Estrogen-induced synthesis of riboflavin-binding protein in immature chicks kinetics and hormonal specificity

The kinetics of estrogen-induced elevation in the plasma concentration of riboflavin-binding protein, a minor yolk constituent, was investigated in immature male chicks, using a specific and sensitive radioimmunoassay proceudre. Following a single injection of the hormone, the plasma riboflavin-binding protein content was enhanced several-fold at 6 h. reaching peak levels around 48 h and declining thereafter. A two-fold amplication of the response was evident on secondary stimulation with the hormone. A 4-h lag phase prior to onset of induction was noticed during both primary and secondary stimulat ions with the steroid hormone. The magnitude of the response was dependent on the hormonal dose whereas the initial lag phase and the time of peak riboflavin-binding protein accumulation were unaltered within the range of hormonal doses tested. The half-life of riboflavin-binding protein in the circulation was 10 h, as calculated from measurement of the rate of disappearance of exogenously administered 125I-labelled protein. Simultaneous administration of progestrone did bot affect the kinetics of riboflavin-binding protein production. On the other hand, the antiestrogens, cis- and trans-clomiphene citrates, given 30 min prior to estrogen and cycloheximide, effectively countered the hormone-induced riboflavin-binding protein elaboration. Both progesterone and the anti-esterogens per se were completely ineffective in substituting for estrogen in the inductive ptrocess.

Properties of acetylcholinesterase from Pisum sativum

Acetylcholinesterase (AChE) from Pisum sativum purified 28 fold showed two closely moving protein bands on polyacrylamide gel electrophoresis, both of which have AChE activity. AChE activity occurs in roots, stem and leaves, that in roots varying with age. Activity is optimal at pH 9 and at 30”. The energy of activation is 9.82 x lo3 J per mol and MW is greater than 200000. Although the enzyme can hydrolyze both choline and non-choline esters, it has greater affinity for acetylthiocholine (ATCh) and acetylcholine (ACh). ATCh inhibits the enzyme at higher concentrations and the K, is 0.2 mM with this as substrate. The enzyme is not as sensitive to Eserine as it is to Neostigmine. It is also inhibited by organophosphorus pesticides such as Fensulfothion, Parathion and Dimethoate. Treatment of the seeds with Fensulfothion [O, O-diethyl (p-methylsulfinylphenyl) phosphorothioate] affects growth and secondary root development. This might be explained by its inhibition of AChE and the consequent increase of endogenous levels of ACh.

Putrescine-Sensitive (Artifactual) and Insensitive (Biosynthetic) S-Adenosyl-L-Methionine Decarboxylase Activities of Lathyrus sativus Seedlings

The crude extracts of 3-day-old etiolated seedlings of Lathyrus sativus contained two S-adenosyl-L-methionine decarboxylase activities. The artifactual putrescine-dependent activity was due to the H2O2 generated by diamine oxidase (EC 1.4.3.6) of this plant system and was inhibited by catalase. This observation was confirmed by using an electrophoretically and immunologically homogeneous preparation of L. sativus diamine oxidase. In the presence of putrescine, diamine oxidase, in addition to S-adenosylmethionine, decarboxylated L-lysine, L-arginine, L-ornithine, L-methionine and L-glutamic acid to varying degrees. The decarboxylation was not metal-ion dependent. The biosynthetic S-adenosylmethionine decarboxylase (EC 4.1.1.21) was detected after removing diamine oxidase specifically from the crude extracts by employing an immunoaffinity column. This Mg2+ -dependent decarboxylase was not stimulated by putrescine or inhibited by catalase. The enzyme activity was inhibited by semicarbazide, 4-bromo-3-hydroxybenzoylamine dihydrogen phosphate and methylglyoxal-bis (guanylhydrazone). It was largely localized in the shoots of the etiolated seedlings and was purified 40-fold by employing a p-hydroxymercuribenzoate/AH-Sepharose affinity column, which also separated the decarboxylase activity from spermidine synthase.

