Purification of N-acetyllactosamine-binding activity from the porcine sperm membrane: possible involvement of an ADAM complex in the carbohydrate-binding activity of sperm

Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads-one recognizes N-acetyllactosamine (Galβ1-4GlcNAc-), and the other recognizes the Lewis X structure (Galβ1-4(Fucα1-3)GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane.     

Mapping of quantitative trait loci controlling seedling vigor in rice (Oryza sativa L.) under submergence

Submergence-induced suppression of seedling vigor is a serious constraint particularly in the direct seeding rice cultivation system. To identify quantitative trait loci (QTLs) associated with seedling vigor in rice under submergence, a mapping population of 98 Backcross Inbred Lines derived from a cross of Nipponbare/Kasalath//Nipponbare was used. Phenotypic evaluation of seedling vigor under submergence was based on shoot length (SHL), root length (RTL) and shoot fresh weight (SFW) using a test tube bioassay method. Thirty-two putative QTLs were detected among which 7 were for SHL, 11 for RTL and 14 for SFW. Phenotypic evaluation was also made of the parental lines and a set of 54 chromosome segment substitution lines in which Nipponbare segments were substituted for by their homologous Kasalath segments covering the entire rice genome. Two QTLs with more than 10 % contribution to the total phenotypic variance were verified for SHL, and at least one for RTL and six for SFW on chromosomes 1, 3, 4, 6 and 7 at the 1 % significance level. Among these, all but two showed reductions in one, two or all three traits. Our present and previous results suggest that the Nipponbare genome has a potential to improve seedling vigor under submergence and that japonica germplasms can be used to breed for this important trait in indica rice.     

B‐cell intestinal lymphoma with Mott cell differentiation in a 1‐year‐old miniature Dachshund

Abstract: A 1‐year‐old intact female miniature Dachshund was presented with hematochezia, vomiting, and diarrhea of more than 1‐week duration. An abdominal mass was palpated, which at exploratory surgery was found to be a 7‐cm‐long thickened section of ileum. The thickened ileum was resected. Impression smears revealed numerous small‐ to medium‐sized lymphocytes, with a smaller number of cells resembling Mott cells. The Mott‐like cells contained multiple pale vacuoles that were positive for periodic acid‐Schiff (PAS) in wet‐fixed smears, consistent with Russell bodies. Histologic evaluation of the surgically excised ileum revealed 2 populations of neoplastic lymphoid cells. The majority were uniform medium‐sized lymphocytes with hyperchromatic oval or round nuclei and inconspicuous nucleoli. The remaining cells resembled Mott cells, which contained several PAS‐positive eosinophilic globules in the cytoplasm, occasionally compressing the nucleus. The majority of neoplastic cells stained positively for vimentin, CD20, CD79a, and Pax‐5, but were negative for CD3 and lysozyme; 43.5% of cells stained positively for Ki‐67. The Mott cells were strongly positive for immunoglobulin but were negative for Pax‐5. Using electron microscopy, a homogenous substance of intermediate electron density was observed frequently in the cisternae of rough endoplasmic reticulum in the cytoplasm of the Mott cells, and rarely in the perinuclear cisternae of the lymphoid cells, corresponding to the site of immunoglobulin staining. Monoclonal rearrangement of immunoglobulin heavy‐chain (IgH) gene was observed by PCR testing for lymphocyte–antigen receptor rearrangement. The morphologic features, immunophenotype, and IgH gene rearrangement verified the lymphoid cells were neoplastic (mature cell type) and had a B‐cell phenotype, with evidence of immunoglobulin production and differentiation into Mott cells. This case was unusual because of the age of the dog and because most intestinal lymphomas are T‐cell phenotype. The Mott cell morphology also differed from typical mature B‐cell lymphoma types and may be a unique B‐cell lymphoma variant.     

