Recombinant porcine zona pellucida glycoproteins expressed in Sf9 cells bind to bovine sperm but not to porcine sperm

The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal alpha-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.     

Presence of several cellulose-binding proteins in culture supernatant and cell lysate of Eubacterium cellulosolvens 5

Attempts were made to separate and characterize cellulose-binding proteins (CBPs) from both the culture supernatant and cell lysate of Eubacterium cellulosolvens 5. Once the CBPs were bound to Avicel cellulose, they were then effectively eluted with the solution containing 3.2 or 5% sodium dodecyl sulfate (SDS), but not eluted with the solution containing various kinds of carbohydrates and reagents. Namely, CBPs in both the culture supernatant and cell lysate of the bacterium bound tightly and strongly to cellulose. The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted CBPs indicated that the CBPs contained the two major proteins having the molecular weights of approximately 160 and 84 kilodaltons (kDa) and one sub-major protein having a molecular weight of approximately 140 kDa. Zymogram analysis after the SDS-PAGE of the eluted CBPs showed that two proteins exhibited the highest levels of carboxymethyl cellulase (CMCase) activity corresponding to the molecular weights of approximately 160 and 90 kDa. A major protein having the molecular weight of approximately 160 kDa exhibited a distinct CMCase activity and was designated as CBPE1. Western immunoblot analysis indicated that the proteins prepared from 16 representative strains of rumen bacteria did not cross-react with rabbit antiserum raised against CBPE1. Thus, CBPE1 may be a unique CBP that plays an important role in the adhesion of the bacterium to cellulose.     

Advanced Application of Porcine Intramuscular Adipocytes for Evaluating Anti-Adipogenic and Anti-Inflammatory Activities of Immunobiotics

We previously established a clonal porcine intramuscular preadipocyte (PIP) line and we were able to establish a protocol to obtain functional mature adipocytes from PIP cells. We hypothesized that both PIP cells and mature adipocytes are likely to be useful in vitro tools for increasing our understanding of immunobiology of adipose tissue, and for the selection and study of immunoregulatory probiotics (immunobiotics) able to modulate adipocytes immune responses. In this study, we investigated the immunobiology of PIP cells and mature adipocytes in relation to their response to TNF-α stimulation. In addition, we evaluated the possibility that immunobiotic microorganisms modify adipogenesis and immune functions of porcine adipose tissue through Peyer’s patches (PPs) immune-competent cells. We treated the porcine PPs immune cells with different probiotic strains; and we evaluated the effect of conditioned media from probiotic-stimulated immune cells in PIP cells and mature adipocytes. The Lactobacillus GG and L. gasseri TMC0356 showed remarkable effects, and were able to significantly reduce the expression of pro-inflammatory factors and negative regulators (A20, Bcl-3, and MKP-1) in adipocytes challenged with TNF-α. The results of this study demonstrated that the evaluation of IL-6, and MCP-1 production, and A20 and Bcl-3 down-regulation in TNF-α-challenged adipocytes could function as biomarkers to screen and select potential immunobiotic strains. Taking into consideration that several in vivo and in vitro studies clearly demonstrated the beneficial effects of Lactobacillus GG and L. gasseri TMC0356 in adipose inflammation, the results presented in this work indicate that the PIP cells and porcine adipocytes could be used for the screening and the selection of new immunobiotic strains with the potential to functionally modulate adipose inflammation when orally administered.     

Social interactions of juvenile brown boobies at sea as observed with animal-borne video cameras

While social interactions play a crucial role on the development of young individuals, those of highly mobile juvenile birds in inaccessible environments are difficult to observe. In this study, we deployed miniaturised video recorders on juvenile brown boobies Sula leucogaster, which had been hand-fed beginning a few days after hatching, to examine how social interactions between tagged juveniles and other birds affected their flight and foraging behaviour. Juveniles flew longer with congeners, especially with adult birds, than solitarily. In addition, approximately 40% of foraging occurred close to aggregations of congeners and other species. Young seabirds voluntarily followed other birds, which may directly enhance their foraging success and improve foraging and flying skills during their developmental stage, or both.     

KEG1/YFR042w encodes a novel Kre6-binding endoplasmic reticulum membrane protein responsible for beta-1,6-glucan synthesis in Saccharomyces cerevisiae

KEG1/YFR042w of Saccharomyces cerevisiae is an essential gene that encodes a 200-amino acid polypeptide with four predicted transmembrane domains. The green fluorescent protein- or Myc(6)-tagged Keg1 protein showed the typical characteristics of an integral membrane protein and was found in the endoplasmic reticulum by fluorescence imaging. Immunoprecipitation from the Triton X-100-solubilized cell lysate revealed that Keg1 binds to Kre6, which has been known to participate in beta-1,6-glucan synthesis. To analyze the essential function of Keg1 in more detail, we constructed temperature-sensitive mutant alleles by error-prone polymerase chain reaction. The keg1-1 mutant cells showed a common phenotype with Deltakre6 mutant including hypersensitivity to Calcofluor white, reduced sensitivity to the K1 killer toxin, and reduced content of beta-1,6-glucan in the cell wall. These results suggest that Keg1 and Kre6 have a cooperative role in beta-1,6-glucan synthesis in S. cerevisiae.