Complete genome sequence of the halophilic and highly halotolerant Chromohalobacter salexigens type strain (1H11(T))

Chromohalobacter salexigens is one of nine currently known species of the genus Chromohalobacter in the family Halomonadaceae. It is the most halotolerant of the so-called 'moderately halophilic bacteria' currently known and, due to its strong euryhaline phenotype, it is an established model organism for prokaryotic osmoadaptation. C. salexigens strain 1H11(T) and Halomonas elongata are the first and the second members of the family Halomonadaceae with a completely sequenced genome. The 3,696,649 bp long chromosome with a total of 3,319 protein-coding and 93 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2004.     

Effect of litter size and bacitracin administration on tissue protein synthesis of lactating rabbit does

Bacitracin is an antibiotic used in rabbit husbandry to control microbial digestive pathologies. Collateral effects on absorption and mucosal development have been reported and these may impact on protein metabolism. This study aims to analyse the effect of the antibiotic on protein synthesis in lactating does because mammary gland metabolism and milk output should provide a sensitive index of any undesirable action of bacitracin. Rates of protein synthesis were measured in mammary gland, liver, intestinal mucosa and muscle of lactating rabbits does by injecting a flooding dose of [(2)H(5)]phenylalanine into the auricular artery of two groups (each n = 8) of New Zealand White does fed different experimental diets. The control group (C) received the basal diet and the bacitracin group (B) ingested the same diet but supplemented with bacitracin (100 mg/kg). Animals received the experimental diet from day 28 of pregnancy until day 26 of lactation when they were slaughtered. Just after birth, litter size was adjusted by cross-fostering either to five or nine pups (four does per dietary treatment). The relative weight of the liver tended to be greater in those females receiving the B diet (27 v. 22.5 g/kg BW; P < 0.07), while diet did not affect mammary gland weight (255.7 ± 10.59 g). Fractional protein synthesis rate (FSR) was higher for intestinal mucosa (duodenum; 51.7% ± 2.09%/day) followed by mammary gland and liver (38.29 ± 2.62%/day and 40.2 ± 1.98%/day, respectively), and the lowest value was observed in muscle (2.92 ± 0.26%/day; P < 0.0001). Bacitracin treatment lowered FSR in the mammary gland by 23% (P = 0.024) and this was independent of litter size. Conversely, FSR in the duodenum was not affected by antibiotic treatment but reduced by 15% (P = 0.021) for the larger litter size.     

A combinatorial approach to the synthesis of cystine based organogelators.

A solid-phase approach was used to prepare 20 cystine amide derivatives with disulfide bond formation resulting from an intra-site reaction between neighbouring cysteine residues. Library members were screened as potential organogelators in a range of solvent mixtures and resulted in the identification of a potent gelator able to rigidify water/DMSO mixtures at concentrations as low as 1.3 mM.     

Synthesis of hepcidin derivatives in order to develop standards for immune adsorption method

MeCN, acetonitrile; ECL, enhanced chemiluminescence; EDT, 1,2‐ethanedithiole; HEPC12‐A, rabbit anti‐human hepcidin IgG, affinity purified; HEPC13‐A, rabbit anti‐mouse/human hepcidin IgG, affinity purified; HEPC61‐P, human hepcidin‐25 control/blocking synthetic peptide; HRP, horseradish peroxidase; IL‐6, interleukin‐6; KLH, keyhole limpet hemocyanin; LEAP, liver‐expressed antimicrobial peptide; NEM, N‐ethylmaleimide; NMP, N‐methyl‐pirrolidone; PBS, phosphate buffered saline; PVDF, polyvinylidene difluoride; SELDI‐TOF‐MS, surface‐enhanced laser desorption ionization–time‐of‐flight mass spectrometry; TMB, tetramethylbenzidin; TNF‐α, tumor necrosis factor‐α Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Enzymatic formation of intermediates in the biosynthests of ajmalicine: Strictosidine and cathenamine

Pre-purified enzymes isolated from Catharanthus roseus suspension cultures synthesize strictosidine and cathenamine from tryptamine and secologanin. Whereas strictosidine showed metabolic activity, cathenamine accumulates during the cell-free incubations in the absence of reduced pyridine nucleotides. In the presence of δ-d-gluconolactone (0.1 M), strictosidine accumulates in a yield of ca 50%. Optimum conditions for its accumulation in crude extracts were found to be at pH 4.1, 0.25 mM tryptamine and 1.25 mM secologinin. Strictosidine synthase is stable for more than 1.5 months at 4°. The optimum conditions for the enzymatic synthesis of cathenamine are 1.54 mM tryptamine and 7.7 mM secologanin at pH 7.5. In the presence of NH₄⁺ the formation of the latter alkaloid decreases due to the synthesis of unidentified compounds.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Catalytic Activities Of [GADV]-Peptides

We have previously postulated a novel hypothesis for the origin of life, assuming that life on the earth originated from “[GADV]-protein world”, not from the “RNA world” (see Ikehara's review, 2002). The [GADV]-protein world is constituted from peptides and proteins with random sequences of four amino acids (glycine [G], alanine [A], aspartic acid [D] and valine [V]), which accumulated by pseudo-replication of the [GADV]-proteins. To obtain evidence for the hypothesis, we produced [GADV]-peptides by repeated heat-drying of the amino acids for 30 cycles ([GADV]-P₃₀) and examined whether the peptides have some catalytic activities or not. From the results, it was found that the [GADV]-P₃₀ can hydrolyze several kinds of chemical bonds in molecules, such as umbelliferyl-β-D-galactoside, glycine-p-nitroanilide and bovine serum albumin. This suggests that [GADV]-P₃₀ could play an important role in the accumulation of [GADV]-proteins through pseudo-replication, leading to the emergence of life. We further show that [GADV]-octapaptides with random sequences, but containing no cyclic compounds as diketepiperazines, have catalytic activity, hydrolyzing peptide bonds in a natural protein, bovine serum albumin. The catalytic activity of the octapeptides was much higher than the [GADV]-P₃₀ produced through repeated heat-drying treatments. These results also support the [GADV]-protein-world hypothesis of the origin of life (see Ikehara's review, 2002). Possible steps for the emergence of life on the primitive earth are presented.     

Carnosine and Homocarnosine,the Forgotten,Enigmatic Peptides of the Brain

Carnosine (beta-alanyl-histidine) and homocarnosine (gamma-aminobutyryl-histidine) are major constituents of excitable tissues, brain and skeletal muscles, but their physiological functions are yet unknown. Using primary cell culture systems, synthesis and uptake of carnosine exclusively by glial cells could be demonstrated. Uptake of carnosine was found to be mediated by a high affinity, energy-dependent dipeptide transport system, subsequently identified as the peptide transporter PepT2. With the synthesis of beta-Ala-Lys-Nepsilon-AMCA as a fluorescent reporter molecule, accumulation of this dipeptide derivative could be monitored under viable conditions not only in astroglia cells but also in folliculostellate cells of the anterior pituitary and in gonadal resident macrophages. This reporter dipeptide provided a most valuable tool to identify an intrapituitary communication system by tracing folliculostellate cells in acute slice preparation. Moreover, this substance could also be used to prepare pituitary cell cultures enriched with or depleted of folliculostellate cells that are needed for further studies.