Absence of N addition facilitates B cell development,but impairs immune responses

The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT^−/−) and wild-type (TdT^+/+) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT^−/− cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP₁₉CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT^−/− bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgM^a and congenic TdT-sufficient CB17 IgM^b bone marrow were placed in competition. TdT^−/− cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.     

Angiotensin II-mediated oxidative stress promotes myocardial tissue remodeling in the transgenic (mRen2) 27 Ren2 rat

Angiotensin II (ANG II) contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. ANG II stimulation of the ANG type 1 receptor (AT(1)R) generates reactive oxygen species via NADPH oxidase, which facilitates this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo AT(1)R blockade (AT(1)B) (valsartan) or superoxide dismutase/catalase mimetic (tempol) treatment in a rodent model of chronically elevated tissue levels of ANG II, the transgenic (mRen2) 27 rat (Ren2). Ren2 rats overexpress the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6-7 wk old) male Ren2 and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (1 mmol/l), or placebo for 3 wk. Heart tissue NADPH oxidase (NOX) activity and immunohistochemical analysis of subunits NOX2, Rac1, and p22(phox), heart tissue malondialdehyde, and insulin-stimulated protein kinase B (Akt) activation were measured. Structural changes were assessed with cine MRI, transmission electron microscopy, and light microscopy. Increases in septal wall thickness and altered systolic function (cine MRI) were associated with perivascular fibrosis and increased mitochondria in Ren2 on light and transmission electron microscopy (P < 0.05). AT(1)B, but not tempol, reduced blood pressure (P < 0.05); significant improvements were seen with both AT(1)B and tempol on NOX activity, subunit expression, malondialdehyde, and insulin-mediated activation/phosphorylation of Akt (each P < 0.05). Collectively, these data suggest cardiac oxidative stress-induced structural and functional changes are driven, in part, by AT(1)R-mediated increases in NADPH oxidase activity.     

Winter wheat (Triticum aestivum) response to Barley yellow dwarf virus at various nitrogen application rates in the presence and absence of its aphid vector,Rhopalosiphum padi

Barley yellow dwarf (BYD) is one of the most common diseases of cereal crops, caused by the phloem‐limited, cereal aphid‐borne Barley yellow dwarf virus (BYDV) (Luteoviridae). Delayed planting and controlling aphid vector numbers with insecticides have been the primary approaches to manage BYD. There is limited research on nitrogen (N) application effects on plant growth, N status, and water use in the BYDV pathosystem in the absence of aphid control. Such information will be essential in developing a post‐infection management plan for BYDV‐infected cereals. Through a greenhouse study, we assessed whether manipulation of N supply to BYDV‐infected winter wheat, Triticum aestivum L. (Poaceae), in the presence or absence of the aphid vector Rhopalosiphum padi L. (Hemiptera: Aphididae), could improve N and/or water uptake, and subsequently promote plant growth. Similar responses of shoot biomass and of water and N use efficiencies to various N application rates were observed in both BYDV‐infected and non‐infected plants, suggesting that winter wheat plants with only BYDV infection may be capable of outgrowing infection by the virus. Plants, which simultaneously hosted aphids and BYDV, suffered more severe symptoms and possessed higher virus loads than those infected with BYDV only. Moreover, in plants hosting both BYDV and aphids, aphid pressure was positively associated with N concentration within plant tissue, suggesting that N application and N concentration within foliar tissue may alter BYDV replication indirectly through their influence on aphid reproduction. Even though shoot biomass, tissue N concentration, and water use efficiency increased in response to increased N application, decision‐making on N fertilization to plants hosting both BYDV and aphids should take into consideration the potential of aphid outbreak and/or the possibility of reduced plant resilience to environmental stresses due to decreased root growth.     

Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1

The mannose-binding capability of recombinant wild-type boar spermadhesin AQN-1 and of its site-directed mutants in the highly-conserved region around of the single glycosylation site (asparagine 50) of some spermadhesins, where the carbohydrate binding site has been proposed to be located, was checked using a solid-phase assay and a biotinylated mannose ligand. Substitution of glycine 54 by amino acids bearing an unipolar side chain did not cause significant decrease in the mannose-binding activity. However, amino acids with uncharged polar side chains or having a charged polar side chain abolished the binding of biotinylated mannose to the corresponding AQN-1 mutants. The results suggest that the higher surface accessibility of amino acids possessing polar side chains compared to those bearing nonpolar groups may sterically interfere with monosaccharide binding. The location of the mannose-binding site in AQN-1 appears to be topologically conserved in other heparin-binding boar spermadhesins, i.e., AQN-3 and AWN, but departs from the location of the mannose-6-phosphate-recognition site of PSP-II. This indicates that different spermadhesin molecules have evolved non-equivalent carbohydrate-binding capabilities, which may underlie their distinct patterns of biological activities.     

