Regulation of bovine papillomavirus replicative helicase e1 by the ubiquitin-proteasome pathway

Papillomaviruses maintain their genomes in a relatively constant copy number as stable extrachromosomal plasmids in the nuclei of dividing host cells. The viral initiator of replication, E1, is not detected in papillomavirus-infected cells. Here, we present evidence that E1 encoded by bovine papillomavirus type 1 is an unstable protein that is degraded through the ubiquitin-proteasome pathway. In a cell-free system derived from Xenopus egg extracts, E1 degradation is regulated by both cyclin E/Cdk2 binding and E1 replication activity. Free E1 is readily ubiquitinated and degraded by the proteasome, while it becomes resistant to this degradation pathway when bound to cyclin E/Cdk2 complexes before the start of DNA synthesis. This stabilization is reversed in a process involving E1-dependent replication activity. In transiently transfected cells, E1 is also polyubiquitinated and accumulates when proteasome activity is inhibited. Thus, the establishment and maintenance of a stable number of papillomavirus genomes in latently infected cells are in part a function of regulated ubiquitin-mediated degradation of E1.     

The present state of the human lactotransferrin sequence. Study and alignment of the cyanogen bromide fragments and characterization of N- and C-terminal domains

Human lactotransferrin contains six residues of methionine per mol. Seven different fragments were characterized after treatment with cyanogen bromide (CNBr) and large parts of their sequences were determined. The alignment of the CNBr fragments was established by the determination of N- and C-terminal sequences, by the study of the C-terminal domain obtained by peptic digestion of the protein and by taking into account the internal homology as well as homology with human serum transferrin. The two glycopeptides were situated in the N- and C-terminal parts of the protein, respectively, a situation quite different from that encountered in serum transferrin. The sequence studies allowed us to suggest a 4- and perhaps 6-fold internal homology.     

An 88 amino acid long C-terminal sequence of human lactotransferrin

The terminal oxidoreductase of nitrous oxide respiration in the marine, denitrifying bacterium, Pseudomonas perfectomarinus, was identified as multi-copper protein and purified to electrophoretic homogeneity. The enzyme reduced N₂O to N₂ with hydrogen, clostridial hydrogenase, and methyl viologen as electron-donating system. The copper content of the reductase corresponded to ～ 8 copper atoms/120 000 M_r. The subunit structure was dimeric with two peptides of equal size. Manganese, iron and zinc were absent, or were not found in stoichiometric amounts. The oxidized chromophore had absorption maxima at 350, 480, 530, 620 and 780 nm; addition of dithionite produced a blue protein form with maxima at 470, 635 and 740 nm. Both forms of the enzyme were paramagnetic. The same copper protein was also isolated from Pseudomonas stutzeri.     

Variability in secondary structure of the antimicrobial peptide Cateslytin in powder,solution, DPC micelles and at the air–water interface

Cateslytin (bCGA ³⁴⁴RSMRLSFRARGYGFR³⁵⁸), a five positively charged 15 amino-acid residues arginine-rich antimicrobial peptide, was synthesized using a very efficient procedure leading to high yields and to a 99% purity as determined by HPLC and mass spectrometry. Circular dichroism, polarized attenuated total reflectance fourier transformed infrared, polarization modulation infrared reflection Absorption spectroscopies and proton two-dimensional NMR revealed the flexibility of such a peptide. Whereas being mostly disordered as a dry powder or in water solution, the peptide acquires a α-helical character in the “membrane mimicking” solvent trifuoroethanol. In zwitterionic micelles of dodecylphophatidylcholine the helical character is retained but to a lesser extent, the peptide returning mainly to its disordered state. A β-sheet contribution of almost 100% is detected at the air–water interface. Such conformational plasticity is discussed regarding the antimicrobial action of Cateslytin. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Population stratification may bias analysis of PGC-1α as a modifier of age at Huntington disease motor onset

Huntington’s disease (HD) is an inherited neurodegenerative disorder characterized by motor, cognitive and behavioral disturbances, caused by the expansion of a CAG trinucleotide repeat in the HD gene. The CAG allele size is the major determinant of age at onset (AO) of motor symptoms, although the remaining variance in AO is highly heritable. The rs7665116 SNP in PPARGC1A, encoding the mitochondrial regulator PGC-1α, has been reported to be a significant modifier of AO in three European HD cohorts, perhaps due to affected cases from Italy. We attempted to replicate these findings in a large collection of (1,727) HD patient DNA samples of European origin. In the entire cohort, rs7665116 showed a significant effect in the dominant model (p value?=?0.008) and the additive model (p value?=?0.009). However, when examined by origin, cases of Southern European origin had an increased rs7665116 minor allele frequency (MAF), consistent with this being an ancestry-tagging SNP. The Southern European cases, despite similar mean CAG allele size, had a significantly older mean AO (p?<?0.001), suggesting population-dependent phenotype stratification. When the generalized estimating equations models were adjusted for ancestry, the effect of the rs7665116 genotype on AO decreased dramatically. Our results do not support rs7665116 as a modifier of AO of motor symptoms, as we found evidence for a dramatic effect of phenotypic (AO) and genotypic (MAF) stratification among European cohorts that was not considered in previously reported association studies. A significantly older AO in Southern Europe may reflect population differences in genetic or environmental factors that warrant further investigation.     

