Binding of the anti-human sperm monoclonal antibody HS-11 to bull spermatozoa is correlated with fertility in vitro

The present study aimed to determine if there is bull to bull variation in the binding of the anti-human sperm monoclonal antibody (MAb) HS-11 to bull spermatozoa, and to investigate if there is any correlation between HS-11 binding to spermatozoa and in vitro fertility of the bulls tested. Semen samples of a single collection (split frozen in 0.5-ml straws) from 8 dairy bulls were used. Swim-up separated motile spermatozoa were incubated in 90-microl drops of capacitation medium (TALP+10 microg/ml heparin) at 39 degrees C, 5% CO2, 95% air. At 0, 2, 4 and 6 h of incubation HS-11 was added (1:1000 final concentration), and the MAb binding was assessed by indirect immunofluorescence assay (IIFA). The HS-11 binding was indicated by a bright green fluorescence of the sperm acrosome region. In vitro-matured, good quality bovine oocytes were randomly allocated to spermatozoa of each bull for in vitro fertilization. Sperm samples of 2 to 3 bulls were used in each trial until 4 replicates per bull were attained for IVF (n approximately 100 oocytes/bull) and IIFA experiments. Sperm capacitation status was assessed simultaneously using an egg yolk lysophosphatidylcholine- (LC) induced acrosome reaction assay. The binding of HS-11 to spermatozoa was maximum at 4 h of incubation in most (6/8) of the bull semen samples. Significant (P < 0.01) differences were observed between bulls in the binding of HS-11 to their spermatozoa (range 22 +/- 8 to 52 +/- 5%) at 4 h, but not within replicates. Similarly, variations (P < 0.05) in the cleavage rate were also seen (range 22 +/- 9 to 58 +/- 7%) between bulls. The HS-11 binding and cleavage were significantly correlated (r = 0.43; n = 32; P < 0.05). The highest percentage of spermatozoa underwent acrosome reaction in response to LC treatment at the 4-h incubation period. This and the linear relationship between HS-11 binding and the cleavage rate observed in the present study together strengthen our earlier suggestion that the binding of the monoclonal antibody HS-11 to bull spermatozoa on a time-dependent manner, may indicate capacitation changes. We conclude that 1) between-bull differences exist in HS-11 binding to spermatozoa, and in the cleavage rate, and 2) HS-11 binding to spermatozoa is correlated with fertility, as determined by the cleavage of bovine oocytes matured and fertilized in vitro.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus transformants harbouring cloned phbCAB genes

Summary Three transformants of Alcaligenes eutrophus harbouring the recombinant plasmids containing phbCAB, phbAB, and phbC genes, were cultivated to investigate the effect of cloned genes on cell growth and poly--hydroxybutyrate accumulation. Both in the nutrient-rich and minimal media, the increased PHB accumulation in the transformants was observed compared to the parent strain, and this was the result of the increased enzyme activities in the transformants. Low carbon concentration and high C/N molar ratio favored higher PHB accumulations in the transformants. The transformant harbouring the phbC gene showed the highest PHB accumulation, which indicated that PHB synthase was the most critical enzyme for PHB biosynthesis in the transformant.     

Production of cyclodextrins using moderately heat-treated cornstarch

Cyclodextrins are very useful compounds for the food, cosmetic, pharmaceutic, and plastic industries. We developed a process for the production of cyclodextrins from moderately heat-treated cornstarch. This method had many merits. First, the cyclodextrins were not degraded by cyclodextrin glucanotransferase, because low molecular weight maltodextrins were hardly produced. Second, it was possible to remove the residual cornstarch by a simple method such as filtration or centrifugation. Third, the amount of cyclodextrin glucanotransferase used for cyclodextrin production was less than that using the traditional method. Fourth, the pretreating method was simple. And fifth, the residual starch could be used as substrate for the production of other compounds. Cyclodextrins were produced at optimum conditions: heating temperature was 65°C; heating time was 1 h; concentration of substrate was 7.5%; amount of enzyme loaded was 48 U g⁻¹ of substrate. Using these conditions, the results were as follows: the content of cyclodextrins, 50%; the conversion yield of substrate, 25%; the productivity per enzyme unit, 5.22 mg of cyclodextrins.     

Lipid structure and Ca2+-ATPase function

Effects of lipid structure on the function of the Ca²⁺-ATPase of skeletal muscle of sarcoplasmic reticulum are reviewed. Binding of phospholipids to the ATPase shows little specificity. Phosphatidylcholines with short (C14) or long (C24) fatty acyl chains have marked effects on the activity of the ATPase, including a change in the stoichiometry of Ca binding. Low ATPase activity in gel phase lipid follows from low rate of phosphorylation. Phosphatidylinositol 4-phosphate increases ATPase activity by increasing the rate of dephosphorylation of the phosphorylated ATPase. Stimulation is not seen with other anionic phospholipids; phosphatidic acid decreases ATPase activity in a Mg²⁺-dependent manner.Abbreviations di(C141)PC dimyristoleoylphosphatidycholine - di(C160)PC dipalmitoylphosphatidylcholine - di(C181)PC dioleoylphosphatidylcholine - di(Br₂C180)PC dibromostearoylphosphatidylcholine - di(C241)PC dinervonylphosphatidylcholine - di(C181)PA dioleoylphosphatidic acid - di(C181)PE dioleoylphosphatidylethanolamine - Ptdlns phosphatidylinositol - PtdIns-4P phos-phatidylinositol 4-phosphate     

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia-Tolerant Reptiles Contrasted with Levels in Anoxia-Intolerant Species

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g^?1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g^?1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g^?1). Regionally distinct levels (2–4 µmol g^?1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2–3 µmol g^?1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.     

Potential role of natural killer cells in controlling tumorigenesis by human T-cell leukemia viruses.

