Identification of sn-glycerol-1-phosphate dehydrogenase activity from genomic information on a hyperthermophilic archaeon, Sulfolobus tokodaii strain 7

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.     

Purification and characterization of a novel isozyme of chitinase from Bombyx mori

75-kDa chitinase, which showed potential as a biocontrol agent against Japanese pine sawyer, was characterized after purification from the integument of the fifth instar larvae of Bombyx mori by chromatography on diethylaminoethyl (DEAE)-Toyoperal 650 (M), hydroxylapatite, and Fractogel EMD DEAE 650 (M) columns. The optimum pH was 6.0 toward N-acetylchitopentaose (GlcNAc5) and 10 toward glycolchitin. The optimum temperature was 60 degrees C toward GlcNAc5 and 25 degrees C toward glycolchitn. The enzyme was stable at pH 7-10 and below 40 degrees C. Kinetic analysis and reaction-pattern analysis using glycolchitin and N-acetylchitooligosacchraides as substrates indicated that 75-kDa chitinase is an endo- or random-type hydrolytic enzyme to produce the beta anomeric product and that it prefers the longer N-acetylchitooligosaccharides, suggesting, together with the N-terminal amino acid sequence, that the 75-kDa chitinase belongs to family 18 of glycosyl hydrolases.     

Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice

MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1(-/-) mice with Muc1(+/+) littermates following intranasal instillation of PA or flagellin. Compared with Muc1(+/+) mice, Muc1(-/-) mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-alpha and KC in bronchoalveolar lavage fluid, higher levels of TNF-alpha in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.     

Crystal structure and structure-based mutational analyses of RNase HIII from Bacillus stearothermophilus: a new type 2 RNase H with TBP-like substrate-binding domain at the N terminus

Ribonuclease HIII (Bst-RNase HIII) from the moderate thermophile Bacillus stearothermophilus is a type 2 RNase H but shows poor amino acid sequence identity with another type 2 RNase H, RNase HII. It is composed of 310 amino acid residues and acts as a monomer. Bst-RNase HIII has a large N-terminal extension with unknown function and a unique active-site motif (DEDE), both of which are characteristics common to RNases HIII. To understand the role of these N-terminal extension and active-site residues, the crystal structure of Bst-RNase HIII was determined in both metal-free and metal-bound forms at 2.1-2.6 angstroms resolutions. According to these structures, Bst-RNase HIII consists of the N-terminal domain and C-terminal RNase H domain. The structures of the N and C-terminal domains were similar to those of TATA-box binding proteins and archaeal RNases HII, respectively. The steric configurations of the four conserved active-site residues were very similar to those of other type 1 and type 2 RNases H. Single Mn and Mg ions were coordinated with Asp97, Glu98, and Asp202, which correspond to Asp10, Glu48, and Asp70 of Escherichia coli RNase HI, respectively. The mutational studies indicated that the replacement of either one of these residues with Ala resulted in a great reduction of the enzymatic activity. Overproduction, purification, and characterization of the Bst-RNase HIII derivatives with N and/or C-terminal truncations indicated that the N-terminal domain and C-terminal helix are involved in substrate binding, but the former contributes to substrate binding more greatly than the latter.     

Alpha B-crystallin mutation in dilated cardiomyopathy

Mutations in genes for sarcomeric proteins such as titin/connectin are known to cause dilated cardiomyopathy (DCM). However, disease-causing mutations can be identified only in a small proportion of the patients even in the familial cases, suggesting that there remains yet unidentified disease-causing gene(s) for DCM. To explore the novel disease gene for DCM, we examined CRYAB encoding alphaB-crystallin for mutation in the patients with DCM, since alphaB-crystallin was recently reported to associate with the heart-specific N2B domain and adjacent I26/I27 domain of titin/connectin, and we previously reported a N2B mutation, Gln4053ter, in DCM. A missense mutation of CRYAB, Arg157His, was found in a familial DCM patient and the mutation affected the evolutionary conserved amino acid residue among alpha-crystallins. Functional analysis revealed that the mutation decreased the binding to titin/connectin heart-specific N2B domain without affecting distribution of the mutant crystallin protein in cardiomyocytes. In contrast, another CRYAB mutation, Arg120Gly, reported in desmin-related myopathy decreased the binding to both N2B and striated muscle-specific I26/27 domains and showed intracellular aggregates of the mutant protein. These observations suggest that the Arg157His mutation may be involved in the pathogenesis of DCM via impaired accommodation to the heart-specific N2B domain of titin/connectin and its disease-causing mechanism is different from the mutation found in desmin-related myopathy.     

