Detection of Ancestry Informative HLA Alleles Confirms the Admixed Origins of Japanese Population

The polymorphisms in the human leukocyte antigen (HLA) region are powerful tool for studying human evolutionary processes. We investigated genetic structure of Japanese by using five-locus HLA genotypes (HLA-A, -B, -C, -DRB1, and -DPB1) of 2,005 individuals from 10 regions of Japan. We found a significant level of population substructure in Japanese; particularly the differentiation between Okinawa Island and mainland Japanese. By using a plot of the principal component scores, we identified ancestry informative alleles associated with the underlying population substructure. We examined extent of linkage disequilibrium (LD) between pairs of HLA alleles on the haplotypes that were differentiated among regions. The LDs were strong and weak for pairs of HLA alleles characterized by low and high frequencies in Okinawa Island, respectively. The five-locus haplotypes whose alleles exhibit strong LD were unique to Japanese and South Korean, suggesting that these haplotypes had been recently derived from the Korean Peninsula. The alleles characterized by high frequency in Japanese compared to South Korean formed segmented three-locus haplotype that was commonly found in Aleuts, Eskimos, and North- and Meso-Americans but not observed in Korean and Chinese. The serologically equivalent haplotype was found in Orchid Island in Taiwan, Mongol, Siberia, and Arctic regions. It suggests that early Japanese who existed prior to the migration wave from the Korean Peninsula shared ancestry with northern Asian who moved to the New World via the Bering Strait land bridge. These results may support the admixture model for peopling of Japanese Archipelago.     

Effects of Detergents on Lysis of Micrococcus lysodeikticus by Lysozyme

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments.

None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.     

Chitin Coated Cellulose as an Adsorbent of lysozyme like Enzymes Preparation and Properties

As a new adsorbent of lysozyme-like enzymes, chitin coated (CC-)cellulose was prepared. CC-cellulose was stable and had good flow properties for use in column chromatography. Affinity chromatography with CC-cellulose showed that 3~5 mg of lysozyme/ml resin was adsorbed specifically and desorbed quantitatively under mild conditions. The utilities of the method of affinity chromatography with CC-cellulose are discussed.     

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.     

Crystal structure of the rac activator, Asef, reveals its autoinhibitory mechanism

The Rac-specific guanine nucleotide exchange factor (GEF) Asef is activated by binding to the tumor suppressor adenomatous polyposis coli mutant, which is found in sporadic and familial colorectal tumors. This activated Asef is involved in the migration of colorectal tumor cells. The GEFs for Rho family GTPases contain the Dbl homology (DH) domain and the pleckstrin homology (PH) domain. When Asef is in the resting state, the GEF activity of the DH-PH module is intramolecularly inhibited by an unidentified mechanism. Asef has a Src homology 3 (SH3) domain in addition to the DH-PH module. In the present study, the three-dimensional structure of Asef was solved in its autoinhibited state. The crystal structure revealed that the SH3 domain binds intramolecularly to the DH domain, thus blocking the Rac-binding site. Furthermore, the RT-loop and the C-terminal region of the SH3 domain interact with the DH domain in a manner completely different from those for the canonical binding to a polyproline-peptide motif. These results demonstrate that the blocking of the Rac-binding site by the SH3 domain is essential for Asef autoinhibition. This may be a common mechanism in other proteins that possess an SH3 domain adjacent to a DH-PH module.     

Histochemical,ultrastructural, and three-dimensional observation of smooth muscle cells in human gastric mucosa

The present study was undertaken to clarify the histochemical and ultrastructural properties and the three-dimensional distribution of the smooth muscle cells (SMCs) located in the lamina propria (LP) of the human gastric mucosa. Standard paraffin sections obtained from stomachs surgically resected for gastric cancer were immunostained for alpha-smooth muscle actin (alpha-SMA), vimentin, desmin, laminin, and type IV collagen. In addition, 100-m-thick sections were fluorostained for alpha-SMA and CD34, while three-dimensional images were prepared by confocal laser scanning microscope. Ultrastructural studies were carried out using normal gastric biopsy specimens. The results indicated that SMCs in the LP differed between the upper and lower regions, SMCs in the lower LP being fairly typical SMCs, whereas those in the upper LP had apparently lost reactivity for desmin but gained that for vimentin. The basal lamina became sparser, but a fibronexus was occasionally seen in SMCs in the upper LP. Three-dimensional images revealed bundles of SMCs in the upper LP encircling several foveolae to form acinus-like structures and, in the upper LP, SMCs branching into fine fibrils with a brush-like (corpus) or besom-like (i.e., a twiggy witchs broom) appearance (antrum).     

Salt-inducible multidrug efflux pump protein in the moderately halophilic bacterium Chromohalobacter sp

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.