Human T-cell leukemia virus (HTLV) is the etiologic agent of adult T-cell leukemia (ATL), a malignancy of T lymphocytes that is characterized by a long latency period after virus exposure. Intraperitoneal inoculation of severe combined immunodeficient (SCID) mice with HTLV-transformed cell lines and ATL tumor cells was employed to investigate the tumorigenic potential of HTLV type I (HTLV-I)-infected cells. In contrast to inoculation of ATL (RV-ATL) cells into SCID mice, which resulted in the formation of lymphomas, inoculation of HTLV-I- and HTLV-II-transformed cell lines (SLB-I and JLB-II cells, respectively) did not result in tumor formation. Immunosuppression of SCID mice, either by whole-body irradiation or by treatment with an antiserum, anti-asialo GM1 (alpha-AGM1), which transiently abrogates natural killer cell activity in vivo, was necessary to establish the growth of tumors derived from HTLV-transformed cell lines. PCR and flow cytometric studies reveal that HTLV-I-transformed cells are eliminated from the peritoneal cavities of inoculated mice by 3 days postinoculation; in contrast, RV-ATL cells persist and are detected until the mice succumb to lymphoma development. The differing behaviors of HTLV-infected cell lines and ATL tumor cells in SCID mice suggest that ATL cells have a higher tumorigenic potential in vivo than do HTLV-infected cell lines because of their ability to evade natural killer cell-mediated cytolysis.     

Essential role of the Vp2 and Vp3 DNA-binding domain in simian virus 40 morphogenesis.

Both a DNA-binding domain and a Vp1 interactive determinant have been mapped to the carboxy-terminal 40 residues of the simian virus 40 (SV40) minor capsid proteins, Vp2 and Vp3 (Vp2/3), with the last 13 residues being necessary for these activities. The role of this DNA-binding domain in SV40 morphogenesis and the ability to separate these two signals were investigated by mutagenesis and assessment of the activity and viability of the mutants. The carboxy-terminal 40 residues of Vp2/3 were expressed as a polyhistidine fusion protein, and five basic residues at the extreme carboxy terminus (Vp3 residues K226, R227, R228, R230, and R233) were mutagenized. The wild-type fusion protein bound DNA with a Kd of 3 x 10(-8) identical to that of the full-length Vp3. Mutant proteins containing either one to three or four amino acid substitutions bound DNA 4- to 7-fold or 20- to 30-fold less well, respectively, than the wild-type protein did. The most severe point mutants showed residual DNA binding similar to that of a truncated protein which lacks the entire 13 carboxy-terminal residues. All of the point mutants were able to interact with Vp1, indicating that the two signals within this region are mediated by different residues. When the mutations were placed into the context of the viral DNA and introduced into cells, all the structural proteins were expressed and localized correctly. Not all, however, were viable: mutant genomes whose Vp2/3 bound DNA with intermediate affinities formed plaques just as well as wild-type SV40 DNA did, but three mutants showing greatly reduced DNA binding failed to form plaques at all. These results are consistent with the hypothesis that Vp2/3 plays an essential role in SV40 virion assembly in the nucleus.     

Conservation of an intact human immunodeficiency virus type 1 vif gene in vitro and in vivo.

Replication of vif-negative human immunodeficiency virus type 1 (HIV-1) is attenuated in certain cell lines and highly impaired in peripheral blood lymphocytes in vitro. To determine whether intact vif is positively selected during natural HIV-1 infection and to determine vif sequence variability, we employed PCR amplification, cloning, and sequencing to investigate the vif region of replicating virus in short-term-passage HIV-1 primary isolates from five asymptomatic individuals and from five persons with AIDS. A total of 46 vif clones were obtained and analyzed. Recombinant proviruses were constructed from selected vif clones from one patient and found to be fully infectious. We found that 38 of the 46 clones sequenced carried open vif reading frames and that there was a low degree of heterogeneity of vif genes within isolates from the same individual and among isolates from different donors. The cysteines previously found to be essential for vif protein function were conserved in all clones. A phylogenetic tree constructed from all available vif nucleotide sequences resulted in a virus grouping similar to those of gag and env. Direct sequencing of vif amplified by PCR from uncultured lymphocytes of 15 individuals at various stages of progression toward AIDS demonstrated vif open reading frames in 13 of 15 samples tested. There was no obvious correlation between disease status and the presence of an intact vif within this sample group at the time of sample procurement. The conservation of the vif open reading frame in vitro and in vivo and its limited variability following virus transmission in vitro are consistent with a role for vif in natural HIV-1 infection.     

SEXUAL DIFFERENTIATION OF GRIFFITHSIA (CERAMIALES, RHODOPHYTA): NUCLEAR PLOIDY LEVEL OF MIXED-PHASE PLANTS IN G. JAPONICA1

Mixed-phase plants of Griffithsia japonica Okamura spontaneously occurred in a laboratory culture. Four female plants produced tetrasporangia and spermatangia in addition to their normal female reproductive structures (bisexual/mixed-phase plants), and four male plants produced tetrasporangia as well as spermatangia (male/mixed-phase plants). To determine the nuclear ploidy level of these mixed-phase plants, relative nuclear sizes of male, female, tetrasporangial, and mixed-phase plants were measured using a microscopic image analysis system. Haploid gametophytes could be distinguished from diploid tetrasporophytes by relative nuclear sizes, with the later having nuclei twice the size of the former. Relative nuclear sizes of the mixed-phase plants were similar to those of the haploid plants. Thus, the mixed-phase plants were determined to be haploid. Haploid mixed-phase plants of G. japonica have a potential to produce male, female and tetrasporangial reproductive structures. Sex determination models are discussed to explain "haploid" mixed-phase phenomena in red algae .