Peptide mapping of β-casein by capillary zone electrophoresis

A method to the study of -casein proteolysis by aspartic proteinases is developed. The 3% trichloroacetic acid-soluble peptides of -casein digested with cardosin A were separated by capillary zone electrophoresis under different experimental conditions in an uncoated fused silica capillary. The best separation was at pH 3.01, 30 kV and 30 °C.     

Capillary electrophoresis of caseino-peptides: influence of applied voltage and column temperature

The peptides formed upon action of purified cardoon rennet on the Ala₁₈₉-Phe₁₉₀-Leu₁₉₁-Leu₁₉₂-Tyr₁93 -casein fragment were separated by capillary electrophoresis in an uncoated fused silica capillary. There was a linear correlation between electrophoretic mobility and Z/M^2/3 (Z, net charge; M, molecular mass) under all experimental conditions tested; under the optimal condicitions, 25 kV and 40 °C, the correlation coefficient was 0.994. The reported method is fast (migration times less than 7 min) and may be used to study the action of aspartic proteinases on the C-terminal domain of -casein and thus to help elucidate their effect on cheese quality.     

Constitutional Mutations of the hSNF5/INI1 Gene Predispose to a Variety of Cancers

Biallelic, truncating mutations of the hSNF5/INI1 gene have recently been documented in malignant rhabdoid tumor (MRT), one of the most aggressive human cancers. This finding suggests that hSNF5/INI1 is a new tumor-suppressor gene for which germline mutations might predispose to cancer. We now report the presence of loss-of-function mutations of this gene in the constitutional DNA from affected members but not from healthy relatives in cancer-prone families. Furthermore, a constitutional mutation is documented in a patient with two successive primary cancers. In agreement with the two-hit model, the wild-type hSNF5/INI1 allele is deleted in the tumor DNA from mutation carriers. In all tested cases, DNA from parents demonstrated normal hSNF5/INI1 sequences, therefore indicating the de novo occurrence of the mutation, which was shown to involve the maternal allele in one case and the paternal allele in two other cases. These data indicate that constitutional mutation of the hSNF5/INI1 gene defines a new hereditary syndrome predisposing to renal or extrarenal MRT and to a variety of tumors of the CNS, including choroid plexus carcinoma, medulloblastoma, and central primitive neuroectodermal tumor. This condition, which we propose to term "rhabdoid predisposition syndrome," may account for previous observations of familial and multifocal cases of the aforementioned tumor types. It could also provide the molecular basis for cases of Li-Fraumeni syndrome without p53 germline mutations.     

Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein–based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia

Purpose: Infection with oncogenic human papillomavirus (HPV) and HPV-16 in particular is a leading cause of anogenital neoplasia. High-grade intraepithelial lesions require treatment because of their potential to progress to invasive cancer. Numerous preclinical studies have demonstrated the therapeutic potential of E7-directed vaccination strategies in mice tumour models. In the present study, we tested the immunogenicity of a fusion protein (PD-E7) comprising a mutated HPV-16 E7 linked to the first 108 amino acids of Haemophilus influenzae protein D, formulated in the GlaxoSmithKline Biologicals adjuvant AS02B, in patients bearing oncogenic HPV-positive cervical intraepithelial neoplasia (CIN). Methods: Seven patients, five with a CIN3 and two with a CIN1, received three intramuscular injections of adjuvanted PD-E7 at 2-week intervals. Three additional CIN1 patients received a placebo. CIN3 patients underwent conization 8 weeks postvaccination. Cytokine flow cytometry and ELISA were used to monitor antigen-specific cellular and antibody responses from blood taken before and after vaccine or placebo injection. Results: Some patients had preexisting systemic IFN- CD4⁺ (1/10) and CD8⁺ (5/10) responses to PD-E7. Vaccination, not placebo injection, elicited systemic specific immune responses in the majority of the patients. Five vaccinated patients (71%) showed significantly increased IFN- CD8⁺ cell responses upon PD-E7 stimulation. Two responding patients generated long-term T-cell immunity toward the vaccine antigen and E7 as well as a weak H. influenzae protein D (PD)–directed CD4⁺ response. All the vaccinated patients, but not the placebo, made significant E7- and PD-specific IgG. Conclusions: The encouraging results obtained from this study performed on a limited number of subjects justify further analysis of the efficacy of the PD-E7/AS02B vaccine in CIN patients.     

Vascular biomaterials: from biomedical engineering to tissue engineering

Biomaterials are already widely used in medical sciences. The field of biomaterials began to shift to produce materials able to stimulate specific cellular responses at the molecular level. The combined efforts of cell biologists, engineers, materials scientists, mathematicians, geneticists, and clinicians are now used in tissue engineering to restore, maintain, or improve tissue functions or organs. This rapidly expanding approach combines the fields of material sciences and cell biology for the molecular design of polymeric scaffolds with appropriate 3D configuration and biological responses. Future developments for new blood vessels will require improvements in technology of materials and biotechnology together with the increased knowledge of the interactions between materials, blood, and living tissues. Biomaterials represent a crucial mainstay for all these studies.     

Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence

Listeria monocytogenes is an opportunistic food-borne human and animal pathogen. Several surface proteins expressed by this intracellular pathogen are critical for the infectious process. By in silico analysis we compared the surface protein repertories of L. monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L. monocytogenes absent in L. innocua. This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules. We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity. We provide evidence that Auto is required for entry of L. monocytogenes into cultured non-phagocytic eukaryotic cells. The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs. During infection, the autolytic activity of Auto may also be critical. Auto appears thus as a novel type of L. monocytogenes virulence factor.     

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).     

NMR of Redox Proteins of Plants, Yeasts and Photosynthetic Bacteria

NMR spectroscopy has evolved dramatically over the past 15 years, establishing a new, reliable methodology for studying biomacromolecules at atomic resolution. The three-dimensional structure and dynamics of a biomolecule or a biomolecular complex is only one of the main types of information available using NMR. The spectral assignment to the specific nuclei of a biostructure is a very precise reflection of their electronic environment. Any change in this environment due to a structural change, the binding of a ligand or the redox state of a redox cofactor, will be very sensitively reported by changes in the different NMR parameters. The capabilities of the NMR method are currently expanding dramatically and it is turning into a powerful means to study biosystems dynamically in exchange between different conformations, exchanging ligands, transient complexes, or the activation/inhibition of regulated enzymes. We review here several NMR studies that have appeared during the past 5 or 6 years in the field of redox proteins of plants, yeasts and photosynthetic bacteria. These new results illustrate the recent biomolecular NMR evolution and provide new physiological models for understanding the different types of electron transfer, including glutaredoxins, thioredoxins and their dependent enzymes, the ferredoxin-NADP oxidoreductase complex, flavodoxins, the plastocyanin-cytochrome f complex, and cytochromes c.     

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.