Hypovitaminosis D in a normal, apparently healthy urban European population

Serum 25 OH Vitamin D (25 OH D) concentrations generally vary with latitude, season, and the composition of the population studied. There is a growing recognition that rather than a seasonal specific decline in serum 25 OH Vitamin D, a significant proportion of the population may exhibit asymtomatic subclinical Vitamin D insufficiency. Vitamin D insufficiency has been described in populations at risk, such as nursing home residents and the homebound elderly. We assessed a population of normal, apparently healthy volunteers at a single European urban center for 25 OH Vitamin D sufficiency. Serum 25 OH D concentrations were determined using an automated LIAISON((R)) 25 OH Vitamin D assay. For the purposes of this study, Vitamin D insufficiency was defined as a serum 25 OH Vitamin D concentration of <15 ng/mL. Of the total population (n = 126) 34% exhibited 25 OH Vitamin D concentrations of <15 ng/ml. The mean +/- S.D. serum 25 OH Vitamin D concentration among the total, sufficient, and insufficient populations was 19.4 +/- 7.7, 23.6 +/- 6.4, and 12.1 +/- 2.3 ng/mL. From these data, we conclude that 25 OH Vitamin D insufficiency is more common than previously thought, and is not restricted to high-risk groups.     

Assessment of the effect of alpha-galactosides injected during embryogenesis on selected chicken traits

The effect of different doses of alpha-galactoside (RFOs) preparations from Pisum sativum L. cv. Opal, injected into eggs during embryogenesis, on maintaining a high number of bifidobacteria, selected chicken broiler traits and the lipoprotein level of blood were studied. Two independent experiments were conducted. In the first, Ringer water solution containing 1.763 mg/egg of fructooligosaccharides (FOS) (I group), 2.1158 mg of pea RFO preparation containing 20% sucrose (II group) and 0.4232 mg of sucrose (III group) were injected into Hubbard broiler breeder eggs containing 12-day old embryos. Only Ringer water solution was applied to the eggs of the control group (IV group). The number of bifidobacteria determined in faeces of two-day old chicken of groups I and II was significantly higher in comparison with the sucrose and control groups. The high level ofbifidobacteria of groups I and II was maintained during 6 weeks. The dose of both preparations had no influence on the body weight, carcass, breast muscle, leg and abdominal fat ratio, total cholesterol, HDL and LDL serum concentrations. Broiler mortality and breast muscle cholesterol concentration was highest (P < 0.05) for the control group. On the other hand, the European Production Index, as well as serum triglycerides, were the lowest for this group. The second experiment was performed on Hybro G chicken breeder eggs. 0.69, 3.43 and 6.87 mg/egg of pea RFO preparation doses containing 20% sucrose were injected into the experimental groups. The number of bifidobacteria in the caecum and selected meat traits of broilers were determined. The results of this experiment confirmed that RFO injection in ovo causes the long-time maintenance of a high level ofbifidobacteria. The dose of the preparations does not have any effect on the selected broiler meat traits, except that the highest dose increases the percent of carcase in body weight. However, this dose reduced the hatchability of the treated embryos.     

Suppression of histone H1 genes in Arabidopsis results in heritable developmental defects and stochastic changes in DNA methylation

Histone H1 is an abundant component of eukaryotic chromatin that is thought to stabilize higher-order chromatin structures. However, the complete knock-out of H1 genes in several lower eukaryotes has no discernible effect on their appearance or viability. In higher eukaryotes, the presence of many mutually compensating isoforms of this protein has made assessment of the global function of H1 more difficult. We have used double-stranded RNA (dsRNA) silencing to suppress all the H1 genes of Arabidopsis thaliana. Plants with a >90% reduction in H1 expression exhibited a spectrum of aberrant developmental phenotypes, some of them resembling those observed in DNA hypomethylation mutants. In subsequent generations these defects segregated independently of the anti-H1 dsRNA construct. Downregulation of H1 genes did not cause substantial genome-wide DNA hypo- or hypermethylation. However, it was correlated with minor but statistically significant changes in the methylation patterns of repetitive and single-copy sequences, occurring in a stochastic manner. These findings reveal an important and previously unrecognized link between linker histones and specific patterns of DNA methylation.     

A novel metabolic pathway of melatonin: oxidation by cytochrome C

The indoleamine melatonin is ubiquitously distributed, and because of its small size and amphiphilic nature, it is able to reach easily all cellular compartments. The highest intracellular melatonin concentrations are found in the mitochondria, suggestive of local metabolism and/or direct participation in organelle function. In mitochondria cytochrome c (cyt c) could represent a melatonin target since it has the capability to oxidize organic molecules in the presence of H2O2, and mitochondria are the main site of H2O2 production in nonphagocytic cells. Therefore, we investigated oxidation of melatonin by cyt c/H2O2 couple as a potential pathway for its metabolism in the mitochondria. We found melatonin conversion into N(1)-acetyl-N(2)-formyl-5-methoxykynuramine via sequential steps that generate the intermediates 2-hydroxymelatonin and 2,3-dihydroxymelatonin. We experimentally excluded mediation by a Fenton/Haber-Weiss-type reaction and documented the dependence on oxoferryl heme for melatonin oxidation. Given the high mitochondrial concentrations of both melatonin and cyt c as well as the continuous generation of H2O2 during respiration, it is entirely possible that mitochondrial cyt c-mediated oxidation of melatonin may be a plausible pathway of its biotransformation in vivo.     

