Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.     

Brucella abortus lipopolysaccharide in murine peritoneal macrophages acts as a down-regulator of T cell activation

Macrophages play a central role in host immune responses against pathogens by acting as both professional phagocytic cells and as fully competent APCs. We report here that the LPS from the facultative intracellular Gram-negative bacteria Brucella abortus interferes with the MHC class II Ag presentation pathway. LPS inhibits the capacity of macrophages to present hen egg lysozyme (HEL) antigenic peptides to specific CD4(+) T cells but not those of OVA to specific CD8(+) T cells. This defect was neither related to a decrease of MHC class II surface expression nor to a deficient uptake or processing of HEL. In addition, B. abortus LPS did not prevent the formation of SDS-resistant MHC class II complexes induced by HEL peptides. At the cell surface of macrophages, we observed the presence of LPS macrodomains highly enriched in MHC class II molecules, which may be responsible for the significant down-regulation of CD4(+) T cell activation. This phenomenon may account for the avoidance of the immune system by certain bacterial pathogens and may explain the immunosuppression observed in individuals with chronic brucellosis.     

Inventory and comparative analysis of rice and Arabidopsis ATP-binding cassette (ABC) systems

ATP-binding cassette (ABC) proteins constitute a large superfamily found in all kingdoms of living organisms. The recent completion of two draft sequences of the rice (Oryza sativa) genome allowed us to analyze and classify its ABC proteins and to compare to those in Arabidopsis thaliana. We identified a similar number of ABC proteins in rice and Arabidopsis (121 versus 120), despite the rice genome being more than three times the size of Arabidopsis. Both Arabidopsis and rice have representative members in all seven major subfamilies of ABC ATPases (A to G) commonly found in eukaryotes. This comparative analysis allowed the detection of 29 potential orthologous sequences in Arabidopsis and rice. However, plant share with prokaryotes a specific set of ABC systems that is not detected in animals. These ABC systems might be inherited from the cyanobacterial ancestor of chloroplasts. The present work provides the first complete inventory of rice ABC proteins and an updated inventory of those proteins in Arabidopsis.     

Heterogeneity of Shiga Toxin-Producing Escherichia coli Strains Isolated from Hemolytic-Uremic Syndrome Patients, Cattle, and Food Samples in Central France

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx₂-restriction fragment length polymorphism [RFLP], stx₂ gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx₂-RFLP and stx₂ variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx₂-RFLP, stx₂ variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.     

High concentration of cadmium induces AtPCS2 gene expression in Arabidopsis thaliana (L.) Heynh ecotype Wassilewskija seedlings

In Arabidopsis thaliana, two genes encoding phytochelatin synthase (PCS; EC 2.3.2.15), AtPCS1 and AtPCS2, have been identified. Until now, only AtPCS1 was shown to play a role in response to Cd. To gain insight into the putative role of AtPCS2, three Cd concentrations (50, 100 and 200 μM) and long-term exposure (7 days) were tested on 1-week-old A. thaliana ecotype Wassilewskija (Ws) seedlings. Since 100 μM Cd did not alter seedling metabolism, as shown by unchanged total soluble protein and free proline contents, we investigated plantlet response to this concentration in addition to Cd accumulation. Seedlings accumulated Cd in roots and shoots. As phytochelatins and glutathione (GSH) contents increased in treated seedlings, we suggested that Cd might be translocated via the phytochelatin pathway. Specific enzymatic activities of γ-glutamylcysteine synthetase (GCS; EC 6.3.2.2), glutathione synthetase (GS; EC 6.3.2.3) and PCS were twice much more stimulated in shoots and roots after Cd exposure except GS that remained constant in shoots. As expression of genes encoding GCS and GS was unchanged in response to Cd, we suggested a regulation at translational or post-translational level. Surprisingly, AtPCS1 and AtPCS2 were differentially up-regulated after Cd treatment: AtPCS1 in shoots and AtPCS2 in whole plantlets. This last result suggests that PCS2 could be involved in plant response to high concentration of Cd in Ws ecotype and supports a putative role of PCS2, not redundant with PCS1, in a long-term response to Cd.     

A model of the cerebellar pathways applied to the control of a single-joint robot arm actuated by McKibben artificial muscles

 This article describes an expanded version of a previously proposed motor control scheme, based on rules for combining sensory and motor signals within the central nervous system. Classical control elements of the previous cybernetic circuit were replaced by artificial neural network modules having an architecture based on the connectivity of the cerebellar cortex, and whose functioning is regulated by reinforcement learning. The resulting model was then applied to the motion control of a mechanical, single-joint robot arm actuated by two McKibben artificial muscles. Various biologically plausible learning schemes were studied using both simulations and experiments. After learning, the model was able to accurately pilot the movements of the robot arm, both in velocity and position. Received: 4 September 2000 / Accepted in revised form: 7 November 2001     

Flow cytometric DNA index and karyotype in childhood lymphoblastic leukemia.

Flow cytometric DNA-index (DI(FCM)) and karyotype were analysed in 82 consecutive children with acute lymphoblastic leukemia (ALL) during a 10 year period. A statistically significant correlation existed between modal chromosome number and DI(FCM) (p = 0.009). DI(FCM) could reliably identify leukemias with >51 chromosomes, whereas only three out of 12 cases with modal chromosome numbers between 47-51 were classified as aneuploid by DI(FCM). In the pseudodiploid group only one out of 20 leukemias had a DI(FCM) > 1.0. Five leukemias with a diploid karyotype showed an aneuploid DI(FCM) and in three patients the flow cytometric measurement revealed biclonality undetected by karyotyping. During treatment aneuploid clones could be detected by DI(FCM) in a substantial number of cases where the cytogenetic analysis was normal, and the opposite was also demonstrated in one case. DI(FCM) gave prognostic information, showing that cases with a DI > 1.12 (corresponding to 51 chromosomes) had a superior outcome with treatment protocols today in use.     

Spectrum of mutations in the COL4A5 collagen gene in X-linked Alport syndrome.

Alport syndrome is a mainly X-linked hereditary disease of basement membranes that is characterized by progressive renal failure, deafness, and ocular lesions. It is associated with mutations of the COL4A5 gene located at Xq22 and encoding the alpha5 chain of type IV collagen. We have screened 48 of the 51 exons of the COL4A5 gene by SSCP analysis and have identified 64 mutations and 10 sequence variants among 131 unrelated Alport syndrome patients. This represents a mutation-detection rate of 50%. There were no hot-spot mutations and no recurrent mutations in our population. The identified mutations were 6 nonsense mutations, 12 frameshift mutations, 17 splice-site mutations, and 29 missense mutations, 27 of the latter being glycine substitutions in the collagenous domain. Two of these occurred on the same allele in one patient and segregated with the disease in the family. We showed that some of the glycine substitutions could be associated with the lack of immunological expression of the alpha3(IV)-alpha5(IV) collagen chains in the glomerular basement membrane.