Comparative transcriptomics of the bacteriome and the spermalege of the bedbug Cimex lectularius (Hemiptera: Cimicidae)

The bedbug Cimex lectularius is notorious as a blood-feeding exoparasite of human and other warm-blooded animals. In addition to its medical and hygienic importance, C. lectularius is known for its unique biological traits, including obligate nutritional mutualism with a vitamin-provisioning Wolbachia endosymbiont and a peculiar mating habit called traumatic insemination wherein male sperm is injected into the female body cavity, migrates to the ovary, and fertilizes eggs therein. For these unique traits, novel insect organs, the bacteriome for endosymbiosis and the spermalege for traumatic insemination, have evolved in the lineage of bedbugs. We constructed cDNA libraries of the bacteriome and the spermalege of C. lectularius, and performed expressed sequence tag (EST) analyses of these peculiar insect organs. In total 4480 ESTs were compiled, which were assembled into 2989 unique sequences (USs). The following ESTs and USs were identified from the different organs: 1151 ESTs and 901 USs from the female whole body; 1098 ESTs and 879 USs from the female bacteriome; 1145 ESTs and 783 USs from the male bacteriome; and 1086 ESTs and 920 USs from the female spermalege. These EST data will provide a valuable genetic resource for understanding the developmental and evolutionary aspects of these novel insect organs.     

Evolution of symbiotic organs and endosymbionts in lygaeid stinkbugs

We investigated seed bugs of the genus Nysius (Insecta: Hemiptera: Lygaeidae) for their symbiotic bacteria. From all the samples representing 4 species, 18 populations and 281 individuals, specific bacterial 16S rRNA gene sequences were consistently identified, which formed a distinct clade in the Gammaproteobacteria. In situ hybridization showed that the bacterium was endocellularly localized in a pair of large bacteriomes that were amorphous in shape, deep red in color, and in association with gonads. In the ovary of adult females, the endosymbiont was also localized in the ‘infection zone'' in the middle of each germarium and in the ‘symbiont ball'' at the anterior pole of each oocyte, indicating vertical transmission of the endosymbiont through the ovarial passage. Phylogenetic analyses based on bacterial 16S rRNA, groEL and gyrB genes consistently supported a coherent monophyly of the Nysius endosymbionts. The possibility of a sister relationship to ‘Candidatus Kleidoceria schneideri'', the bacteriome-associated endosymbiont of a lygaeid bug Kleidocerys resedae, was statistically rejected, indicating independent evolutionary origins of the endosymbionts in the Lygaeidae. The endosymbiont genes consistently exhibited AT-biased nucleotide compositions and accelerated rates of molecular evolution, and the endosymbiont genome was only 0.6 Mb in size. The endosymbiont phylogeny was congruent with the host insect phylogeny, suggesting strict vertical transmission and host–symbiont co-speciation over evolutionary time. Based on these results, we discuss the evolution of bacteriomes and endosymbionts in the Heteroptera, most members of which are associated with gut symbiotic bacteria. The designation ‘Candidatus Schneideria nysicola'' is proposed for the endosymbiont clade.     

Higher alcohol intake may modify the association between mammographic density and breast cancer: An analysis of three case-control studies

Alcohol consumption and mammographic density are established risk factors for breast cancer. This study examined whether the association of mammographic density with breast cancer varies by alcohol intake. Mammographic density was assessed in digitized images for 1207 cases and 1663 controls from three populations (Japan, Hawaii, California) using a computer-assisted method. Associations were estimated by logistic regression. When comparing ever to never drinking, mean density was similar and consumption was not associated with breast cancer risk. However, within the Hawaii/Japan subset, women consuming >1 drink/day had a non-significantly elevated relative risk compared to never drinkers. Also in the Hawaii/Japan population, alcohol intake only modified the association between mammographic density and breast cancer in women consuming >1 drink/day (p(interaction)=0.05) with significant risk estimates of 3.65 and 6.58 for the 2nd and 3rd density tertiles as compared to 1.57 and 1.61 for never drinkers in Hawaii/Japan. Although these findings suggest a stronger association between mammographic density and breast cancer risk for alcohol consumers, the small number of cases requires caution in interpreting the results.