Transcriptomic profile reveals molecular events associated to focal adhesion and invasion in canine mammary gland tumour cell lines

The prevalence of cancer in animals has increased significantly over the years. Mammary tumours are the most common neoplasia in dogs, in which around 50% are presented in the malignant form. Hence, the development and characterization of in vitro models for the study of canine tumours are important for the improvement of cancer diagnosis and treatment. Thus, the aim of this study was to characterize cell lines derived from canine mammary gland neoplasias which could be further used for basic and applied oncology research. Samples of canine mammary carcinomas were taken for cell culture and 2 cell lines were established and characterized in terms of cell morphology, tumourigenicity and global gene expression. Both cell lines presented spindle‐shape morphology and shown common malignant features as in vitro invasion potential and expression of epithelial and mesenchymal proteins. Also, we found gene expression patterns between the 2 cell cultures in comparison to the normal mammary gland tissue. Cells from M25 culture showed a higher invasion and in vivo tumourigenic potential, associated to the overexpression of genes involved in focal adhesion and extracellular matrix communication, such as FN1, ITGA8 and THBS2. The phenotypic characterization of these cells along with their global gene expression profile potentially determine new therapeutic targets for mammary tumours.     

Effect of dietary lipid-lowering drugs upon plasma lipids and egg yolk cholesterol levels of laying hens.

To evaluate the effect of lipid-lowering agents upon egg quality, reproductive performance, plasma lipids, and egg yolk cholesterol levels, 30-week-old Shaver laying hens were fed a basal diet (commercial ration) supplemented with 0.1% probucol (PROB), 0.025% gemfibrozil (GEMF), or lovastatin at 0.0005% (LOV1), 0.001% (LOV2), or 0.0015% (LOV3) for a 12-week experimental period. It was observed that the supplementation of the drugs did not impair albumen and shell quality. Hen performance was not adversely affected. The depression in triglyceride concentrations approached statistical significance only in LOV2 (38.5%), and total cholesterol was significantly depressed in LOV2 (36.0%), LOV3 (36.8%), PROB (29.6%), and GEMF (30.4%) treatments. Egg cholesterol content, expressed per gram of yolk, was significant lowered in LOV1 (7.5%) and LOV3 (12. 7%).     

Superoxide dismutase and catalase activities in Photobacterium damselae ssp. piscicida

The ability of a set of Photobacterium damselae ssp. piscicida strains isolated from different fish species to produce different superoxide dismutase (SOD) and catalase enzymes was determined. Unlike other bacterial pathogens, P. damselae ssp. piscicida is not able to produce different isoforms of SOD or catalase containing different metal cofactors when cultured under oxidative stress induced by hydrogen peroxide or methyl viologen, or under iron depleted conditions. However, iron content of the growth medium influenced the levels of SOD and catalase activity in cells, these levels decreasing with iron availability of the medium. Comparison of virulent and non-virulent strains of P. damselae ssp. piscicida showed similar contents of SOD, but higher levels of catalase were detected in cells of the virulent strain. Incubation of bacteria with sole, Solea senegalensis (Kaup), phagocytes has shown that survival rates range from 19% to 62%, these rates being higher for the virulent strain. The increased levels of catalase activity detected in the virulent strain indicates a possible role for this enzyme in bacterial survival.     

Effect of liquid paraffin on antibody responses and local adverse reactions of bivalent oil adjuvanted vaccines containing newcastle disease virus and infectious bronchitis virus

Effects of liquid paraffin on antibody responses and local adverse reactions after intramuscular injection of oil adjuvanted vaccines containing Newcastle disease (ND) and infectious bronchitis (IB) virus were investigated in chickens. Each vaccine was prepared with a liquid paraffin such as Carnation, Crystol 52 and Lytol. These vaccines induced sustained antibody responses against ND and IB. Among local adverse reactions, Lytol induced granulomatous reactions and abscesses, but Carnation and Crystol 52 did not. The residual weight of liquid paraffin at the injection site decreased in the order Carnation, Crystol 52, Lytol. Crystol 52 was composed of relatively few short-chain hydrocarbons (i.e., n-C20H42). The vaccine with liquid paraffin mainly composed of n-C16H34-n-C20H42 was suggested to induce fewer adverse reactions.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.