The spacer region of XPG mediates recruitment to nucleotide excision repair complexes and determines substrate specificity

XPG has structural and catalytic roles in nucleotide excision repair (NER) and belongs to the FEN-1 family of structure-specific nucleases. XPG contains a stretch of over 600 amino acids termed the "spacer region" between the conserved N- and I-nuclease regions. Its role is unknown, and it is not similar to any known protein. To investigate its possible functions, we generated and analyzed several deletion mutants of XPG. The spacer region is not required for endonuclease activity, but amino acids 111-550 contribute to the substrate specificity of XPG, and they are required for interaction with TFIIH and for NER activity in vitro and in vivo. Deletion of residues 184-210 and 554-730 leads only to a partial defect in NER activity and a weakened interaction with TFIIH. XPGDelta184-210 and XPGDelta554-730 are not observed at sites of local UV damage in living cells by immunofluorescence, suggesting that the weakened interaction between XPG and TFIIH results in an NER reaction with altered kinetics. This study demonstrates that the N-terminal portion of the spacer region is particularly important for NER progression by mediating the XPG-TFIIH interaction and XPG substrate specificity.     

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.     

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.     

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).     

Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.     

The evaluation of mixtures of yeast and potato extracts in growth media for biomass production of lactic cultures

The effectiveness of yeast extracts (YE) and potato extracts (PE) to promote growth of seven lactic cultures was evaluated by automated spectrophotometry (AS). Two aspects of the growth curve were analysed: (1) maximum biomass obtained (using ODmax) and (2) highest specific growth rate mu(max)) Eleven lots from the same PE-manufacturing process were examined for lot-to-lot variability. The ODmax values of three of the seven strains were significantly affected by lot source, but mu(max) was not significantly affected. The growth of bacteria was systematically lower in base medium containing 100% PE than in base medium containing 100% YE for both ODmax or mu(max) data, which could be related to the lower content in nitrogen-based compounds in PE. In AS assays, highest OD values for Lactobacillus casei EQ28, Lactobacillus rhamnosus R-011, Lactobacillus plantarum EQ12, and Streptococcus thermophilus R-083 were obtained with a mixture of PE and YE. Fermentations (2 L) were also carried out to determine the accuracy of AS to predict biomass levels obtained under fermentation trials. In these fermentations, replacement of 50% YE with PE was shown to enable good growth of S. thermophilus. With L. rhamnosus R-011, a high correlation (R2 = 0.95) was found between ODmax data obtained in the AS assays and that of the 2-L bioreactor when the same growth medium was used for both series of fermentations. However, AS was not as efficient when industrial media were used for the bioreactor assays. The relationship was still good for ODmax between AS data and that of the bioreactor data with L. rhamnosus R-011 in industrial LBS medium (R2 = 0.87), but was very poor with the S. thermophilus R-083 on Rosell #43 industrial medium (R2 = 0.33). Since PE cost 40% less than YE, there are strong economic advantages in considering such a partial replacement of YE by PE.