Epitope analysis and molecular modeling reveal the topography of the C-terminal peptide of the beta-subunit of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones that share a common alpha-subunit and a hormone-specific beta-subunit. Among the gonadotropin beta-subunits, greater than 85% homology exists between lutropin (hLH)beta and hCGbeta in their first 114 amino acid residues. However, unlike hLHbeta, hCGbeta contains a 31-amino acid hydrophilic stretch at its carboxyl end (CTPbeta: C-terminal peptide). Although the crystal structure of deglycosylated hCG has been solved, the topography of CTPbeta remains unknown. In this study, we have attempted to define the topology of CTPbeta using mAb probes. We investigated three epitopes on hCGalpha, which are hidden in the hCGalphabeta dimer. However, these epitopes are not hidden in hLH, which has a similar subunit interface to that of hCG, but lacks CTPbeta. This suggested that these epitopes are not masked at the subunit interface of hLH or hCG. Hence, we hypothesized that, in the case of hCG, these epitopes are masked by the CTPbeta. Consistent with this view, several treatments of hCG that removed CTPbeta unmasked these epitopes and enhanced their reactivity with the corresponding mAbs. In order to localise the position of CTPbeta on the alpha-subunit, we used an epitope-mapping strategy [N. Venkatesh & G. S. Murthy (1997) J. Immunol. Methods 202, 173-182] based on differential susceptibility of epitopes to covalent modifications. This enabled us to predict the possible topography of CTPbeta. Further, we were also able to build a model of CTPbeta, completely independently of the epitope-mapping studies, using a homology-based modeling approach [S. Krishnaswamy, I. Lakshminarayanan & S. Bhattacharya (1995) Protein Sci. 4 (Suppl. 2), 86-97]. Results obtained from these two different approaches (epitope analysis and homology modeling) agree with each other and indicate that portions of CTPbeta are in contact with hCGalpha in the native hCG dimer.     

The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.     

Haploid plant regeneration from unpollinated ovule cultures of niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.)

Haploid plants were regenerated in vitro from unpollinated ovules of niger (Guizotia abyssinica (L. f.) (Cass.) on Murashige and Skoog nutrient medium (MS) supplemented with 10 μM naphthaleneacetic acid or 10 μM NAA + 1.5 μM kinetin and 30 g/l sucrose. Gamborg (B5) medium was the best for plant regeneration (in comparison with MS, Nitsch and Nitsch (NN), and Chu (N6) media) from cultured ovules, and 6.66 and 7.33 ovules of JNC-6 and Ootacamund cultivars were involved in direct plant regeneration on this medium. Matured ovules (ovules collected one day before anthesis or on the day of anthesis) only responded to cultural regimes and involved in direct plantlet development. Cytological preparation of root tips and chloroplast counts in the guard cells of leaf stomata of regenerated plants confirmed their haploid nature. This text was submitted by the authors in English.     

A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.     

Phosphorylation of AMP by inorganic phosphorylating agents

AMP was phosphorylated by inorganic phosphorylating agents: cyclo-triphosphate and diphosphonate, in aqueous solution (70-80 degrees C, pH 6-12). The molecular structures of phosphorylated products were established by use of phosphorus-31 NMR and high-performance liquid chromatography (HPLC). The OH groups on AMP were phosphorylated by both phosphorylating agents to form 2'- or 3'-phosphate but an OH group on dAMP was not phosphorylated. Phosphorylation of OH group proceeds in two steps: formation of hydrogen bond between OH group and phosphorylating agent; subsequent nucleophilic attack of OH group on a phosphorus atom. Phosphate group on AMP was phosphorylated by diphosphonate but not by cyclo-triphosphate. The difference in the reactivities is explained in terms of charge repulsion between AMP and agents.     

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.