Molecular basis of cellulose biosynthesis disappearance in submerged culture of Acetobacter xylinum

Acetobacter xylinum strains are known as very efficient producers of bacterial cellulose which, due to its unique properties, has great application potential. One of the most important problems faced during cellulose synthesis by these bacteria is generation of cellulose non-producing cells, which can appear under submerged culture conditions. The reasons of this remain unknown. These studies have been undertaken to compare at the molecular level wild-type, cellulose producing (Cel(+)) A. xylinum strains with Cel(-) forms of cellulose-negative phenotype. Comparison of protein profiles of both forms of A. xylinum by 2D electrophoresis allowed for the isolation of proteins which were produced exclusively by either Cel+ or Cel- cells. Sequences of peptides derived from these proteins were aligned with those of proteins deposited in databases. This analysis revealed that Cel(-) cells lacked two enzymes: phosphoglucomutase and glucose-1-phosphate uridylyltransferase, which generates UDP-glucose being the substrate for cellulose synthase. DNA was analyzed by ligation-mediated PCR carried out at low denaturation temperature (PCR-MP). Two DNA fragments of different thermal stability (218 and 217 bp) were obtained from the DNA of Cel(+) and Cel(-) forms, respectively. The only difference between these Cel(-) and Cel(+) DNA fragments is deletion of one T residue. Alignment of those two sequences with those deposited in the GenBank database revealed that similar fragments are present in the genomes of some bacterial cellulose producers and are located downstream from open reading frames (ORF) encoding phosphoglucomutase. The meaning of this observation is discussed.     

Some structural properties of plant serine:glyoxylate aminotransferase?

The structural properties of photorespiratory serine:glyoxylate aminotransferases (SGAT, EC 2.6.1.45) from maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves were examined. By means of molecular sieving on Zorbax SE-250 column and filtration through centrifugal filters it was shown that dimers of wheat enzyme (molecular mass of about 90 kDa) dissociate into component monomers (molecular mass of about 45 kDa) upon decrease in pH value (from 9.1 or 7.0 to 6.5). At pH 9.1 a 50-fold decrease of ionic strength elicited a similar effect. Under the same conditions homodimers of the maize enzyme (molecular mass similar to that of the wheat enzyme) remained stable. Immunoblot analysis with polyclonal antiserum against wheat seedling SGAT on leaf homogenates or highly purified preparations of both enzymes showed that the immunogenic portions of the wheat enzyme are divergent from those of the maize enzyme. The sequence of 136 amino acids of the maize enzyme and 78 amino acids of the wheat enzyme was established by tandem mass spectrometry with time of flight analyzer. The two enzymes likely share similarity in tertiary and quaternary structures as well as high level of hydrophobicity on their molecular surfaces. They likely differ in the mechanism of transport from the site of biosynthesis to peroxisomes as well as in some aspects of secondary structure.     

Surface changes of the mechanosensitive channel MscS upon its activation, inactivation, and closing

MscS is a bacterial mechanosensitive channel that shows voltage dependence. The crystal structure of MscS revealed that the channel is a homoheptamer with a large chamber on the intracellular site. Our previous experiments indicated that the cytoplasmic chamber of the channel is not a rigid structure and changes its conformation upon the channel activation. In this study, we have applied various sized cosolvents that are excluded from protein surfaces. It is well known that such cosolvents induce compaction of proteins and prevent thermal fluctuations. It is also known that they shift channel equilibrium to the state of lower volume. We have found that large cosolvents that cannot enter the channel interior accelerate channel inactivation when applied from the cytoplasmic side, but they slow down inactivation when applied from the extracellular side. We have also found that small cosolvents that can enter the channel cytoplasmic chamber prevent the channel from opening, unlike the large ones. These data support our idea that the channel cytoplasmic chamber shrinks upon inactivation but also give new clues about conformational changes of the channel upon transitions between its functional states.     

Synthesis of bis-(methyl 3,4,6-tri-O-acetyl-D-glucopyranosid-2-yl)-oxamides

The synthesis of a new bis-(D-glucopyranosid-2-yl)oxamides via the key intermediate, N-acetyl N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl) oxamic acid chloride (2alpha) is described. Treatment of compound 2alpha with methyl 3,4,6-tri-O-acetyl-2-amino-2-deoxy-beta-D-glucopyranoside afforded N-(methyl 3,4,6-tri-O-acetyl-alpha-D-glucopyranosid-2-yl)-N'-(methyl 3,4,6-tri-O-acetyl-beta-D-glucopyranosid-2-yl)-oxamide. Reaction of 2alpha with 1,2-diaminoethane afforded 1,2-bis-[N,N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)]ethyloxamide as a main product, while 2-N-[N'-(methyl 3',4',6'-tri-O-acetyl-alpha-D-glucopyranosid-2'-yl)oxamide]-ethyl acetamide was formed as a side product. Reaction of 2alpha with 1,3-diamino-2-hydroxypropane gave only 1,3-bis-N,N-[N'-(methyl 3',4',6'-tri-O-acetyl-2'-deoxy-alpha-D-glucopyranosid-2'-yl)-oxamido]-2-propanol.     

N-Alkyl derivatives of 2-amino-2-deoxy-D-glucose

Mono- and di-N-alkylated derivatives of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-beta-D-glucose (alkyl=methyl, ethyl, propyl, butyl, pentyl, hexyl, benzyl) were synthesised by the reductive alkylation of per-O-acetyl-d-glucosamine. (N-ethyl, N-propyl, N-butyl, N-pentyl and N-hexyl)-1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-beta-D-glucoses were deacetylated in order to attempt an enzymatic phosphorylation. All products were characterised by means of IR, NMR and MS spectra. N-Ethyl- and N-pentyl-d-glucosamines were found to exhibit weak antifungal activity.