The Lactococcus lactis sex-factor aggregation gene cluA

A gene, cluA, was cloned from the chromosomally located sex factor of Lactococcus lactis MG1363. Sequence analysis revealed significant homology with previously described aggregation proteins in Enterococcus and Streptococcus species. The possibility that cluA was an equivalent protein involved in cell aggregation between donor and recipient bacteria during lactococcal conjugation was confirmed by its expression under the control of a heterologous promoter in L. lactis. Analysis of the homology between the CluA protein and the related proteins of Enterococcus and Streptococcus allowed a common structure for these proteins to be postulated. This consisted of five domains. Functionally conserved domains I and V act respectively as a secretary leader and C-terminal membrane anchor. Domains II and IV are conserved at the amino acid level and probably have common structural roles whereas domain III is variable and may control binding specificity.     

Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay

Abstract— Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human, as a capturing antibody. With this technique, the detection limit for was less than 5 pg/ml of CSF. Using ATS, which recognizes abnormally phosphorylated ser-ines 199–202 in, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactiv-ity in CSF, suggesting that only a small fraction of CSF contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF levels are significantly increased in Alzheimer's disease. Also, CSF levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF levels in Alzheimer's disease.     

Detection of a Novel Sepiapterin Reductase mRNA: Assay of mRNA in Various Cells and Tissues of Various Species

Fragments of cDNA coding for rat, murine, and human sepiapterin reductase (SR) were amplified by PCR via primer positioning close to the reported 3′-end of the coding region in the rat enzyme. They were sequenced and used as probes for mRNA detection. Northern blot analysis detected two mRNA species for SR. Their sizes were 1.3 and 2.1 kb for rat, 1.3 and 2.3 kb for mouse, and 1.6 and 2.1 kb for human cell lines. Comparison of rat cell lines and rat tissues indicated that in tissues only the 1.3-kb species is present. Washing of the Northern blots under different stringency conditions indicated a more stable interaction of the 1.3-kb mRNA species with the cDNA probe as compared to the 2.3-kb species. The 1.3-kb species corresponds to the reported 28.2-kDa molecular mass of rat SR monomer. SR mRNA expression is absent in the human NK-like cell line YT and in the murine erythroleukemia subclone B8/3, which both lack SR activity. Moreover, the relative mRNA expression correlates with the enzymatic activities of different cell lines within the same species. This indicates that SR activity is regulated by its steady state mRNA levels.     

Membrane inlet—ion mobility spectrometry with automatic spectra evaluation as online monitoring tool for the process control of microalgae cultivation

The cultivation of algae either in open raceway ponds or in closed bioreactors could allow the renewable production of biomass for food, pharmaceutical, cosmetic, or chemical industries. Optimal cultivation conditions are however required to ensure that the production of these compounds is both efficient and economical. Therefore, high-frequency analytical measurements are required to allow timely process control and to detect possible disturbances during algae growth. Such analytical methods are only available to a limited extent. Therefore, we introduced a method for monitoring algae release volatile organic compounds (VOCs) in the headspace above a bioreactor in real time. This method is based on ion mobility spectrometry (IMS) in combination with a membrane inlet (MI). The unique feature of IMS is that complete spectra are detected in real time instead of sum signals. These spectral patterns produced in the ion mobility spectrum were evaluated automatically via principal component analysis (PCA). The detected peak patterns are characteristic for the respective algae culture; allow the assignment of the individual growth phases and reflect the influence of experimental parameters. These results allow for the first time a continuous monitoring of the algae cultivation and thus an early detection of possible disturbances in the biotechnological process.     

Localization of the intrachain disulfide bonds of the envelope glycoprotein 71 from Friend murine leukemia virus.

Envelope glycoprotein 71 from Friend murine leukemia virus was purified to homogeneity by reversed-phase HPLC. It could be shown that all 20 cysteine residues of the molecule are linked by disulfide bonds. After complete tryptic digestion, peptides containing cystine were identified by comparison of the reversed-phase HPLC profile of the digest with that of a reduced aliquot which had been subjected to affinity chromatography on thiol-Sepharose. The locations of the 10 disulfide bonds were determined by isolation, further digestion and analysis of peptides containing cystine. The first cysteine residue of the sequence (Cys46) was shown to be coupled to the sixth (Cys98), leading to a large loop containing four additional cysteine residues. Computer model building and energy calculations led to the assignment of Cys72 to Cys87 and Cys73 to Cys83. The following four cysteine residues of the sequence also constitute a structural unit, with Cys121 bonded to Cys141 and Cys133 to Cys146, and the last two cysteine residues in the amino-terminal domain of glycoprotein 71 form a small loop (Cys178 to Cys184). The first two cysteine residues of the carboxy-terminal domain produce a very small hydrophobic loop (Cys312-Cys315). Cys361 is bound to Cys373, Cys342 to Cys396 and Cys403 to Cys416. A model for the folding pattern of the viral glycoprotein is proposed.     

Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.     

Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs

Nearly 7000 Arabidopsis thaliana -expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.     

Mapping of a Gene for Long QT Syndrome to Chromosome 4q25-27

Long QT syndrome (LQTS) is a heterogeneous inherited disorder causing syncope and sudden death from ventricular arrhythmias. A first locus for this disorder was mapped to chromosome 11p15.5. However, locus heterogeneity has been demonstrated in several families, and two other loci have recently been located on chromosomes 7q35-36 and 3p21-24. We used linkage analysis to map the locus in a 65-member family in which LQTS was associated with more marked sinus bradycardia than usual, leading to sinus node dysfunction. Linkage to chromosome 11p15.5, 7q35-36, or 3p21-24 was excluded. Positive linkage was obtained for markers located on chromosome 4q25-27. A maximal LOD score of 7.05 was found for marker D4S402. The identification of a fourth locus for LQTS confirms its genetic heterogeneity. Locus 4q25-27 is associated with a peculiar phenotype within the LQTS entity.