Effect of melatonin on norepinephrine,epinephrine, and corticosterone contents in the adrenal gland of three avian species

Summary A single melatonin injection was administered intraperitoneally to three avian species in two doses (250 and 500 g · 100 g body wt^–1). Norepinephrine (NE), epinephrine (E), and corticosterone (C) contents of the adrenal gland were measured spectrofluorometrically 0.5, 2, and 24 h after injection. The results showed that melatonin at the lower dose caused a decrease of NE content in bulbul (42%), babbler (52%), and pigeon (39%), while at the higher dose it resulted in a decrease of NE only in bulbul (51%) 0.5 h after treatment. Melatonin at the lower dose also caused a decrease of NE in bulbul (45%) and babbler (53%) 2 and 24 h, respectively, after treatment, while at the higher dose it resulted in an increase of NE in bulbul (82%) and a decrease of NE in babbler (44%) 24 h after injection.Both low and high doses of melatonin resulted in a decrease of E content in bulbul (32–43%), babbler (34–43%), and pigeon (44–56%) 0.5 h after treatment, and a 34–46% decrease of E in bulbul and a 32–33% decrease of E in babbler 2 h after treatment. A 57% decrease of E was evident in pigeon with the lower dose of melatonin 2 h after injection. Melatonin at the higher dose caused a 67% increase of E in babbler and a 41% decrease of E in pigeon 24 h after administration. Melatonin at the lower dose resulted in an increase of C content in bulbul (70%), babbler (132%), and pigeon (69%) 0.5 h after treatment. A 60% increase of C was evident in pigeon following the lower dose of melatonin 24 h after injection. Melatonin at the higher dose resulted in an increase of C in the bulbul of 72% and 86% at 0.5 and 24 h, respectively, after treatment. The results indicate that melatonin produces significant changes of NE, E, and C contents in three avian species. The lower dose appears to be more effective in changing NE and C content.Abbreviations C corticosterone - CA catecholamine - DBH dopamine -hydroxylase - E epinephrine - NE norepinephrine     

Cytogenetic investigations in three cell types of a Saudi family with ataxia telangiectasia

Summary Chromosomal analyses were performed on lymphocytes, fibroblasts and lymphoblastoid cell lines derived from a Saudi family with ataxia telangiectasia (AT). The three siblings of a consanguineous marriage were all affected. The lymphocytes of the AT homozygotes (probands) showed an increase of 2- to 6-fold and 4- to 8-fold respectively, in the frequency of spontaneous and X-ray-induced chromosomal aberrations compared with controls, while the parents (obligate heterozygotes) of the patients showed no notable difference. The unirradiated lymphocytes from the oldest AT sibling, an 11-year-old boy (AT1), showed specific rearrangements involving chromosomes 7 and 14 [t(7;14)(q35;q12)] and 12 and 14 [t(12;14)(q23;q12)] in two different clones. The most severely affected sibling was a 9-year-old girl (AT2) who presented with a clone showing a novel rearrangement involving chromosomes 14 and 17, namely: del(14) (q31q32) and dup(17)(q21–q24). The lymphocytes from the third sibling, a 2-year-old boy (AT3), showed a t(2;14)(p24;q12). In addition, an inv(14)(q12q32) was observed in all three AT patients, while inv(7)(p14q35) was found only in patients 2 and 3. The lymphocytes from the AT parents and controls showed normal karyotypes. The breakpoints involving chromosomes 2,12 and 17, observed in our studies, have rarely been reported in other series of AT patients. No non-random chromosomal rearrangements were observed either in the skin fibroblasts or in the lymphoblastoid cell lines derived from the AT patients, although all cell lines showed an increase in both spontaneous and radiation-induced chromosomal breaks per cell. The present study constitutes the first report on a cytogenetic analysis of a Saudi family with three AT siblings.     

Influence of age and splanchnic nerve on the action of melatonin in the adrenomedullary catecholamine content and blood glucose level in the avian group

