Circular dichroic study of conformational changes in ovalbumin induced by modification of sulfhydryl groups and disulfide reduction

Sulfhydryl groups of ovalbumin were chemically modified under denaturing conditions in the absence and presence of dithiothreitol, and effects on the secondary structure of the protein were investigated by circular dichroic (CD) measurements. The contents of -helix, -structure, and random coil (unordered, nonrepetitive structure) were estimated by simulation of the CD spectra and using the parameters established by Chen et al. The principal findings were these: (1) Modification of the four free sulfhydryl groups [with 5,5-dithiobis(2-nitrobenzoic acid), iodoacetate, or iodoacetamide] caused ovalbumin molecule to unfold partially and to undergo primarily helix-to- structure transition. (2) Cleavage of the disulfide bond did not lead to a further conformational change in the sulfhydryl-modified ovalbumin. (3) The remaining helical structure existed in a destabilized state with increased chain flexibility, as the modified protein was very susceptible to denaturation by guanidine and urea. (4) Further evidence for increased chain flexibility was provided by the finding that sodium dodecyl sulfate (SDS) induced helix formation in the sulfhydryl-modified, but not native, ovalbumin. And (5), since both nonreduced and reduced proteins, with their sulfhydryl groups blocked, displayed similar transitions in solutions of guanidine, urea, and SDS suggested that the single disulfide bond did not physically constrain the ovalbumin molecule.     

Complete Nucleotide Sequences of 93-kb and 3.3-kb Plasmids of an Enterohemorrhagic Escherichia coli O157:H7 Derived from Sakai Outbreak

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, derived froman outbreak in Sakai city, Japan in 1996, possesses two kindsof plasmids: a 93-kb plasmid termed pO157, found in clinicalEHEC isolates world-wide and a 3.3-kb plasmid termed pOSAK1,prevalent in EHEC strains isolated in Japan. Complete nucleotidesequences of both plasmids have been determined, and the putativefunctions of the encoded proteins and the cis-acting DNA sequenceshave been analyzed. pO157 shares strikingly similar genes andDNA sequences with F-factor and the transmissible drug-resistantplasmid R100 for DNA replication, copy number control, plasmidsegregation, conjugative functions and stable maintenance inthe host, although it is defective in DNA transfer by conjugationdue to the truncation and deletion of the required genes andDNA sequences. In addition, it encodes several proteins implicatedin EHEC pathogenicity such as an EHEC hemolysin (HlyA), a catalase-peroxidase(KatP), a serine protease (EspP) and type II secretion system.pOSAK1 possesses a ColE1-like replication system, and the DNAsequence is extremely similar to that of a drug-resistant plasmid,NTP16, derived from Salmonella typhimurium except that it lacksdrug resistance transposons.     

Identification of the gene variations in human CD22

 CD22, a member of the immunoglobulin superfamily, is a B-cell transmembrane glycoprotein that acts as an accessory-signaling component of the B-cell antigen receptor (BCR). Recent evidence indicating the role of CD22 as a negative regulator of BCR signal transduction prompted us to test the possibility that genetic variations of human CD22 may be associated with autoimmune diseases. In this study, variation screening of the entire CD22 coding region was performed, and possible association with rheumatic diseases was tested, using the genomic DNA from 207 healthy Japanese individuals, 68 patients with systemic lupus erythematosus (SLE), and 119 patients with rheumatoid arthritis (RA). Through the variation screening, seven non-synonymous and four synonymous substitutions were identified. In addition, single base substitutions were found in two introns flanking exon-intron junctions. Among these variations, Q152E substitution within the second extracellular domain was observed with a marginally higher frequency in the patients with SLE (3/68, 4.4%) than that in healthy individuals (1/207, 0.5%) (P=0.048. SLE vs healthy individuals), although this difference was no longer significant after correction for the number of comparisons (Pc=0.62). No significant association was observed between any of the variations and RA. These findings indicate that a number of genetic variants are present in CD22, and suggest that CD22 could be considered a candidate for the susceptibility genes to autoimmune diseases. Received: 14 July 1998 / Revised: 7 September 1998     

Comparative Evaluation of Quantitative Polymerase Chain Reaction Methods for Routine Enumeration of Specific Bacterial DNA in Aquatic Samples