A single point mutation disrupts the capsid assembly in Sesbania Mosaic Virus resulting in a stable isolated dimer

Protein-protein interactions play a Crucial role in Virus assembly and stability. With the view of disrupting capsid assembly and capturing smaller oligomers, interfacial residue mutations were carried Out in the coat protein gene of Sesbania Mosaic Virus, a T=3 ss (+) RNA plant virus. A single point mutation of a Trp 170 present at the five-fold interface of the virus to a charged residue (Glu or Lys) arrested assembly of virus like particles and resulted in stable Soluble dimers of the capsid Protein. The X-ray crystal structure of one of the isolated dimer mutants - rCP Delta N65W170K was determined to a resolution of 2.65 angstrom. Detailed analysis of the dimeric mutant protein structure revealed that a number of Structural changes take place, especially in the loop and interfacial regions during the course of assembly. The isolated chiller was ``more relaxed'' than the dimer found in the T=3 or T=1 capsids. The isolated dimer does not bind Ca2+ ion and consequently four C-terminal residues are disordered. The FG loop, which interacts with RNA in the Virus, has different conformations in the isolated dimer and the intact Virus Suggesting its flexible nature and the conformational changes that accompany assembly. The isolated choler mutant was much less stable when compared to the assembled capsids, suggesting the importance of inter-subunit interactions and Ca2+ mediated interactions in the stability of the capsids. With this study, SeMV becomes the first icosahedral virus for which X-ray crystal Structures of T=3, T=1 capsids as well as a smaller oligomer of the capsid protein have been determined.

Dynamics of Circulating Concentrations of Gonadotropins and Ovarian Hormones Throughout the Menstrual Cycle in the Bonnet Monkey: Role of Inhibin A in the Regulation of Follicle-Stimulating Hormone Secretion

In higher primates, increased circulating follicle-stimulating hormone (FSH) levels seen during late menstrual cycle and during menstruation has been suggested to be necessary for initiation of follicular growth, recruitment of follicles and eventually culminating in ovulation of a single follicle. With a view to establish the dynamics of circulating FSH secretion with that of inhibin A (INH A) and progesterone (P-4)secretions during the menstrual cycle, blood was collected daily from bonnet monkeys beginning day 1 of the menstrual cycle up to 35 days. Serum INH A levels were low during early follicular phase, increased significantly coinciding with the mid cycle luteinizing hormone (LH) surge to reach maximal levels during the mid luteal phase before declining at the late luteal phase, essentially paralleling the pattern Of P-4 secretion seen throughout the luteal phase. Circulating FSH levels were low during early and mid luteal phases, but progressively increased during the late luteal phase and remained high for few days after the onset of menses. In another experiment, lutectomy performed during the mid luteal phase resulted in significant decrease in INH A concentration within 2 hr (58.3 +/- 2 vs. 27.3 +/- 3 pg/mL), and a 2- to 3-fold rise in circulating FSH levels by 24 hr (0.20 +/- 0.02 vs. 0.53 +/- 0.14 ng/mL) that remained high until 48 hr postlutectomy. Systemic administration of Cetrorelix (150 mu g/kg body weight), a gonadotropin releasing hormone receptor antagonist, at mid luteal phase in monkeys led to suppression of serum INH A and P-4 concentrations 24 hr post treatment, but circulating FSH levels did not change. Administration of exogenous LH, but not FSH, significantly increased INH A concentration. The results taken together suggest a tight coupling between LH and INH A secretion and that INH A is largely responsible for maintenance of low FSH concentration seen during the luteal phase. Am. J. Primatol. 71:817-824, 2009.

Two forms of methionyl-transfer RNA synthetase from Mycobacterium Smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from Image . The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNAmMet and tRNAfMet; enzyme B exhibited much higher specificity for tRNAfMet. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 106 as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.

