Construction of a new shuttle vector for Lactobacillus.

To clone the malolactic enzyme gene from Lactobacillus sp. 89, construction of a shuttle vector able to express itself in Lactobacillus sp. 89 and Escherichia coli was undertaken. The shuttle plasmid pLE16 resulted from the union of pBR328 and of the pLB10 plasmid extracted from Lactobacillus bulgaricus 10. The bacterial transformation in Lactobacillus sp. 89 was performed by electroporation, and the clones were selected on MRS medium with 30 micrograms.mL-1 chloramphenicol added. Fifty percent of the clones from Lactobacillus sp. 89 lost their resistance to chloramphenicol following 28 generations when the selection pressure was not maintained. The restriction map of pLE16 (7600 bp) was established using several restriction enzymes.     

Monoclonal Anti-Conjugated Acetylcholine Antibody and Immunohistochemical Applications in Rat Nervous System

Acetylcholine (ACh) conjugates were injected into AKR and DBA mice over a period of 10 weeks. The polyclonal antisera were tested at various immunization times for affinity and specificity using an enzyme-linked immunosorbent assay (ELISA). The most immunoreactive compound was found to be choline-glutaryl-bovine serum albumin (or conjugated ACh). The AKR and DBA mice yielding the highest apparent affinity were killed, and the spleen cells were fused with X63 or SP2/O/Ag mouse myeloma cells. Supernatants of confluent cultures were tested for the presence of anti-conjugated ACh antibodies using the same ELISA method. The best results were obtained with the hybridomas from AKR spleen cells and X63 mouse myeloma cells. Monoclonal antibody affinity and specificity were then evaluated by a radioimmunological procedure using iodinated monoclonal anti-conjugated ACh antibody. From competition experiments, the most immunoreactive compound was choline-glutaryl-protein. The other related compounds were recognized either poorly or not at all. The high affinity and specificity of our monoclonal antibody enabled us to visualize ACh molecules on fixed rat brain sections. ACh was fixed with a mixture of nitrobenzyl alcohol and glutaraldehyde. Many ACh-immunoreactive cell bodies and fibers were seen on sections from the basal forebrain and spinal cord. Preadsorption and other immunohistochemical tests demonstrated that the ACh staining was highly specific.     

A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut

Members of the genus Lactobacillus are common inhabitants of the gut, yet little is known about the traits that contribute to their ecological performance in gastrointestinal ecosystems. Lactobacillus reuteri 100-23 persists in the gut of the reconstituted Lactobacillus-free mouse after a single oral inoculation. Recently, three genes of this strain that were specifically induced (in vivo induced) in the murine gut were identified (38). We report here the detection of a gene of L. reuteri 100-23 that encodes a high-molecular-mass surface protein (Lsp) that shows homology to proteins involved in the adherence of other bacteria to epithelial cells and in biofilm formation. The three in vivo-induced genes and lsp of L. reuteri 100-23 were inactivated by insertional mutagenesis in order to study their biological importance in the murine gastrointestinal tract. Competition experiments showed that mutation of lsp and a gene encoding methionine sulfoxide reductase (MsrB) reduced ecological performance. Mutation of lsp impaired the adherence of the bacteria to the epithelium of the mouse forestomach and altered colonization dynamics. Homologues of lsp and msrB are present in the genomes of several strains of Lactobacillus and may play an important role in the maintenance of these bacteria in gut ecosystems.     

Adaptation of a South American malaria vector to laboratory colonization suggests faster-male evolution for mating ability

Background  

Anopheles (Nyssorhynchus) albitarsis (Diptera: Culicidae) is one of the very few South American mosquito vectors of malaria successfully colonized in the laboratory. These vectors are very hard to breed because they rarely mate in artificial conditions. A few years ago a free-mating laboratory colony of An. albitarsis sensu stricto was established after about 30 generations of artificial-mating. To begin to understand the process of adaptation of these malaria vectors to the laboratory we have compared the insemination rates of colony mosquitoes to those from the original population in both artificial and free-mating crosses. We also carried out crossing experiments between the two types of mosquitoes for a preliminary analysis of the genetic basis of such adaptation.     

