Expression and purification of Clostridium perfringens beta-toxin glutathione S-transferase fusion protein.

The beta-toxin gene from Clostridium perfringens type C was cloned and expressed as a glutathione S-transferase fusion protein in Escherichia coli. The DNA sequence was determined and compared to the type B sequence. Two nucleotide differences were found in the protein coding sequence, resulting in one amino acid difference between the two proteins. The purified beta-toxin fusion protein is not toxic in mice, but rabbit antiserum raised against it neutralises the toxic effect of C. perfringens type C culture filtrate in mice.

Site-directed mutagenesis of Clostridium perfringens beta-toxin: expression of wild-type and mutant toxins in Bacillus subtilis.

Recombinant beta-toxin has been expressed and secreted from Bacillus subtilis. Biological activity was tested in vivo and in vitro. The lethal dose in mice was determined. Hemolysis of rabbit and sheep erythrocytes was tested but no effect was observed. Seven mutant proteins were produced. Targets for mutagenesis were mostly selected on the basis of the similarity between beta-toxin and alpha-toxin from Staphylococcus aureus, a pore-forming toxin. Mutations of two amino acids affected the lethal dose in mice. Both residues have counterparts in the membrane binding region of alpha-toxin. Alteration of the single cysteine residue did not affect protein function, contrary to previous suggestions.

A common biological basis of obesity and nicotine addiction.

Smoking influences body weight such that smokers weigh less than non-smokers and smoking cessation often leads to weight increase. The relationship between body weight and smoking is partly explained by the effect of nicotine on appetite and metabolism. However, the brain reward system is involved in the control of the intake of both food and tobacco. We evaluated the effect of single-nucleotide polymorphisms (SNPs) affecting body mass index (BMI) on smoking behavior, and tested the 32 SNPs identified in a meta-analysis for association with two smoking phenotypes, smoking initiation (SI) and the number of cigarettes smoked per day (CPD) in an Icelandic sample (N=34,216 smokers). Combined according to their effect on BMI, the SNPs correlate with both SI (r=0.019, P=0.00054) and CPD (r=0.032, P=8.0 × 10(-7)). These findings replicate in a second large data set (N=127,274, thereof 76,242 smokers) for both SI (P=1.2 × 10(-5)) and CPD (P=9.3 × 10(-5)). Notably, the variant most strongly associated with BMI (rs1558902-A in FTO) did not associate with smoking behavior. The association with smoking behavior is not due to the effect of the SNPs on BMI. Our results strongly point to a common biological basis of the regulation of our appetite for tobacco and food, and thus the vulnerability to nicotine addiction and obesity.

Enteric adenovirus type 40:E1B transcription map and identification of novel E1A-E1B cotranscripts in lytically infected cells.

Adenovirus 40 (Ad40) is defective for growth in tissue culture but is complemented when the Ad2/5 or Ad12 E1B 55K protein is supplied in trans. Ad40 E1B mRNA has not been detected in E1-transformed cells, or at early times in lytically infected cells. In cells constitutively expressing the E1B region of Ad2, Ad40 E1B mRNAs are detected at late times in infection, after the onset of DNA replication. We have determined the Ad40 E1B transcription map from RNA produced at late times in infected KB16 cells, using S1 nuclease, primer extension, PCR-cDNA analysis, and Northern blotting. E1B transcripts corresponding to Ad2 14 S, 22 S, and 9 S mRNAs were identified but no 13 S mRNA equivalent was detected, a pattern similar to that seen in the Ad12 transcription map. The coding potential for E1B 19K, 55K, and 15K proteins and for ppIX is retained in the Ad40 transcripts. In addition we find novel E1A-E1B cotranscript counterparts of the 14 S and 22 S mRNAs. These contain the first 40 codons of the E1A first exon linked to a site 4-5 nt downstream of the E1B cap site, retaining all the coding potential of the E1B mRNAs. No new open reading frames are created by the junction, and the E1A ORF terminates with one codon added after the junction. Each E1A-E1B cotranscript is present in abundance comparable to that of its authentic E1B counterpart. The E1A-E1B junction is unusual in that it does not conform to splice consensus sequences and thus may not be generated by a conventional splicing mechanism.

Complementation of enteric adenovirus type 40 for lytic growth in tissue culture by E1B 55K function of adenovirus types 5 and 12.

The enteric adenovirus type 40 strain Dugan (Ad40) cannot be passaged in HeLa cells, but will grow in 293 cells, which express Ad5 E1 functions. To determine the reason for this limited host range, KB cell lines expressing Ad2 E1A, E1B, or E1A + E1B (L. E. Babiss, C. S. H. Young, P. B. Fisher, and H. S. Ginsberg, 1983, J. Virol. 46, 454-465) have been tested for their ability to support Ad40 replication. Only cell lines which supply E1B functions, but not those expressing E1A alone, are permissive for Ad40, suggesting that Ad40 may require some function supplied by E1B or induced in E1B-containing cells. In coinfection assays Ad40 complements Ad5 dl312 (delta E1A) but not Ad5 dl313 (delta E1B) and is itself complemented by dl312 but not by dl313. Mutants of Ad2 and Ad12 with lesions in E1B 55K or 19K protein have been used to further delineate the requirements for Ad40 growth in HeLa cells. For mutants lacking 55K function there is minimal complementation in either direction, whereas those lacking only the 19K product are able to complement Ad40.