Summary A single intraperitoneal (IP) melatonin injection (0.5 mg/100 g body wt.) caused an increase in norepinephrine (NE) fluorescence and elevation of NE content in newly-hatched pigeons (Columba livia), but a reduction of NE fluorescence and depletion of NE content in the adrenal medulla of newly-hatched crows (Corvus splendens) after 0.5 h of treatment. In contrast, in adults melatonin caused increase in NE fluorescence and elevation of NE content only in the parakeet (Psittacula krameri).Half an hour of IP melatonin treatment (0.5 mg/100 g body wt.) induced release of epinephrine (E) from the adrenal medulla of newly-hatched pigeon and parakeet. In contrast, in the adults melatonin caused more than a two-fold increase in E in the pigeon, and a significant increase in the crow.Single IP melatonin injection (0.5 mg/100 g body wt.) caused hypoglycemia in the newly-hatched parakeet and adult pigeon, and hyperglycemia in newly-hatched pigeon after 0.5 h of treatment. Melatonin failed to regulate glucose homoeostasis in newly-hatched and adult crow.Splanchnic denervation of the left adrenal gland was performed in the adult pigeon. The right adrenal served as the innervated gland. Melatonin-induced modulation of catecholamines following a single IP injection (0.5 mg/100 g body wt.) revealed significant increases in NE fluorescence and NE content at 4 and 12 h after treatment in the denervated gland only, which gradually approached normal levels 9 days after treatment. In contrast, E content showed more than a two-fold increase over the control value in both the innervated and denervated glands 0.5 and 24 h after treatment. At 9 days after treatment, E content showed significant depletion in the innervated gland.The results of this study indicate that melatonin modulates catechol hormone content in avian adrenal medulla, and also regulates glucose homoeostasis (except in the crow). The splanchnic nerve plays a vital role in the synthesis of NE but has no effect on E.     

Influence of ions on cyclization of the amino terminal glutamine residues of tryptic peptides of streptococcal PepM49 protein. Resolution of cyclized peptides by HPLC and characterization by mass spectrometry

RPHPLC of the tryptic digest of lysine blocked group A streptococcal PepM49 protein (DHP-PepM49) consistently yielded, among others, two pairs of peptides which were well resolved, eluted in tandem, and had identical amino acid compositions. In each pair, the earlier eluting peptide was readily amenable to sequencing and yielded an amino-terminal glutamine whereas the later eluting peptide could not be sequenced. Mass spectral analysis revealed that each of these pairs of peptides differed in mass corresponding to the loss of ammonia. These data suggested that the later eluting peptide in each pair is a result of cyclization of the amino-terminal glutamine residue to pyroglutamic acid, which apparently leads to an increase in the hydrophobicity of the peptide. A kinetic analysis of the tryptic digestion of the DHP-PepM49 protein revealed that the cyclized form of the peptides were essentially absent during the initial time and increased with time of incubation, with a concomitant decrease in the uncyclized form. In 0.2 M ammonium bicarbonate at 37 degrees, nearly 44% conversion of the glutaminyl peptides to the pyroglutamyl peptides was observed in 24 h. This conversion was accelerated in sodium phosphate buffer relative to that in ammonium bicarbonate whereas it had a significantly lower rate in ammonium acetate buffer. The conversion was also temperature dependent, with essentially no cyclization at 0 degree, in all the three buffers. Thus, an extended digestion at 0 degree or a brief digestion at 37 degrees in ammonium acetate was found to be a suitable condition for limiting the cyclization of amino-terminal glutamine residues of PepM49 peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Swainsonine production in fed-batch fermentations of Metarhizium anisopliae

Summary Extended and enhanced production of swainsonine was achieved from fed-batch fermentations of the fungus Metarhizium anisopliae. A complex medium based on oatmeal extract was intermittently fed with D-glucose and/or lysine. Swainsonine titres were improved eleven-fold and the duration of production extended was from 240 to 550 hours compared with a batch culture under the same conditions.     

Dihydroxypropylation of amino groups of proteins: use of glyceraldehyde as a reversible agent for reductive alkylation

The mode of derivatization of amino groups of proteins by glyceraldehyde, an aldotriose, depends on the presence or absence of reducing agent. In the presence of sodium cyanoborohydride, the Schiff base adducts of the aldehyde with the amino groups are reduced, and dihydroxypropylation of amino groups takes place (reductive mode). The reductively glycated lysine residue, N epsilon-(2,3-dihydroxypropyl)lysine, is a substituted alpha-amino alcohol. This alpha-amino alcoholic function of the derivatized lysine should be susceptible to periodate oxidation, and this oxidation is anticipated to result in the regeneration of the lysine residue. This aspect has been now investigated. Indeed, on mild periodate oxidation (15 mM periodate, 15 min at room temperature) of dihydroxypropylated ribonuclease A, nearly 95% of its N epsilon-(2,3-dihydroxypropyl)lysine residues were regenerated to lysine residues. The removal of the dihydroxypropyl groups by periodate oxidation could be accomplished within a wide pH range with little variation in the recovery of lysines. The possible usefulness of this reversible chemical modification procedure in the primary structural studies of proteins was investigated with a tryptic peptide of dihydroxypropylated streptococcal M5 protein, namely, DHP-T4. This 12-residue tryptic peptide contains one internal N epsilon-(dihydroxypropyl)lysine. The dihydroxypropylated peptide released most of its dihydroxypropyl groups on mild periodate oxidation. Redigestion of the periodate-treated peptide with trypsin generated the two expected peptides, demonstrating the generation of a trypsin-susceptible site. Reductive dihydroxypropylation of amino groups of RNase A resulted in the loss of its enzyme activity, the extent of inactivation increasing with the concentration of the glyceraldehyde used.(ABSTRACT TRUNCATED AT 250 WORDS)     

An interpretation of the apparent dual specificity of some murine myeloma immunoglobulins with inulinbinding activity.