Interactions of chromogranin A-derived vasostatins and monolayers of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine

Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 degrees C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure-area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5-10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine.     

Construction of a genomic library of Erwinia chrysanthemi and molecular cloning of cellulase gene

Summary A genomic library of Erwinia chrysanthemi has been constructed using cosmid pMMB34 as the cloning vector. Among 1,500 clones many Erwinia markers have been found. One of the 9 recombinant cosmids carrying a structural gene for endo--1,4-glucanase, cel, was studied: after subcloning and restriction analysis, a physical map of the 10 kb insert is given and the location of the cel gene is proposed.     

Loss of heterozygosity of the L-myc oncogene in human breast tumors

Summary Recent studies suggest that loss of heterozygosity may play an important role in various human neoplasia. Cytogenetic abnormalities detected in primary breast tumors led us to examine breast tumor DNAs for deletions. In the present study, we demonstrate, using restriction fragment length polymorphism (RFLP) analysis at the L-myc proto-oncogene (chromosome 1p32), a frequent loss of heterozygosity in primary breast tumor DNAs (55 out of 152 informative tumor DNAs). Most of these deletions appear to be limited to chromosome 1p. No correlation was observed between this genetic alteration and several parameters of each patient's history or characteristics of the tumor. However, a significantly (P = 0.011) shorter survival period after relapse was observed for patients with loss of heterozygosity at L-myc in primary tumor DNAs compared with patients with tumor DNAs lacking this alteration.     

Extending the Bacillus cereus group genomics to putative food-borne pathogens of different toxicity

The Bacillus cereus group represents sporulating soil bacteria containing pathogenic strains which may cause diarrheic or emetic food poisoning outbreaks. Multiple locus sequence typing revealed a presence in natural samples of these bacteria of about 30 clonal complexes. Application of genomic methods to this group was however biased due to the major interest for representatives closely related to Bacillus anthracis. Albeit the most important food-borne pathogens were not yet defined, existing data indicate that they are scattered all over the phylogenetic tree. The preliminary analysis of the sequences of three genomes discussed in this paper narrows down the gaps in our knowledge of the B. cereus group. The strain NVH391-98 is a rare but particularly severe food-borne pathogen. Sequencing revealed that the strain should be a representative of a novel bacterial species, for which the name Bacillus cytotoxis or Bacillus cytotoxicus is proposed. This strain has a reduced genome size compared to other B. cereus group strains. Genome analysis revealed absence of sigma B factor and the presence of genes encoding diarrheic Nhe toxin, not detected earlier. The strain B. cereus F837/76 represents a clonal complex close to that of B. anthracis. Including F837/76, three such B. cereus strains had been sequenced. Alignment of genomes suggests that B. anthracis is their common ancestor. Since such strains often emerge from clinical cases, they merit a special attention. The third strain, KBAB4, is a typical facultative psychrophile generally found in soil. Phylogenic studies show that in nature it is the most active group in terms of gene exchange. Genomic sequence revealed high presence of extra-chromosomal genetic material (about 530kb) that may account for this phenomenon. Genes coding Nhe-like toxin were found on a big plasmid in this strain. This may indicate a potential mechanism of toxicity spread from the psychrophile strain community. The results of this genomic work and ecological compartments of different strains incite to consider a necessity of creating prophylactic vaccines against bacteria closely related to NVH391-98 and F837/76. Presumably developing of such vaccines can be based on the properties of non-pathogenic strains such as KBAB4 or ATCC14579 reported here or earlier. By comparing the protein coding genes of strains being sequenced in this project to others we estimate the shared proteome, or core genome, in the B. cereus group to be 3000+/-200 genes and the total proteome, or pan-genome, to be 20-25,000 genes.     

Peptides from the N-terminal domain of chromogranin A (vasostatins) exert negative inotropic effects in the isolated frog heart

The negative inotropic effects of synthetic peptides derived from the N-terminus of chromogranin A (CgA) were studied in an avascular model of the vertebrate myocardium, the isolated working frog heart (Rana esculenta). The peptides were frog and bovine CgA(4-16) and CgA(47-66), and bovine CgA(1-40) with (CgA(1-40SS)) and without an intact disulfide bridge (CgA(1-40SH)). Under basal cardiac conditions, four of the peptides caused a concentration-dependent negative inotropism that was comparable to the negative inotropy reported for human recombinant vasostatin I (CgA(1-78)) and bovine CgA(7-57). By comparison of the structural characteristics of the bovine and frog sequences with their minimally effective concentrations ranging from 68 to 125 nM of peptide, the results were consistent with the natural structure (CgA(17-38SS)) being essential for the negative inotropism. In addition, the partial sequences of the frog and bovine vasostatin I were effective in counteracting the characteristic positive inotropism exerted by isoproterenol (1 nM) at minimally effective concentrations ranging from 45 to 272 nM. Taken together, these results extend the first evidence for a cardiosuppressive role of the N-terminal domain of chromogranin A known for its co-storage with catecholamines in the sympathoadrenal system of vertebrates.