Clostridium perfringens beta-toxin forms multimeric transmembrane pores in human endothelial cells.

Beta-toxin is one of the lethal toxins of Clostridium perfringens. It shares sequence homology with the pore-forming alpha-toxin of Staphylococcus aureus and structural homology has been indicated by mutagenesis studies. Human endothelial cells are sensitive to the toxic effect of alpha-toxin and in order to investigate the function of beta-toxin we have looked at the effect of the protein on human umbilical vein endothelial cells. We show that like alpha-toxin beta-toxin induces release of arachidonic acid in a dose dependent manner. In addition we show that both toxins cause leakage of inositol from the cells, consistent with the formation of transmembrane pores. The effect of toxin mutants on endothelial cells correlates with the lethal dose of each mutant in mice. Furthermore, we demonstrate the formation of heat stable toxin multimers in the cell membrane. Multimer formation was not observed on other cell types tested. We conclude that beta-toxin is a cell specific pore-forming toxin, structurally and functionally related to alpha-toxin of Staphylococcus aureus.

Properties of the adenovirus type 40 E1B promoter that contribute to its low transcriptional activity.

The adenovirus type 5 (Ad5) E1B promoter contains two elements essential for maximal activity, a TATA box and a GC box. The enteric adenovirus type 40 (Ad40) E1B promoter has a TATA box sequence identical to that of Ad5 and a GC box that fits the Sp1 binding site consensus. Nevertheless, Ad40 E1B RNA synthesis is severely impaired in HeLa cells, attributable in part at least to the weak transactivating activity of Ad40 E1A. However, the responsiveness of Ad40 early promoters to E1A transactivation has not been directly demonstrated. Using a transient expression assay with a chloramphenicol acetyl transferase (CAT) reporter gene, the Ad40 E1B promoter was very poorly transactivated by E1A of both Ad40 and Ad5 and showed only a limited response to the promiscuous varicella zoster virus transactivator p140. Construction of Ad5 recombinant viruses expressing the CAT gene under the control of the Ad5 or Ad40 E1B promoter allowed detection and measurement of expression from the Ad40 E1B promoter in a well-defined background and showed that overall activity is some 100-fold lower than for the Ad5 E1B promoter. Deletion analysis revealed that sequences upstream of the Sp1 binding site down-modulated Ad40 E1B promoter responsiveness, and two protein binding sites, identified by DNase footprinting and gel retardation assay, may be implicated in this effect. Gel shift analysis also showed that the Ad40 Sp1 binding site had a reduced affinity for Sp1 protein, relative to the Ad5 site, and that the context as well as the core sequence had an influence on Sp1 recognition.

Genetic factors contribute to the risk of developing endometriosis.

BACKGROUND: Endometriosis is known to cluster within nuclear families. The extent of familial clustering can be evaluated in Iceland with its large population-based genealogical database. METHODS AND RESULTS: Applying several measures of familiality we demonstrated that 750 women with endometriosis were significantly more interrelated than matched control groups. The risk ratio for sisters was 5.20 (P < 0.001) and for cousins 1.56 (P = 0.003). The average kinship coefficient for the patients was significantly higher than that calculated for 1000 sets of 750 matched controls (P < 0.001) and this remained significant when contribution from first-degree relatives was excluded (P < 0.05). The minimum number of ancestors required to account for the group of patients was compared with the minimum number of ancestors required to account for the control groups at different time points in the past. The minimum number of founders for the group of patients was significantly smaller than for the control groups. Affected cousin pairs were as likely to be paternally connected as maternally connected. CONCLUSIONS: This is the first population-based study using an extensive genealogy database to examine the genetic contribution to endometriosis. A genetic factor is present, with a raised risk in close and more distant relatives, and a definite kinship factor with maternal and paternal inheritance contributing.

Identification of a novel neuregulin 1 at-risk haplotype in Han schizophrenia Chinese patients, but no association with the Icelandic/Scottish risk haplotype.

To determine if neuregulin 1 (NRG1) is associated with schizophrenia in Asian populations, we investigated a Han Chinese population using both a family trio design and a case-control design. A total of 25 microsatellite markers and single nucleotide polymorphisms (SNPs) were genotyped spanning the 1.1 Mb NRG1 gene including markers of a seven-marker haplotype at the 5' end of the gene found to be in excess in Icelandic and Scottish schizophrenia patients. The alleles of the individual markers forming the seven marker at-risk haplotype are not likely to be causative as they are not in excess in patients in the Chinese population studied here. However using unrelated patients, we find a novel haplotype (HAP(China 1)), immediately upstream of the Icelandic haplotype, in excess in patients (11.9% in patients vs 4.2% in controls; P=0.0000065, risk ratio (rr) 3.1), which was not significant when parental controls were used. Another haplotype (HAP(China 2)) overlapping the Icelandic risk haplotype was found in excess in the Chinese (8.5% of patients vs 4.0% of unrelated controls; P=0.003, rr 2.2) and was also significant using parental controls only (P=0.0047, rr 2.1). A four-marker haplotype at the 3' end of the NRG1 gene, HAP(China 3), was found at a frequency of 23.8% in patients and 13.7% in nontransmitted parental haplotypes (P=0.000042, rr=2.0) but was not significant in the case-control comparison. We conclude that different haplotypes within the boundaries of the NRG1 gene may be associated with schizophrenia in the Han Chinese.