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.     

Molecular analysis of tlrD, an MLS resistance determinant from the tylosin producer, Streptomyces fradiae

The macrolide antibiotic, tylosin (Ty), is produced by Streptomyces fradiae. Two resistance determinants (tlrA, synonym ermSF, and tlrD) conferring resistance to macrolide, lincosamide and streptogramin B type (MLS) antibiotics were previously isolated from this strain, and their products shown to methylate 23S ribosomal RNA (rRNA) at a common site, thereby rendering the ribosomes MLS resistant. However, the T1rA and T1rD proteins differ in their action; the former dimethylates, and the latter monomethylates, the target nucleotide. Here, 2.2 kb of DNA from the tylLM region of the tylosin biosynthetic gene cluster of S. fradiae has been sequenced and shown to encompass tlrD. Comparison of the sequences of tlrA and tlrD (and of their deduced products) with those of related (‘erm-type’) genes from other actinomycetes suggests that the combined presence of tlrA and tlrD in S. fradiae is not the result of recent gene duplication.     

Metabolic differentiation of homologous leg muscles of two aquatic birds at the level of enzymatic organization

The quantitative determination of succinic dehydrogenase (SDH), hexokinase (HK), phosphorylase, phosphofructokinase (PFK), glycerol-3-phosphate dehydrogenase (G-3-PDH) and lactate dehydrogenase (LDH) was carried out in the homologous leg muscles of two aquatic Birds. It appears that the leg muscle fibres of the coot, a surface swimmer are more oxidative in nature and appear to utilize glucose as source of energy. The leg muscles of the dabchick, a diving Bird, on the other hand, seem to depend on glycogen as source of energy. The relative activity levels of HK, phosphorylase and PFK support the accepted r?le of glycogen as primary substrate of carbohydrate catabolism in the leg muscles. The ratio of G-3-PDH/LDH in the leg muscles revealed that glycerol 3-phosphate cycle appears to be insufficient to account for the major part of NADH oxidation. However, the LDH activity is quite high in all the muscles. These results led us to believe that glycerol 3-phosphate cycle may function during rest, when the rate of glycolysis will be low.     

Characterization of affinity-purified juvenile hormone esterase from the plasma of the tobacco hornworm, Manduca sexta

Juvenile hormone (JH) esterase found primarily in the hemolymph and tissues of insects is a low abundance protein involved in the ester hydrolysis of insect juvenile hormones, JHs. The enzyme was purified from the larval plasma of wild-type Manduca sexta using an affinity column prepared by binding the ligand, 3-[(4'-mercapto)butylthio]-1,1,1-trifluoropropan-2-one (MBTFP), to epoxy-activated Sepharose. The purification was greater than 700-fold with a 72% recovery, and the purified enzyme appeared as a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis, reverse phase high performance liquid chromatography, and amino acid sequence analysis. The molecular weight was 66,000. The plasma JH esterase in wild-type, black, and white strains of M. sexta was similar when analyzed by immunotitration, wide range (pH 3.5-9.0) isoelectric focusing, and inhibition with MBTFP and 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP). Inhibition studies revealed a sensitive and insensitive form (I50 = 10(-9) and 10(-6) M, respectively) in these three biotypes. Narrow range isoelectric focusing (pH 4.0-7.0) indicated the presence of two major isoelectric forms with pI values of 6.0 and 5.5, but their inhibition kinetics with OTFP and O,O-diisopropyl phosphorofluoridate were identical.     

The role of male accessory-gland substance on female reproduction with some observations of spermatogenesis in the stable fly

The bulbous tubular portion of the median ejaculatory duct functions as the accessory gland in the male stable fly, Stomoxys calcitrans. The gland started to enlarge after emergence when the fly was fed on sugar-water or blood. Implants of accessory glands from sugar-water or blood-fed males were effective in stimulating oviposition in virgin females. The injection into females of accessory-gland extracts from males fed blood prevented insemination; this extract was effective in concentration as low as 0.25 gland per female. Accessory-gland extracts from sugar-water fed males were only partially effective in preventing female insemination. However, the accessory-gland extracts of male stable flies had little effect on insemination when injected into three other species of female dipterans.Spermatogenesis was completed by the time of emergence, or shortly thereafter, a process independent of blood feeding. Three types of spermatids were identified, forming a continuum of spermiogenesis stages. Fat, fusiform spermatozoa elongated to become thin, elongate and mature spermatozoa.     

Genetic variability, heritability and genetic advance of growth and yield components of Jatropha (Jatropha curcas Linn.) genotypes

A total of 113 Jatropha curcas clonal accessions collected from different regions of India were studied to quantify the magnitude of genetic variability present in the test population and to identify important yield-attributing characters useful for developing high-yielding Jatropha cultivars. High heritability was observed for fruits per plant, seeds per plant, 100-seed weight, seed/kernel (S/K) ratio and kernel oil percentage coupled with high genetic advance suggesting that the accessions can be considered improvement. The significant positive association of seed oil content (%) with 100-seed weight suggested the effectiveness of indirect selection for seed oil content through 100-seed weight. Accessions 76, 120, 29, 86 and 84 showed above average higher values for all yield attributes (viz. fruit and seed yield, 100-seed weight, S/K ratio and oil content) suggesting these as best out of the test accessions. Accessions showing higher values for one or the other yield attributes could be selected as parents for further improvement.