Rapid PCR-based procedure to identify lactic acid bacteria: application to six common Lactobacillus species

The goal of this study was to develop a method allowing rapid identification of the lactic acid bacteria strains in use in the laboratory (Lactobacillus plantarum NCIMB8826; L. fermentum KLD; L. reuteri 100-23; L. salivarius UCC43321; L. paracasei LbTGS1.4; L. casei ATCC393), based on PCR amplification of 16S RNA coding sequences. First, specific forward oligonucleotides were designed in the variable regions of 16S RNA coding sequences of six Lactobacillus strains. The reverse oligonucleotide was designed in the region where the sequences were homologous for the six strains. The expected size of the amplification product was +/-1000 bp. The specificity of the method was tested on total chromosomal DNA. For five out of the six strains, the amplification of the fragment was strain-specific and the method was directly applicable to colonies. For the strain L. casei ATCC393, an additional argument to the classification of this bacteria in the paracasei group could be proposed. Validation of the developed method was performed by applying it to six Lactobacillus reference strains and to various species of bacteria.     

The Association of the Vanin-1 N131S Variant with Blood Pressure Is Mediated by Endoplasmic Reticulum-Associated Degradation and Loss of Function

High blood pressure (BP) is the most common cardiovascular risk factor worldwide and a major contributor to heart disease and stroke. We previously discovered a BP-associated missense SNP (single nucleotide polymorphism)–rs2272996–in the gene encoding vanin-1, a glycosylphosphatidylinositol (GPI)-anchored membrane pantetheinase. In the present study, we first replicated the association of rs2272996 and BP traits with a total sample size of nearly 30,000 individuals from the Continental Origins and Genetic Epidemiology Network (COGENT) of African Americans (P = 0.01). This association was further validated using patient plasma samples; we observed that the N131S mutation is associated with significantly lower plasma vanin-1 protein levels. We observed that the N131S vanin-1 is subjected to rapid endoplasmic reticulum-associated degradation (ERAD) as the underlying mechanism for its reduction. Using HEK293 cells stably expressing vanin-1 variants, we showed that N131S vanin-1 was degraded significantly faster than wild type (WT) vanin-1. Consequently, there were only minimal quantities of variant vanin-1 present on the plasma membrane and greatly reduced pantetheinase activity. Application of MG-132, a proteasome inhibitor, resulted in accumulation of ubiquitinated variant protein. A further experiment demonstrated that atenolol and diltiazem, two current drugs for treating hypertension, reduce the vanin-1 protein level. Our study provides strong biological evidence for the association of the identified SNP with BP and suggests that vanin-1 misfolding and degradation are the underlying molecular mechanism.     

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface

Several studies have indicated that olfactory responses are impeded by amiloride. Therefore, it was of interest to see whether, and if so which, olfactory epithelial cellular compartments have amiloride- sensitive structures. Using ultrastructural methods that involved rapid freezing, freeze-substitution and low temperature embedding of olfactory epithelia, this study shows that, in the rat, this tissue is immunoreactive to antibodies against amiloride sensitive Na(+)- channels. However, microvilli of olfactory supporting cells, as opposed to receptor cilia, contained most of the immunoreactive sites. Apices from which the microvilli sprout and receptor cell dendritic knobs had much less if any of the amiloride-antibody binding sites. Using a direct ligand-binding cytochemical method, this study also confirms earlier ones that showed that olfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed that supporting cell microvilli and the receptor cilia themselves have mechanisms, different but likely complementary, that participate in regulating the salt concentration around the receptor cell cilia. In this way, both structures help to provide the ambient mucous environment for receptor cells to function properly. This regulation of the salt concentration of an ambient fluid environment is a function that the olfactory epithelium shares with cells of transporting epithelia, such as those of kidney.      

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope.     

Responses to dipole stimuli of anterior lateral line nerve fibres in goldfish, <Emphasis Type="Italic">Carassius auratus</Emphasis>, under still and running water conditions

We investigated how fibres in the anterior lateral line nerve of goldfish, Carassius auratus, respond to sinusoidal water motions in a background of still or running water. Two types of fibres were distinguished: type I fibres, which most likely innervate superficial neuromasts, were stimulated by running water (10 cm s⁻¹) while type II fibres, which most likely innervate canal neuromasts, were not stimulated by running water. The responses of type I fibres to sinusoidal water motions were masked in running water whereas responses of type II fibres were not masked. These findings are in agreement with previous data obtained from the posterior lateral line nerve of goldfish. Furthermore, we demonstrate here that for type I fibres the degree of response masking increased with increasing flow velocity. Finally, the ratio between responses that were masked in running water (type I) and those that were not masked (type II) increases with increasing flow velocity. Flow fluctuations that were generated by a cylinder in front of the fish did not affect ongoing activity in the flow, nor the dipole-evoked responses. The findings are discussed with respect to particle image velocimetry data of the water motions generated in the experiments.