Evidence for a fucose-binding protein in boar spermatozoa

Summary A fucose binding, protein was detected in boar spermatozoa by means of a specifically developed modified enzyme-linked-lectin-assay using glycosylated peroxidase derivatives. The distribution of the fucose binding protein was assessed by means of fluorescence microscopy with fluoresceinyl-glycosylated peroxidase. Fucose binding was particularly prominent at the apical region of the sperm head. In order to gain more insight into the precise localization of the carbohydrate binding, protein electron microscopical studies were performed using fucosyl peroxidase coupled to colloidal gold. In ultrathin sections as well as in specimens, prepared in toto for TEM an intensive binding of fucosylperoxidase-colloidal gold was predominantly found at the apical part of the acrosome appearing as a crescent-like area. In some cases this binding pattern was replaced by a triangle-like intensive labelling at the equatorial segment as revealed clearly by specimens prepared in toto. By SDS-PAGE of the SDS-extractable sperm-proteins, followed by transblotting to nitrocellulose and visualization with the fucosylperoxidase by enzymatic amplification with 4-chloro-1-naphthol mainly one protein with the reduced molecular weight of approximately 53 kdal and some small proteins with apparent molecular weights less than 20 kdal was found to be responsible for the fucose-binding ability of porcine spermatozoa.     

Multicopy Tn10 tet plasmids confer sensitivity to induction of tet gene expression.

We inserted the Tn10 tetracycline resistance determinant (tet) into the multicopy plasmid pACYC177, and we examined the phenotype of Escherichia coli K-12 strains harboring these plasmids. In agreement with others, we find that Tn10 tet exhibits a negative gene dosage effect. Strains carrying multicopy Tn10 tet plasmids are 4- to 12-fold less resistant to tetracycline than are strains with a single copy of Tn10 in the bacterial chromosome. In addition, we find that multicopy tet strains are 30- to 100-fold less resistant to the tetracycline derivative 5a,6-anhydrotetracycline than are single-copy tet strains. Multicopy tet strains are, in fact, 10- to 25-fold more sensitive to anhydrotetracycline than are strains that lack tet altogether. The hypersensitivity of multi-copy strains to anhydrotetracycline is correlated with the effectiveness of anhydrotetracycline as an inducer of tet gene expression, rather than its effectiveness as an inhibitor of protein synthesis. Anhydrotetracycline is 50- to 100-fold more effective than tetracycline as an inducer of tetracycline resistance and as an inducer of beta-galactosidase in strains that harbor tet-lac gene fusions. In contrast, anhydrotetracycline appears to be two- to fourfold less effective than tetracycline as an inhibitor of protein synthesis. Both anhydrotetracycline and tetracycline induce synthesis of tet polypeptides in minicells harboring multicopy tet plasmids. Differences between E. coli K-12 backgrounds influence the tetracycline and anhydrotetracycline sensitivity of multicopy strains; ZnCl2 enhances the tetracycline and anhydrotetracycline sensitivity of these strains two- to threefold. We propose that the overexpression of one or more Tn10 tet gene products inhibits the growth of multicopy tet strains and accounts for their relative sensitivity to inducers of tet gene expression.     

Effect of dietary fiber on absorption of B-6 vitamers in a rat jejunal perfusion study

Previous research has indicated that dietary fiber may affect the absorption and utilization of certain nutrients. To determine the effect of certain fiber materials on the absorption of B-6 vitamers, jejunal segments from young male adult rats were perfused in situ with a control solution containing 0.02 mM pyridoxine (PN), 0.02 mM pyridoxal (PL), and 0.02 mM pyridoxamine (PM), followed by a test solution containing the same vitamin B-6 mixture and one of five fiber-rich test materials (cellulose, pectin, lignin, homogenized fresh carrot, or carrot homogenized after 10 min boiling) added at a concentration of 1-3%. The mean absorption rates of PL, PN, and PM from the control solution were, respectively, 3.66 +/- 0.23, 2.06 +/- 0.23, and 1.74 +/- 0.37 nmole/min/20 cm jejunal segment. There were no significant differences between the absorption rates of B-6 vitamers from control and test solutions containing cellulose, pectin, and lignin. The absorption rates of PM and PL were significantly depressed (P less than 0.05 and P less than 0.01, respectively) by the presence of fresh or cooked carrot. The absorption rate of PN in presence of cooked carrot was also decreased relative to the control value but the difference was only marginally significant (P less than 0.10). When the concentration of fresh carrot in the test solution was increased to 10% by weight and the perfusion rate was decreased from 1.91 to 0.49 ml/min in a second perfusion experiment, there was a significant increase in variability and the differences between absorption rates of the B-6 vitamers in control and test solutions were not statistically significant. The limited evidence of adverse effect of carrot on absorption of vitamin B-6 suggested the need for further clarification of the influence of dietary fiber in an unrefined state on the bioavailability of vitamin B-6.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Large-scale mapping and chromosome jumping in the q27 region of the human X chromosome

We have used pulsed field gel electrophoresis for further physical mapping studies in the q27 region of the human X chromosome. We show that the DXS 102 locus and the F9 gene are separated by only 300 kb despite a genetic distance of 1.4 cM; this linkage orients our large-scale map and shows that the mcf.2 transforming sequence is telomeric to F9. A BssHII complete-digest jumping library was used to jump toward the DXS 105 locus; a 130-kb jump was achieved and the corresponding "linking clone" was obtained.     

