Study of the infectivity of saline-stored Campylobacter jejuni for day-old chicks

The culturability of three Campylobacter jejuni strains and their infectivity for day-old chicks were assessed following storage of the strains in saline. The potential for colonization of chicks was weakened during the storage period and terminated 3 to 4 weeks before the strains became nonculturable. The results from this study suggest that the role of starved and aged but still culturable campylobacters may be diminutive, but even more, that the role of viable but nonculturable stages in campylobacter epidemiology may be negligible. Even high levels of maternally derived anti-campylobacter outer membrane protein serum antibodies in day-old chicks did not protect the chicks from campylobacter colonization.     

Novel interaction between the M4 muscarinic acetylcholine receptor and elongation factor 1A2

The activation of the muscarinic acetylcholine receptor (mAChR) family, consisting of five subtypes (M1-M5), produces a variety of physiological effects throughout the central nervous system. However, the role of each individual subtype remains poorly understood. To further elucidate signal transduction pathways for specific subtypes, we used the most divergent portion of the subtypes, the intracellular third (i3) loop, as bait to identify interacting proteins. Using a brain pull-down assay, we identify elongation factor 1A2 (eEF1A2) as a specific binding partner to the i3 loop of M4, and not to M1 or M2. In addition, we demonstrate a direct interaction between these proteins. In the rat striatum, the M4 mAChR colocalizes with eEF1A2 in the soma and neuropil. In PC12 cells, endogenous eEF1A2 co-immunoprecipitates with the endogenous M4 mAChR, but not with the endogenous M1 mAChR. In our in vitro model, M4 dramatically accelerates nucleotide exchange of eEF1A2, a GTP-binding protein. This indicates the M4 mAChR is a guanine exchange factor for eEF1A2. eEF1A2 is an essential GTP-binding protein for protein synthesis. Thus, our data suggest a novel role for M4 in the regulation of protein synthesis through its interaction with eEF1A2.     

Prediction of human protein function from post-translational modifications and localization features

We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects such as the length, isoelectric point and composition of the polypeptide chain.     

A new non-linear normalization method for reducing variability in DNA microarray experiments

Background  

Microarray data are subject to multiple sources of variation, of which biological sources are of interest whereas most others are only confounding. Recent work has identified systematic sources of variation that are intensity-dependent and non-linear in nature. Systematic sources of variation are not limited to the differing properties of the cyanine dyes Cy5 and Cy3 as observed in cDNA arrays, but are the general case for both oligonucleotide microarray (Affymetrix GeneChips) and cDNA microarray data. Current normalization techniques are most often linear and therefore not capable of fully correcting for these effects.     

The long-acting GLP-1 derivative NN2211 ameliorates glycemia and increases beta-cell mass in diabetic mice

NN2211 is a long-acting, metabolically stable glucagon-like peptide-1 (GLP-1) derivative designed for once daily administration in humans. NN2211 dose dependently reduced the glycemic levels in ob/ob mice, with antihyperglycemic activity still evident 24 h postdose. Apart from an initial reduction in food intake, there were no significant differences between NN2211 and vehicle treatment, and body weight was not affected. Histological examination revealed that beta-cell proliferation and mass were not increased significantly in ob/ob mice with NN2211, although there was a strong tendency for increased proliferation. In db/db mice, exendin-4 and NN2211 decreased blood glucose compared with vehicle, but NN2211 had a longer duration of action. Food intake was lowered only on day 1 with both compounds, and body weight was unaffected. beta-Cell proliferation rate and mass were significantly increased with NN2211, but with exendin-4, only the beta-cell proliferation rate was significantly increased. In conclusion, NN2211 reduced blood glucose after acute and chronic treatment in ob/ob and db/db mice and was associated with increased beta-cell mass and proliferation in db/db mice. NN2211 is currently in phase 2 clinical development.     

Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIalpha was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.     

Protein synthesis by single ribosomes

The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence. Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis. In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors. Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes. This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.     

Potential rates of fermentation in digesta from the gastrointestinal tract of pigs: effect of feeding fermented liquid feed

Microbial catabolic capacity in digesta from the gastrointestinal tract of pigs fed either dry feed or fermented liquid feed (FLF) was determined with the PhenePlate multisubstrate system. The in vitro technique was modified to analyze the kinetics of substrate catabolism mediated by the standing stock of enzymes (potential rates of fermentation), allowing a quantitative evaluation of the dietary effect on the catabolic capacity of the microbiota. In total, the potential rates of fermentation were significantly reduced in digesta from the large intestine (cecum, P < 0.1; colon, P < 0.01; and rectum, P < 0.0001) of pigs fed FLF compared to pigs fed dry feed. No effect of diet was observed in the stomach (P = 0.71) or the distal part of the small intestine (P = 0.97). The highest rates of fermentation and the most significant effect of diet were observed for readily fermentable carbohydrates like maltose, sucrose, and lactose. Feeding FLF to pigs also led to a reduction in the large intestine of the total counts of anaerobic bacteria in general and lactic acid bacteria specifically, as well as of microbial activity, as determined by the concentration of ATP and short-chain fatty acids. The low-molecular-weight carbohydrates were fermented mainly to lactic acid in the FLF before being fed to the animals. This may have limited microbial nutrient availability in the digesta reaching the large intestine of pigs fed FLF and may have caused the observed reduction in activity and density of the cecal and colonic microbial population. On the other hand, feeding FLF to pigs reduced the viable counts of coliform bacteria (indicator of Escherichia coli and Salmonella spp.) most profoundly in the stomach and the distal part of the small intestine, probably due to the bactericidal effect of lactic acid and low pH. The results presented clearly demonstrate that feeding FLF to pigs had a great impact on the indigenous microbiota, as reflected in bacterial numbers, short-chain fatty acid concentration, and substrate utilization. However, completely different mechanisms may be involved in the proximal and the distal parts of the gastrointestinal tract. The present study illustrates the utility of the PhenePlate system for quantifying the catabolic capacity of the indigenous gastrointestinal tract microbiota.