Bacteriophage P22 tail accessory factor GP26 is a long triple-stranded coiled-coil

P22 is a well characterized tailed bacteriophage that infects Salmonella enterica serovar Typhimurium. It is characterized by a "short" tail, which is formed by five proteins: the dodecameric portal protein (gp1), three tail accessory factors (gp4, gp10, gp26), and six trimeric copies of the tail-spike protein (gp9). We have isolated the gene encoding tail accessory factor gp26, which is responsible for stabilization of viral DNA within the mature phage, and using a variety of biochemical and biophysical techniques we show that gp26 is very likely a triple stranded coiled-coil protein. Electron microscopic examination of purified gp26 indicates that the protein adopts a rod-like structure approximately 210 angstroms in length. This trimeric rod displays an exceedingly high intrinsic thermostability (T(m) approximately 85 degrees C), which suggests a potentially important structural role within the phage tail apparatus. We propose that gp26 forms the thin needle-like fiber emanating from the base of the P22 neck that has been observed by electron microscopy of negatively stained P22 virions. By analogy with viral trimeric coiled-coil class I membrane fusion proteins, gp26 may represent the membrane-penetrating device used by the phage to pierce the host outer membrane.     

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.     

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.     

Synthesis and D(2)-like binding affinity of new derivatives of N-(1-ethyl-2-pyrrolidinylmethyl)-4,5-dihydro-1H-benzo[g]indole-3-carboxamide and related 4H-[1]benzothiopyrano[4,3-b]pyrrole and 5,6-dihydro-4H-benzo[6,7]cyclohepta[b]pyrrole-3-carboxamide analogues

Various new derivatives and structural analogues of N-(1-ethyl-2-pyrrolidinylmethyl)-4,5-dihydro-1H-benzo[g]indole-3-carboxamide (2a), a representative term of a series of 2-aminomethylpyrrolidinyl derived 4,5-dihydrobenzo[g]indolcarboxamides with good D(2)-like affinity, were synthesized and evaluated for their ability to bind to dopamine D(2)-like receptors in vitro. The structural contribution to D(2)-like receptor binding of the 4,5-dihydrobenzo[g]indole portion of the molecule was examined. From these studies, compound 2k, 2-chloro-N-(1-ethyl-2-pyrrolidinylmethyl)-5,6-dihydro-4H-benzo[6,7]cyclohepta[b]pyrrole-3-carboxamide, was found to possess a potent affinity for D(2)-like receptors. Behavioural tests in rats have shown that this compound reduces the hyperactivity induced by amphetamine, a property shared by all antipsychotic drugs, at a dose which failed to induce catalepsy, an effect which is predictive of extrapyramidal side effects in humans. The other compounds demonstrated moderate (2c, 2h, and 2j) or no affinity for D(2)-like receptors.     

Distribution of Serotonin Receptor of Type 6 (5-HT6) in Human Brain Post-mortem. A Pharmacology, Autoradiography and Immunohistochemistry Study

The aim of this study was to investigate the distribution of serotonin (5-HT) receptors of type 6 (5-HT(6)) in postmortem human prefrontal cortex, striatum and hippocampus. The brain samples were obtained from 6 subjects who had died for causes not involving primarily or secondarily the CNS. The 5-HT(6) receptor distribution was explored by the [(125)I]SB-258585 binding to brain membranes followed by the pharmacological characterization, where possible, and by autoradiographic, immunohistochemical and immunofluorescence evaluations. A specific and saturable [(125)I]SB-258585 binding was detected in striatum only, with a pharmacological characterization consistent with that of a 5-HT(6) receptor. The autoradiography showed the presence of a specific [(125)I]SB-258585 binding distributed homogeneously in caudate, putamen and accumbens. The immunohistochemistry, carried out in the striatum only, coupled with the immunofluorescence with glial fibrillary acidic protein (GFAP) and parvalbumin (PV) showed the co-localization of 5-HT(6) receptor with PV, while indicating that this receptor subtype was expressed in neurons and not in astrocytes. Taken together, the present findings showed the presence of a higher density of 5-HT(6) receptors, as labeled by [(125)I]SB-258585, in striatum than in hippocampus and prefrontal cortex, and specifically within the neuronal body. In addition, they would suggest that striatum is one of the major potential CNS targets linked to 5-HT(6) receptor modulation.     