Five murine myeloma immunoglobulins have been studied for their binding with fructose-containing homopolysaccharides. The results indicate that the anti-(2 leads to 1)-D-fructofuranans are capable of binding (2 leads to 6)-D-fructofuranans with reduced affinity. An anti-(2 leads to 6)-D-fructofuranan (U10) was incapable of binding a (2 leads to 1)-D-fructofuranan. By using molecular models of fructofuranans of both linkage types, and a hypothetical molecular model of the combining region of E109, an anti-(2 leads to 1)-D-fructofuranan, a rationalization of these date can be proposed.     

Probing the “fingers” domain binding pocket of Hepatitis C virus NS5B RdRp and D559G resistance mutation via molecular docking,molecular dynamics simulation and binding free energy calculations

The NS5B RdRp polymerase is a prominent enzyme for the replication of Hepatitis C virus (HCV). During the HCV replication, the template RNA binding takes place in the “fingers” sub-domain of NS5B. The “fingers” domain is a new emerging allosteric site for the HCV drug development. The inhibitors of the “fingers” sub-domain adopt a new antiviral mechanism called RNA intervention. The details of essential amino acid residues, binding mode of the ligand, and the active site intermolecular interactions of RNA intervention reflect that this mechanism is ambiguous in the experimental study. To elucidate these details, we performed molecular docking analysis of the fingers domain inhibitor quercetagetin (QGN) with NS5B polymerase. The detailed analysis of QGN-NS5B intermolecular interactions was carried out and found that QGN interacts with the binding pocket amino acid residues Ala97, Ala140, Ile160, Phe162, Gly283, Gly557, and Asp559; and also forms π?π stacking interaction with Phe162 and hydrogen bonding interaction with Gly283. These are found to be the essential interactions for the RNA intervention mechanism. Among the strong hydrogen bonding interactions, the QGN?Ala140 is a newly identified important hydrogen bonding interaction by the present work and this interaction was not resolved by the previously reported crystal structure. Since D559G mutation at the fingers domain was reported for reducing the inhibition percentage of QGN to sevenfold, we carried out molecular dynamics (MD) simulation for wild and D559G mutated complexes to study the stability of protein conformation and intermolecular interactions. At the end of 50?ns MD simulation, the π?π stacking interaction of Phe162 with QGN found in the wild-type complex is altered into T-shaped π stacking interaction, which reduces the inhibition strength. The origin of the D559G resistance mutation was studied using combined MD simulation, binding free energy calculations and principal component analysis. The results were compared with the wild-type complex. The mutation D559G reduces the binding affinity of the QGN molecule to the fingers domain. The free energy decomposition analysis of each residue of wild-type and mutated complexes revealed that the loss of non-polar energy contribution is the origin of the resistance.

Communicated by Ramaswamy H. Sarma     

Truncated human LMP-1 triggers differentiation of C2C12 cells to an osteoblastic phenotype in vitro

LIM mineralization protein-1 （LMP-1） is a novel intracellular osteoinductive protein that has been shown to induce bone formation both in vitro and in viva. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. In this study, we investigated whether a truncated form of human LMP-1 （hLMP-1 [t]）, lacking the three C-terminal LIM domains, triggers the differentiation of pluripotent myoblastic C2C12 cells to the osteoblast lineage. C2C12 cells were transiently transduced with AdS-hLMP-1 （t）-green fluorescent protein or viral vector control. The expression of hLMP-1 （t） RNA and the truncated protein were examined. The results showed that hLMP-1 （t） blocked myotube formation in C2C12 cultures and significantly enhanced the alkaline phosphatase （ALP） activity. In addition, the expressions of ALP, osteocalcin, and bone morphogenetic protein （BMP）-2 and BMP-7 genes were also increased. The induction of these key osteogenic markers suggests that hLMP- 1 （t） can trigger the pluripotent myoblastic C2C12 cells to differentiate into osteoblastic lineage, thus extending our previous observation that LMP-1 and LMP-1 （t） enhances the osteoblastic phenotype in cultures of cells already committed to the osteoblastic lineage. Therefore, C2C12 cells are an appropriate model system for the examination of LMP-1 induction of the osteoblastic phenotype and the study of mechanisms of LMP-1 action.