Protein denaturation during heat shock and related stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase inactivation in mouse cells

In an attempt to question the toxic effect of heat shock and related stress, we have studied the activity of reporter enzymes during stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase were synthesized in mouse and Drosophila cells after transfection of the corresponding genes. Both enzymes are rapidly inactivated during hyperthermia. The corresponding polypeptides are not degraded but become insoluble even in the presence of non-ionic detergents. The heat inactivation is more dramatic in vivo within the living cell than in vitro, in a detergent-free crude cell lysate. The extent of enzyme inactivation at a given temperature depends on the cell type in which the enzyme is expressed. Luciferase is inactivated at lower temperatures within Drosophila cells than within mouse cells, whereas beta-galactosidase is inactivated at higher temperatures in E. coli than in mouse cells. A "priming" heat shock confers a transient increased resistance (thermotolerance) of cells against a second "challenging" heat shock. Enzyme inactivation during heat shock or exposure of the cells to ethanol is attenuated in heat shock-primed cells. A comparable thermoprotection is raised by a priming heat shock for both luciferase activity and protein synthesis. Thus, the study of reporter enzyme inactivation is a promising tool for understanding the molecular basis of the toxicity of heat shock and related stress as well as the mechanisms leading to thermotolerance.     

Characterization of a human homologue of the murine peripheral lymph node homing receptor

Lymphocyte trafficking is a fundamental aspect of the immune system that allows B and T lymphocytes with diverse antigen recognition specificities to be exposed to various antigenic stimuli in spatially distinct regions of an organism. A lymphocyte adhesion molecule that is involved with this trafficking phenomenon has been termed the homing receptor. Previous work (Lasky, L., T. Yednock, M. Singer, D. Dowbenko, C. Fennie, H. Rodriguez, T. Nguyen, S. Stachel, and S. Rosen. 1989. Cell. 56:1045-1055) has characterized a cDNA clone encoding a murine homing receptor that is involved in trafficking of lymphocytes to peripheral lymph nodes. This molecule was found to contain a number of protein motifs, the most intriguing of which was a carbohydrate binding domain, or lectin, that is apparently involved in the adhesive interaction between murine lymphocytes and peripheral lymph node endothelium. In this study, we have used the murine cDNA clone to isolate a human homologue of this peripheral lymph node-specific adhesion molecule. The human receptor was found to be highly homologous to the murine receptor in overall sequence, but showed no sequence similarity to another surface protein that may be involved with human lymphocyte homing, the Hermes glycoprotein. The extracellular region of the human receptor contained an NH2 terminally located carbohydrate binding domain followed by an EGF-like domain and a domain containing two repeats of a complement binding motif. Transient cell transfection assays using the human receptor cDNA showed that it encoded a surface glycoprotein that cross reacted with a polyclonal antibody directed against the murine peripheral lymph node homing receptor. Interestingly, the human receptor showed a high degree of sequence homology to another human cell adhesion glycoprotein, the endothelial cell adhesion molecule ELAM.     

Freezing pattern in apple seeds as affected by the temperature of fruit storage

Storage of apple fruits ( Pyrus malus L. cv. Golden Delicious) at different temperatures (0, 12 and 35°C) markedly altered the pattern of water freezing in the seeds. Higher temperatures of storage brought about a shift from the sequential to the discontinuous type of freezing in seeds, the low temperature exotherm (LTE) being much more pronounced than the high temperature one (HTE). The occurrence of LTE was highly correlated with embryo death. Fruit storage at higher temperatures also caused a decrease in the threshold super cooling temperature of seeds and of naked embryos, removed from the seed coat and endosperm. The decrease was less pronounced in naked embryos than in intact seeds. The results presented show that the frost resistance of apple seeds relies mainly on the supercooling ability of the embryos and is increased by the presence of seed coat and endosperm. The broad peak of the LTE indicates that, in contrast with other seeds and many super cooling organs, massive ice nucleation in apple seeds occurs within the embryo tissues and that extra organ freezing seems to be of less importance. Therefore, the increased super cooling ability of apple seeds, isolated from fruits stored at higher temperatures, seems to rely on those seed properties that protect embryo cells against heterogeneous ice nucleation.