Mannitol production by heterofermentative <Emphasis Type="BoldItalic">Lactobacillus reuteri</Emphasis> CRL 1101 and <Emphasis Type="BoldItalic">Lactobacillus fermentum</Emphasis> CRL 573 in free and controlled pH batch fermentations

Certain lactic acid bacteria, especially heterofermentative strains, are capable to produce mannitol under adequate culture conditions. In this study, mannitol production by Lactobacillus reuteri CRL 1101 and Lactobacillus fermentum CRL 573 in modified MRS medium containing a mixture of fructose and glucose in a 6.5:1.0 ratio was investigated during batch fermentations with free pH and constant pH 6.0 and 5.0. Mannitol production and yields were higher under constant pH conditions compared with fermentations with free pH, the increase being more pronounced in the case of the L. fermentum strain. Maximum mannitol production and yields from fructose for L. reuteri CRL 1101 (122 mM and 75.7 mol%, respectively) and L. fermentum CRL 573 (312 mM and 93.5 mol%, respectively) were found at pH 5.0. Interestingly, depending on the pH conditions, fructose was used only as an alternative external electron acceptor or as both electron acceptor and energy source in the case of the L. reuteri strain. In contrast, L. fermentum CRL 573 used fructose both as electron acceptor and carbon source simultaneously, independently of the pH value, which strongly affected mannitol production by this strain. Studies on the metabolism of these relevant mannitol-producing lactobacilli provide important knowledge to either produce mannitol to be used as food additive or to produce it in situ during fermented food production.     

The metabolic blueprint of Phaeodactylum tricornutum reveals a eukaryotic Entner-Doudoroff glycolytic pathway

Diatoms are one of the most successful groups of unicellular eukaryotic algae. Successive endosymbiotic events contributed to their flexible metabolism, making them competitive in variable aquatic habitats. Although the recently sequenced genomes of the model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana have provided the first insights into their metabolic organization, the current knowledge on diatom biochemistry remains fragmentary. By means of a genome‐wide approach, we developed DiatomCyc, a detailed pathway/genome database of P. tricornutum. DiatomCyc contains 286 pathways with 1719 metabolic reactions and 1613 assigned enzymes, spanning both the central and parts of the secondary metabolism of P. tricornutum. Central metabolic pathways, such as those of carbohydrates, amino acids and fatty acids, were covered. Furthermore, our understanding of the carbohydrate model in P. tricornutum was extended. In particular we highlight the discovery of a functional Entner–Doudoroff pathway, an ancient alternative for the glycolytic Embden–Meyerhof–Parnas pathway, and a putative phosphoketolase pathway, both uncommon in eukaryotes. DiatomCyc is accessible online ( http://www.diatomcyc.org ), and offers a range of software tools for the visualization and analysis of metabolic networks and ‘omics’ data. We anticipate that DiatomCyc will be key to gaining further understanding of diatom metabolism and, ultimately, will feed metabolic engineering strategies for the industrial valorization of diatoms.     

Synthesis and biological evaluation of new active For‐Met‐Leu‐Phe‐OMe analogues containing para‐substituted Phe residues

In the present study, we report synthesis and biological evaluation of the N‐Boc‐protected tripeptides 4a–l and N‐For protected tripeptides 5a–l as new For‐Met‐Leu‐Phe‐OMe (fMLF‐OMe) analogues. All the new ligands are characterized by the C‐terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF‐OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with ‐CH₃ and ‐C(CH₃)₃, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i , containing ‐F and ‐I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Simultaneous Non-Negative Matrix Factorization for Multiple Large Scale Gene Expression Datasets in Toxicology

Non-negative matrix factorization is a useful tool for reducing the dimension of large datasets. This work considers simultaneous non-negative matrix factorization of multiple sources of data. In particular, we perform the first study that involves more than two datasets. We discuss the algorithmic issues required to convert the approach into a practical computational tool and apply the technique to new gene expression data quantifying the molecular changes in four tissue types due to different dosages of an experimental panPPAR agonist in mouse. This study is of interest in toxicology because, whilst PPARs form potential therapeutic targets for diabetes, it is known that they can induce serious side-effects. Our results show that the practical simultaneous non-negative matrix factorization developed here can add value to the data analysis. In particular, we find that factorizing the data as a single object allows us to distinguish between the four tissue types, but does not correctly reproduce the known dosage level groups. Applying our new approach, which treats the four tissue types as providing distinct, but related, datasets, we find that the dosage level groups are respected. The new algorithm then provides separate gene list orderings that can be studied for each tissue type, and compared with the ordering arising from the single factorization. We find that many of our conclusions can be corroborated with known biological behaviour, and others offer new insights into the toxicological effects. Overall, the algorithm shows promise for early detection of toxicity in the drug discovery process.