<Emphasis Type="Italic">Marinococcus halophilus</Emphasis> DSM 20408<Superscript>T </Superscript>encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM

In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium. To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M. halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport. The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM. The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs). The transporter responsible for the uptake of glycine betaine in M. halophilus is encoded by an open reading frame of 1,482 bp called betM. The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family. The affinity of whole cells of E. coli MKH13 for ectoine (K_s=1.6 M) and betaine (K_s=21.8 M) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates. An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E. coli MKH13 cells, demonstrating that both systems are osmoregulated.Communicated by W.D. Grant     

<Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> type III secretion: Evidence for the ability to transport proteins that are folded prior to secretion

Background  

Pathogenic Yersinia species (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis) share a type three secretion system (TTSS) which allows translocation of effector proteins (called Yops) into host cells. It is believed that proteins are delivered through a hollow needle with an inner diameter of 2–3 nm. Thus transport seems to require substrates which are essentially unfolded. Recent work from different groups suggests that the Yersinia TTSS cannot accommodate substrates which are folded prior to secretion. It was suggested that folding is prevented either by co-translational secretion or by the assistance of specific Yop chaperones (called Sycs).     

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.     

Subunit composition and quantitative importance of GABA(A) receptor subtypes in the cerebellum of mouse and rat

In cerebellum, 13 different GABA(A) receptor subunits are expressed. The number of different receptor subtypes formed in this tissue, their subunit composition and their quantitative importance so far has not been determined. In the present study, immunodepletion by immunoaffinity chromatography, as well as immunoprecipitation and western blot analysis was performed using 13 different subunit-specific antibodies to provide an overview on the subunit composition and abundance of GABA(A) receptor subtypes in mouse and rat cerebellum. Results obtained indicate that alpha1betaxgamma2, alpha1alpha6betaxgamma2, alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta are the major GABA(A) receptor subtypes present in the cerebellum. In addition, small amounts of alpha1betaxdelta receptors and a series of minor receptor subtypes containing alpha2, alpha3, alpha4, alpha5, gamma1 or gamma3 subunits are also present in the cerebellum. Whereas the abundance of alpha1alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta receptors is different in mouse and rat cerebellum, that of other receptors is quite similar in these tissues. Data obtained for the first time provide an overview on the GABA(A) receptor subtypes present in the cerebellum and represent the basis for further studies investigating changes in receptor expression and composition under pathological conditions.     

Rho kinase is required for CCR7-mediated polarization and chemotaxis of T lymphocytes

A chemokine receptor, CXCR4, and its endogenous ligand, stromal cell-derived factor-1 (SDF-1), have been recognized to be involved in the metastasis of several types of cancers. T140 analogs are peptidic CXCR4 antagonists composed of 14 amino acid residues that were previously developed as anti-HIV agents having inhibitory activity against HIV-entry through its co-receptor, CXCR4. Herein, we report that these compounds effectively inhibited SDF-1-induced migration of human breast cancer cells (MDA-MB-231), human leukemia T cells (Sup-T1) and human umbilical vein endothelial cells at concentrations of 10–100 nM in vitro. Furthermore, slow release administration by subcutaneous injection using an Alzet osmotic pump of a potent and bio-stable T140 analog, 4F-benzoyl-TN14003, gave a partial, but statistically significant (P≤0.05 (t-test)) reduction in pulmonary metastasis of MDA-MB-231 in SCID mice, even though no attempt was made to inhibit other important targets such as CCR7. These results suggest that T140 analogs have potential use for cancer therapy, and that small molecular CXCR4 antagonists could potentially replace neutralizing antibodies as anti-metastatic agents for breast cancer.     

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.     

A neuropeptide FF-related gene is expressed selectively in neurons of the terminal nerve in Danio rerio

RFamides constitute a large family of neuromodulatory peptides. We have cloned a zebrafish gene, which is presumably a homologue to the mammalian PQRF subfamily of RFamides, and named it zfPQRF for its species and subfamily allocation. We report that in contrast to its mammalian counterparts zfPQRF is expressed in the olfactory bulb and the nucleus olfactoretinalis in the telencephalon, but absent in more caudal regions, including hypothalamus, brain stem and spinal cord. zfPQRF-expressing neurons originate in the vicinity of the olfactory placode and populate the nuclei of the terminal nerve during later development, as demonstrated by co-expression of zebrafish salmon-type gonadotropin releasing hormone, which was found to exclusively label terminal nerve neurons.     

Yeast reporter system for rapid determination of estrogenic activity

An in vitro test system for the determination of estrogens, xeno- and phytoestrogens, based on the activation of human estrogen receptor-alpha, has been examined for ability in monitoring environmental estrogens. The system consists of an expression plasmid for the human estrogen receptor-alpha and a reporter plasmid containing the lacZ gene under the control of the vitellogenin hormone response element. These plasmids have been transformed into S. cerevisae. Cultivation of yeast in the presence of estrogenic substances leads to activation of the estrogen receptor and induces the expression of the reporter lacZ. beta-Galactosidase activity of the translated gene lacZ is a measure of the estrogenic activity of a compound. First, the selectivity of the system was compared to data available in the literature. Then the sensitivity of the system was checked. The detection limit is 0.1 ng 17-beta estradiol or an equivalent activity per liter, if a sample can be concentrated 1000-fold. The system has been further characterized by selected compounds with known and unknown estrogenic activity.     

An european pupil project linked to the scientific aims of the experiment AQUARIUS-XENOPUS on the taxi Soyuz flight Andromede to ISS

The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools.