation occurs. Previously, it was determined that bull sperm binding could be blocked by fucoidan and its component fucose; furthermore, treatment of epithelium with fucosidase prevented binding. The present study was conducted to further characterize binding. Because fucose would probably be present on the epithelium as part of oligosaccharide moieties of glycoproteins and/or glycolipids, competitive binding inhibition activity was tested for fucose in five linkages commonly found in oligosaccharides. Binding inhibition was assayed by determining the concentration of motile, frozen/thawed sperm bound to fresh epithelial explants in the presence of test inhibitors. Initially, 5 monosaccharides were tested at 30 mM (fucose, mannose, sialic acid, glucose, N-acetyl glucosamine, and galactose), and only fucose significantly reduced sperm binding compared to vehicle control (p = 0.03). Of the oligosaccharides tested (lacto-N-fucopentaose I, 3-fucosyllactose, Lewis-X, Lewis-a, and GlcNAcbeta1-4[Fucalpha1-6]GlcNAc-O-Me), only Lewis-a significantly reduced binding, and it did so in a dose-dependent fashion (p = 0.009 at 12.5 mM). Ca2+ dependency of binding was examined. Sperm were incubated with explants in Tyrode's albumin lactate pyruvate (TALP) containing 2 mM CaCl2 or lacking CaCl2 and containing 2 mM EGTA. Sperm-binding density was reduced significantly in EGTA (p < 0.03) but could be restored by readdition of CaCl2. Also, live sperm were labeled with the oligosaccharide ligand Lewis-a conjugated to fluorescein isothiocyanate-tagged polyacrylamide. Sperm exhibited labeling on the head only in the presence of Ca2+. Labeling could be blocked by fucose or Lewis-a-polyacrylamide. It was concluded that bull sperm bind to an oligosaccharide ligand on the oviductal epithelium that resembles Lewis-a and that binding is Ca2+-dependent.

The NADPH oxidase system is a promising target for correcting vasoreparative dysfunction in diabetic EPCs.

p45NF-E2 is a member of the cap 'n' collar (CNC)-basic leucine zipper family of transcriptional activators that is expressed at high levels in various types of blood cells. Mice deficient in p45NF-E2 that were generated by gene targeting have high mortality from bleeding resulting from severe thrombocytopenia. Surviving p45nf-e2 ؊/؊ adults have mild anemia characterized by hypochromic red blood cells (RBCs), reticulocytosis, and splenomegaly. Erythroid abnormalities in p45nf-e2 ؊/؊ animals were previously attributed to stress erythropoiesis caused by chronic bleeding and, possibly, ineffective erythropoiesis. Previous studies suggested that CNC factors might play essential roles in regulating expression of genes that protect cells against oxidative stress. In this study, we found that p45NF-E2-deficient RBCs have increased levels of reactive oxygen species and an increased susceptibility to oxidative-stress-induced damage. Deformability of p45NF-E2-deficient RBCs was markedly reduced with oxidative stress, and mutant cells had a reduced life span. One possible reason for increased sensitivity to oxidative stress is that catalase levels were reduced in mutant RBCs. These findings suggest a role for p45NF-E2 in the oxidative-stress response in RBCs and indicate that p45NF-E2 deficiency contributes to the anemia in p45nf-e2 ؊/؊ mice. IntroductionEukaryotic cells have a battery of mechanisms to defend against the harmful effects of reactive oxygen molecules, 1,2 including antioxidant molecules that function by directly inactivating reactive oxygen molecules and enzymes that metabolically convert toxic compounds to forms that are readily excreted by cells. In many cases, transcriptional activation of genes that play a role in detoxification of xenobiotics and defense against oxidative stress is mediated partly by the antioxidant response element (ARE). For example, AREs were found in promoter sequences of genes including nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase, heme oxygenase, glutathione-S-transferases (GST), and glutamylcysteine synthetase. [3][4][5][6][7] Several different transcription factors, including basic leucine zipper (bZIP) proteins, activator protein 1 (AP-1), and other novel factors, were shown to bind the ARE. 4,8 The ARE consensus sequence is very similar to the NF-E2/AP1-like sequence of the ␤-globin locus control region, which was found to be essential for globin gene expression. Multiple proteins can interact with the NF-E2 consensus sequence. The cap 'n' collar (CNC)-bZIP factor family of proteins was identified from searches for proteins that bind and activate the NF-E2/AP1 site of the ␤-globin locus control region. This multiple-protein family includes p45NF-E2, NF-E2-related factor (Nrf) 1, Nrf2, Nrf3, and the more distantly related Bach1 and Bach2. [9][10][11][12][13][14][15] The similarities among CNC family members are most notable in the basic-DNA binding region and another homology domain spanning 43 amino acids immediately N-terminal to the basic domain. 16 Thi...