Nature of induction of tryptophan pyrrolase in cold exposure

The activity of hepatic tryptophan pyrrolase in rats exposed to cold increased rapidly and reached a maximum of three-fold at 8 h. On continued exposure up to 48 h stress, the activity partly decreased but remained at a level higher than the initial. Withdrawal from the cold stress reversed the change. Adrenalectomy or treatment with inhibitors of protein synthesis abolished the increase in the enzyme activity during cold stress indicating a possible involvement of corticosteroids and de novo protein synthesis. Treatment with drugs known to block autonomic nervous system failed to inhibit the cold-mediated increase in enzyme activity. The results suggest that the increase in enzyme activity obtained on cold exposure is mediated by corticosteroids and not by either indoleaklylamines or autonomic nervous system. The changes in the enzyme obtained under cold stress with respect to the overshoot phenomenon, relationship to the degree of stress and reversibility on withdrawal from the stress indicate the "adaptate" nature of the response.

Conformational analysis of cyclo (l-cystine

Conformational analysis of cyclo(L-cystine) shows that the diketopiperazine ring has to exist only in the boat form. With this geometry, the molecule can adopt two distinct forms differing mainly in the chirality of the disulphide bridge. In both the P- and M-models, corresponding to dihedral angles of nearly + 90° and —90° respectively about the S-S bond, the molecule displays an approximate two-fold symmetry. According to our semi-empirical energy calculations, the minimum energy of the M-model is —9.2 kcal/mol, only 0.3 kcal/mol lower than that of the P-model. Because the difference between the two minima is so small, neither form is clearly superior to the other. However, the number of low energy conformations of the M-model in the allowed conformational space is significantly larger than that of the P-model by a ratio of 3 to 1, and therefore the former is likely to be thermodynamically favoured.

Thermal expansion and a new phase transition in pyroelectric LiKSO4

Single crystal macroscopic thermal expansion coefficient measurements have been made on uniaxial lithium potassium sulphate crystal both along and normal to the six fold axis, employing Fizeau’s interferometer method. Measurements were made in the range of −120°C to 500°C. The results show that lithium potassium sulphate exhibits two major anomalies in its expansion coefficients around −95°C and 422°C respectively, the one at −95°C has been observed for the first time. The nature of dimensional changes of the crystal at the upper and lower transition points are opposite in nature. The crystal shows considerable lattice anisotropy. Megaw’s tilt concept has been invoked to explain the relative magnitudes of expansion coefficients alonga andc directions. Structural features responsible for the absence of ferroelectricity in this crystal have been pointed out.

Aspects of tautomerism. 6. Base-catalyzed hydrolysis of pseudo esters of .gamma.-keto acids

The rates of base-catalyzed hydrolysis of pseudo esters derived from y-keto acids show strikingly poor sensitivity to the nature of the leaving group.la The rates vary in the narrow range of about 12-fold as contrasted to a 103-fold spread of the corresponding benzoate esters. The results presented are consistent with a rate-determining formation of a tetrahedral intermediate (11) and i t s rapid collapse, by the cleavage of the lactone ring in a fast step.

New structural insights and molecular-modelling studies of 4-methyl-5-beta-hydroxyethylthiazole kinase from Pyrococcus horikoshii OT3 (PhThiK)

4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyses the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole. This work reports the first crystal structure of an archaeal ThiK: that from Pyrococcus horikoshii OT3 (PhThiK) at 1.85 angstrom resolution with a phosphate ion occupying the position of the beta-phosphate of the nucleotide. The topology of this enzyme shows the typical ribokinase fold of an alpha/beta protein. The overall structure of PhThiK is similar to those of Bacillus subtilis ThiK (BsThiK) and Enterococcus faecalis V583 ThiK (EfThiK). Sequence analysis of ThiK enzymes from various sources indicated that three-quarters of the residues involved in interfacial regions are conserved. It also revealed that the amino-acid residues in the nucleotide-binding, magnesium ion-binding and substrate-binding sites are conserved. Binding of the nucleotide and substrate to the ThiK enzyme do not influence the quaternary association (trimer) as revealed by the crystal structure of PhThiK.