Summary Three quantitative polymerase chain reaction (PCR) methods, the internal standard method (IS-PCR), competitive PCR (cPCR) and most probable number-PCR (MPN-PCR), were compared in terms of their ability to quantify specific bacterial DNA in environmental samples. Serially diluted Pseudomonas putida BH, the target bacterium, was inoculated into sterilized potassium phosphate buffer (PPB), river water and activated sludge, total DNA was extracted, and the number of pheB genes carried by P. putida BH in each sample was enumerated. IS-PCR and cPCR could not quantify the pheB gene at low concentrations (1.0 × 10³ copies ml^-1 in all samples and 1.0 × 10⁴ copies ml^--1 in some samples) and tended to give overestimations because of differences in amplification efficiencies between pheB gene and the internal standard/competitor in a reaction tube. Although reproducibility of MPN-PCR was slightly lower than that of the other two methods, MPN-PCR was the most sensitive, enabling us to quantify the pheB gene at 1.0 × 10³ copies ml^--1, and it had a good correlation with the inoculum size of P. putida BH. These results suggest that MPN-PCR is the best suited for routine microbial monitoring in natural environmental samples because of the simple handling, the ease of modification as occasion demands and the wide detection range, especially at low cell densities of the target microbe.     

Molecular chaperone Hsp90 stabilizes Pih1/Nop17 to maintain R2TP complex activity that regulates snoRNA accumulation

Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.     

Lack of Association of the HbE Variant with Protection from Cerebral Malaria in Thailand

Hemoglobin E (HbE; beta26Glu --> Lys) is the most common variant of the beta-globin gene in Southeast Asia; it has been suggested that it confers resistance against Plasmodium falciparum malaria. In this study 306 adult patients with P. falciparum malaria (198 mild and 108 cerebral malaria patients) living in northwest Thailand were investigated to examine whether the HbE variant is associated with protection from cerebral malaria. Our results revealed that the sample allele frequency of HbE was not significantly different between mild (7.3%) and cerebral malaria (7.4%) patients. Thus, the HbA/HbE polymorphism would not be a major genetic factor influencing the onset of cerebral malaria in Thailand.     

A replication study confirmed the <Emphasis Type="Italic">EDAR</Emphasis> gene to be a major contributor to population differentiation regarding head hair thickness in Asia

Hair morphology is a highly divergent phenotype among human populations. We recently reported that a nonsynonymous SNP in the ectodysplasin A receptor (EDAR 1540T/C) is associated with head hair fiber thickness in an ethnic group in Thailand (Thai-Mai) and an Indonesian population. However, these Southeast Asian populations are genetically and geographically close, and thus the genetic contribution of EDAR to hair morphological variation in the other Asian populations has remained unclear. In this study, we examined the association of 1540T/C with hair morphology in a Japanese population (Northeast Asian). As observed in our previous study, 1540T/C showed a significant association with hair cross-sectional area (P = 2.7 × 10⁻⁶) in Japanese. When all populations (Thai-Mai, Indonesian, and Japanese) were combined, the association of 1540T/C was stronger (P = 3.8 × 10⁻¹⁰) than those of age, sex, and population. These results indicate that EDAR is the genetic determinant of hair thickness as well as a strong contributor to hair fiber thickness variation among Asian populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Gene flow and natural selection in oceanic human populations inferred from genome-wide SNP typing

It is suggested that the major prehistoric human colonizations of Oceania occurred twice, namely, about 50,000 and 4,000 years ago. The first settlers are considered as ancestors of indigenous people in New Guinea and Australia. The second settlers are Austronesian-speaking people who dispersed by voyaging in the Pacific Ocean. In this study, we performed genome-wide single-nucleotide polymorphism (SNP) typing on an indigenous Melanesian (Papuan) population, Gidra, and a Polynesian population, Tongans, by using the Affymetrix 500K assay. The SNP data were analyzed together with the data of the HapMap samples provided by Affymetrix. In agreement with previous studies, our phylogenetic analysis indicated that indigenous Melanesians are genetically closer to Asians than to Africans and European Americans. Population structure analyses revealed that the Tongan population is genetically originated from Asians at 70% and indigenous Melanesians at 30%, which thus supports the so-called Slow train model. We also applied the SNP data to genome-wide scans for positive selection by examining haplotypic variation and identified many candidates of locally selected genes. Providing a clue to understand human adaptation to environments, our approach based on evolutionary genetics must contribute to revealing unknown gene functions as well as functional differences between alleles. Conversely, this approach can also shed some light onto the invisible phenotypic differences between populations.