Tetraspanins connect several types of Ig proteins: IgM is a novel component of the tetraspanin web on B-lymphoid cells

The tetraspanins form a family of about 30 molecules mainly expressed on the cell surface. They have been reported to be involved in many physiological or pathological processes, such as fertilization, immune response, development of the nervous system, and metastasis, as well as in infectious diseases (HCV, malaria, etc.). The tetraspanins may play a role as organizers of multimolecular complexes on the cell surface associating numerous proteins, the tetraspanin web. To better define the composition of the tetraspanin web, its characterization has been recently performed using mass spectrometry and proteomics. We report the proteomic analysis of tetraspanin complexes on B-lymphoid cells. Immunoprecipitation experiments were performed using mAbs directed against the tetraspanin CD9, and associated molecules were identified by MALDI-TOF (matrix assisted laser desorption ionization time of flight) mass spectrometry. This led to the identification of IgM as a novel component of the complexes. Thus, tetraspanins may connect several types of proteins with Ig domains, including HLA-DR, EWI-2, and IgM, that may play a role in immune responses.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Hereditary deafness: molecular genetics

This article outlines recent advances in explaining hereditary deafness in molecular terms, focusing on isolated (i.e. nonsyndromic) hearing loss. The number of genes identified (36 to date) is growing rapidly. However, difficulties inherent in genetic linkage analysis, coupled with the possible involvement of environmental causes, have so far prevented the characterization of the main genes causative or predisposing to the late-onset forms of deafness.     

Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1

Progress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipCMut1. In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC. cipC is the first gene in a large 'cel' gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component. Physiological and biochemical studies showed that the mutant strain was affected in cellulosome synthesis and severely impaired in its ability to degrade crystalline cellulose. It produced small amounts of a truncated CipC protein (P120), which had functional cohesin domains and assembled complexes which did not contain any of the enzymes encoded by genes of the 'cel' cluster. The mutant cellulolytic system was mainly composed of three proteins designated P98, P105 and P125. Their N-termini did not match any of the known cellulase sequences from C. cellulolyticum. A large amount of entire CipC produced in the cipCMut1 strain by trans-complementation with plasmid pSOScipC did not restore the cellulolytic phenotype, in spite of the assembly of a larger amount of complexes. The complexes produced in the mutant and complemented strains contained at least 12 different dockerin-containing proteins encoded by genes located outside of the 'cel' cluster. The disturbances observed in the mutant and trans-complemented strains were the result of a strong polar effect resulting from the cipC gene disruption. In conclusion, this study provided genetic evidence that the cellulases encoded by the genes located in the 'cel' cluster are essential for the building of cellulosomes efficient in crystalline cellulose degradation.     

Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum

The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.     

Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces

Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 microM) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h(-1)g [dry weight] of cells(-1). Among these strains, C. bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine. Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH. The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s). Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s).