Four base recognition by triplex-forming oligonucleotides at physiological pH

We have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU [2′-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine] recognizes AT base pairs with high affinity, ^MeP (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while ^APP (6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one) and S [N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide)] bind to CG and TA base pairs, respectively. Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, ^MeP and ^APP retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA.     

Development of the chondrocranium in the suckermouth armored catfish Ancistrus cf. triradiatus (Loricariidae, Siluriformes)

The chondrocranium of the suckermouth armored catfish Ancistrus cf. triradiatus was studied. Its development is described based on specimens ranging from small prehatching stages with no cartilage visible, to larger posthatching stages where the chondrocranium is reducing. Cleared and stained specimens, as well as serial sections, revealed a cartilaginous skeleton with many features common for Siluriformes, yet several aspects of A. cf. triradiatus are not seen as such in other catfishes, or to a lesser extent. The skull is platybasic, but the acrochordal cartilage is very small and variably present, leaving the notochord protruding into the hypophyseal fenestra in the earlier stages. The ethmoid region is slender, with a rudimentary solum nasi. A lateral commissure and myodomes are present. The larger posterior myodome is roofed by a prootic bridge. The maxillary barbel is supported by a conspicuous cartilaginous rod from early prehatching stages. The ceratohyal has four prominent lateral processes. Infrapharyngobranchials I-II do not develop. During ontogeny, the skull lengthens, with an elongated ethmoid, pointing ventrally, and a long and bar-shaped hyosymplectic-pterygoquadrate plate. Meckel's cartilages point medially instead of rostrally.     

Rapid protocol for electroporation of Clostridium perfringens

A rapid and simple method has been developed for the electroporation of Clostridium perfringens with plasmid DNA. The new improvements, harvesting cells early in the logarithmic stage of growth, keeping the cells at room temperature and the absence of post-shock incubation on ice increased transformation efficiency by one order of magnitude.     

Short-term insulin and nutritional energy provision do not stimulate muscle protein synthesis if blood amino acid availability decreases

Muscle protein synthesis requires energy and amino acids to proceed and can be stimulated by insulin under certain circumstances. We hypothesized that short-term provision of insulin and nutritional energy would stimulate muscle protein synthesis in healthy subjects only if amino acid availability did not decrease. Using stable isotope techniques, we compared the effects on muscle phenylalanine kinetics across the leg of an amino acid-lowering, high-energy (HE, n = 6, 162 +/- 20 kcal/h) hyperglycemic hyperlipidemic hyperinsulinemic clamp with systemic insulin infusion to a low-energy (LE, n = 6, 35 +/- 3 kcal/h, P < 0.05 vs. HE) euglycemic hyperinsulinemic clamp with local insulin infusion in the femoral artery. Basal blood phenylalanine concentrations and phenylalanine net balance, muscle protein breakdown, and synthesis (nmol.min(-1).100 g leg muscle(-1)) were not different between groups. During insulin infusion, femoral insulinemia increased to a similar extent between groups and blood phenylalanine concentration decreased 27 +/- 3% in the HE group but only 9 +/- 2% in the LE group (P < 0.01 HE vs. LE). Phenylalanine net balance increased in both groups, but the change was greater (P < 0.05) in the LE group. Muscle protein breakdown decreased in the HE group (58 +/- 12 to 35 +/- 7 nmol.min(-1).100 g leg muscle(-1)) and did not change in the LE group. Muscle protein synthesis was unchanged in the HE group (39 +/- 6 to 30 +/- 7 nmol.min(-1).100 g leg muscle(-1)) and increased (P < 0.05) in the LE group (41 +/- 9 to 114 +/- 26 nmol.min(-1).100 g leg muscle(-1)). We conclude that amino acid availability is an important factor in the regulation of muscle protein synthesis in response to insulin, as decreased blood amino acid concentrations override the positive effect of insulin on muscle protein synthesis even if excess energy is provided.     

Postprandial intestinal and whole body nitrogen kinetics and distribution in piglets fed a single meal

Our aim was to characterize the postprandial total and dietary N fluxes in the portal drained viscera (PDV) and whole body after administration of a single meal in young pigs. Seven 4-wk-old piglets, implanted with a portal flow probe and portal, arterial and venous catheters, received a primed constant [(18)O]urea intravenous infusion and were studied for 8 h after a bolus mixed meal ingestion (46 mmol N/kg body wt) intrinsically labeled with (15)N to trace dietary N fluxes. The real cecal digestibility of the formula was 94.3% (SD 1.8). PDV output of dietary N was found principally in the pool of circulating protein (51% of the measured dietary N PDV output), in the free alpha-amino N pool (44%), and to a lesser extent in ammonia (5%). Dietary N release in alpha-amino N and ammonia mainly occurred during the first 3 h. Total and exogenous postprandial urea productions were 5.8 and 2.0 mmol N/kg body wt, respectively. At the end of the postprandial period, losses of dietary N amounted to 10.3% of the dose: 5.7% through ileal losses and 4.6% by deamination and transfer to urea. Net postprandial retention of dietary N was 90.4% (SD 1.3), of which 20% was found in splanchnic zone (small intestine 10%, liver 5%, and plasma protein 3%) and 42% in peripheral zone (muscle 31%, skin 6%). In conclusion, our results show a high efficiency of dietary N utilization for muscular uptake and anabolic utilization. However, the results obtained point out the necessity to further explore the form of dietary N released into the portal blood.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.     

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.     

Organisation of the S10, spc and alpha ribosomal protein gene clusters in prokaryotic genomes

Although it is well known that there is no long range colinearity in gene order in bacterial genomes, it is thought that there are several regions that are under strong structural constraints during evolution, in which gene order is extremely conserved. One such region is the str locus, containing the S10-spc-alpha operons. These operons contain genes coding for ribosomal proteins and for a number of housekeeping genes. We compared the organisation of these gene clusters in 111 sequenced prokaryotic genomes (99 bacterial and 12 archaeal genomes). We also compared the organisation to the phylogeny based on 16S ribosomal RNA gene sequences and the sequences of the ribosomal proteins L22, L16 and S14. Our data indicate that there is much variation in gene order and content in these gene clusters, both in bacterial as well as in archaeal genomes. Our data indicate that differential gene loss has occurred on multiple occasions during evolution. We also noted several discrepancies between phylogenetic trees based on 16S rRNA gene sequences and sequences of ribosomal proteins L16, L22 and S14, suggesting that horizontal gene transfer did play a significant role in the evolution of the S10-spc-alpha gene clusters.