Function of the mammalian oviductal sperm reservoir.

Sperm are stored in the isthmic region of the oviduct under conditions that maintain sperm viability and suppress motility. This region is also the site in which essential steps of the capacitation process are coordinated with the appearance of the ovulated egg. The influx of Ca(2+) and phosphorylation of sperm proteins are features of the ongoing capacitation process. Using a cell-culture system of oviductal epithelial cells, it was found that sperm bound to the epithelial cells showed a reduced Ca(2+) uptake and almost no tyrosine phosphorylation as shown by indirect immunofluorescence. Furthermore, sperm viability, measured as membrane integrity with propidium iodide, is significantly prolonged as compared to sperm in suspension. The formation of the sperm reservoir appears to be mediated by carbohydrate-protein interaction. In the pig, it has been found that mannosyl-oligosaccharides exposed by the epithelial cells are high-affinity ligands for sperm-associated lectins. Ovalbumin and mannopentaose are effective inhibitors of sperm binding to explants of oviductal epithelium. Spermadhesins, a new class of animal lectins and the major secretory products of the porcine seminal vesicle, associate with the sperm surface at ejaculation and are candidate molecules for the receptors of the epithelial carbohydrates.     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.     

Hepatic mechanisms of the Walnut antidiabetic effect in mice

The present study was designed to explore the mechanism of action of walnut (the seed of Juglans regia) leaf and ridge on hepatic glucose metabolism in diabetic mice. Experimental diabetes was induced by intravenous administration of streptozotocin (60 mg/kg)and confirmed with an increase of blood glucose, 90–100% of the control, 72 hours later. Isolated extracts from walnut leaf and ridges were administered in a single effective dose of 400 mg/kg orally. Firstly, blood glucose was determined every 1 hour until 5 hours post administration of extracts. In the second experiment, the liver was surgically removed, 2 hours post treatment of diabetic animals with extracts, homogenized and used for measurement of key enzymes of glycogenolysis (glycogen phosphorylase, GP) and gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPCK). Treatment by both leaf and ridge extracts decreased blood glucose and liver PEPCK activity and increased blood insulin and liver GP activity. It is concluded that walnut is able to lower blood glucose through inhibition of hepatic gluconeogenesis and secretion of pancreatic insulin.     

Structural and molecular characterization of equine sperm-binding fibronectin-II module proteins

Phospholipid-binding proteins in the male genital tract are characterized by differing numbers Fn-2 modules (B-domain) carrying N-terminal extensions (A-domain) of variable length. In the stallion, three different proteins were identified, SP-1, SP-2, and EQ-12. SP-1 and SP-2 of the AA'BB'- and ABB'-type, respectively, are major proteins of the seminal plasma. Here we report the cDNA sequences of SP-1, and of a new member of the SP-2 family (SPnew) and the partial characterization of their iso- and glycoforms. The phosphorylcholine (PC)-binding ability of the long Fn-2 protein, EQ-12, with four tandemly arranged Fn-2 modules was determined by PC-affinity chromatography. Expression patterns of EQ-12, and the SP-proteins were studied by means of RT-PCR, Northern blot analysis and immunological approaches indicating differential expression along the male reproductive tract. The vast majority of the short SP-1 and SP-2 proteins are produced by the ampulla whereas EQ-12 originates from the epididymis. Indirect immunofluorescence microscopy of sperm isolated from different regions of the epididymis and Western blot analysis indicate that both, the long and the short Fn-2 proteins associate to the sperm surface during post-testicular maturation. Sperm binding of Fn-2 proteins at the post-acrosome and midpiece was at first detected in the corpus epididymis. Enhanced fluorescence intensity after ejaculation point to an increased number of molecules bound to the sperm surface. The function of these proteins is discussed in regard to their structure-function relationships.