Unusual structural stability and ligand induced alterations in oligomerization of a galectin

Abstract L-14, a 14-kDa S-type lectin shows the jelly roll tertiary structural fold akin to legume lectins yet, unlike them, it does not dissociate on thermal unfolding. In the absence of ligand L-14 displays denaturation transitions corresponding to tetrameric and octameric entities. The presence of complementary ligand reduces the association of L-14, which is in stark contrast with legume lectins where no alterations in quaternary structures are brought about by saccharides. From the magnitude of the increase in denaturation temperature induced by disaccharides the binding constants calculated from differential scanning calorimetry are comparable with those extrapolated from titration calorimetry indicating that L-14 interacts with ligands essentially in the folded state.

Succinate and malate improve development of hamster eight-cell embryos in vitro: Confirmation of viability by embryo transfer

The in vitro development of hamster preimplantation embryos is supported by non-glucose energy substrates. To investigate the importance of embryonic metabolism, influence of succinate and malate on the development of hamster 8-cell embryos to blastocysts was examined using a chemically defined protein-free modified hamster embryo culture medium-2 (HECM-2m). There was a dose-dependent influence of succinate on blastocyst development; 0.5 mM succinate was optimal (85.1% ± 3.9 vs. 54.5% ± 3.5). In succinate-supplemented HECM-2m, blastocyst development was reduced by omission of lactate (68.5% ± 7.2), but not pyruvate (85.8% ± 6.2) or glutamine (84.1% ± 2.1). Succinate along with either glutamine or lactate or pyruvate poorly supported blastocyst development (28%-58%). Malate also stimulated blastocyst development; 0.01 mM malate was optimal (86.3% ± 2.8). Supplementation of both succinate and malate to HECM-2m supported maximal (100%) blastocyst development, which was inhibited 4-fold by the addition of glucose/phosphate. The mean cell numbers (MCN) of blastocysts cultured in succinate-supplemented HECM-2m was higher (28.3 ± 1.1) than it was for those cultured in the absence of glutamine or pyruvate (range 20-24). The MCN was the highest (33.4 ± 1.6) for blastocysts cultured in succinate-malate-supplemented HECM-2m followed by those in succinate (28.3 ± 1.1) or malate (24.7 ± 0.5) supplemented HECM-2m. Embryo transfer experiments showed that 29.8% (±4.5) of transferred blastocysts cultured in succinate-malate-supplemented HECM-2m produced live births, similar (P > 0.1) to the control transfers of freshly recovered 8-cells (33.5% ± 2.0) or blastocysts (28.9% ± 3.0). These data show that supplementation of succinate and malate to HECM-2m supports 100% development of hamster 8-cell embryos to high quality viable blastocysts and that non-glucose oxidizable energy substrates are the most preferred components in hamster embryo culture medium. Mol. Reprod. Dev. 47:440-447, 1997. © 1997 Wiley-Liss, Inc.

Polypurine/polypyrimidine sequences as cis-acting transcriptional regulators

Genome sequence information has generated increasing evidence for the claim that repetitive DNA sequences present within and around genes could play a important role in the regulation of gene expression. Polypurine/polypyrimidine sequences [poly(Pu/Py)] have been observed in the vicinity of promoters and within the transcribed regions of many genes. To understand whether such sequences influence the level of gene expression, we constructed several prokaryotic and eukaryotic expression vectors incorporating poly(Pu/Py) repeats both within and upstream of a reporter gene, lacZ (encoding β-galactosidase), and studied its expression in vivo. We find that, in contrast to the situation in Escherichia coli, the presence of poly(Pu/Py) sequences within the gene does not significantly inhibit gene expression in mammalian cells. On the other hand, the presence of such sequences upstream of lacZ leads to a several-fold reduction of gene expression in mammalian cells. Similar down-regulation was observed when a structural cassette containing poly(Pu/Py) sequences upstream of lacZ was integrated into yeast chromosome V. Sequence analysis of the nine totally sequenced yeast chromosomes shows that a large number of such sequences occur upstream of ORFs. On the basis of our experimental results and DNA sequence analysis, we propose that these sequences can function as cis-acting transcriptional regulators.

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.