Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe

The recombinant catalytic α-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGIIα) was produced in Escherichia coli. The recombinant SpGIIα exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 °C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGIIα, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 α-glucosidases. The enzyme hydrolyzed not only α-(1→3)- but also α-(1→2)-, α-(1→4)-, and α-(1→6)-glucosidic linkages, and p-nitrophenyl α-glucoside. SpGIIα displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal α-glucosidic residue of Glc₂Man₃-Dansyl was faster than that of Glc₁Man₃-Dansyl. This catalytic α-subunit also removed terminal glucose residues from native N-glycans (Glc₂Man₉GlcNAc₂ and Glc₁Man₉GlcNAc₂) although the activity was low.     

Difference in NaCl tolerance of membrane-bound 5′-nucleotidases purified from deep-sea and brackish water Shewanella species

Shewanella species are widely distributed in sea, brackish, and fresh water areas, growing psychrophilically or mesophilically, and piezophilically or piezo-sensitively. Here, membrane-bound 5′-nucleotidases (NTases) from deep-sea Shewanella violacea and brackish water Shewanella amazonensis were examined from the aspect of NaCl tolerance to gain an insight into protein stability against salt. Both NTases were single polypeptides with molecular masses of ~59 kDa, as determined on mass spectroscopy. They similarly required 10 mM MgCl₂ for their activities, and they exhibited the same pH dependency and substrate specificity for 5′-nucleotides. However, S. violacea 5′-nucleotidase (SVNTase) was active enough in the presence of 2.5 M NaCl, whereas S. amazonensis 5′-nucleotidase (SANTase) exhibited significantly reduced activity with the same concentration of the salt. Although SVNTase and SANTase exhibited high sequence identity (69.7%), differences in the ratio of acidic to basic amino acid residues and the number of potential salt bridges maybe being responsible for the difference in the protein stability against salt. 5′-Nucleotidases from these Shewanella species will provide useful information regarding NaCl tolerance, which may be fundamental for understanding bacterial adaptation to growth environments.

     

The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.     

Menadione-catalyzed O2- production by Escherichia coli cells: application of rapid chemiluminescent assay to antimicrobial susceptibility testing

This study proposes a novel chemiluminescent assay of bacterial activity. Luminol chemiluminescence (LC) was amplified on addition of menadione to Escherichia coli suspension, and it was effectively inhibited by addition of superoxide dismutase rather than catalase. This fact suggests that H2O2 produced from O2 by superoxide dismutase is decomposed by catalase of E. coli. NAD(P)H:menadione reductase activities in periplasm and cytosol corresponded to the amplification of menadione-catalyzed LC, and outer and cytoplasmic membranes were only slightly involved in the LC. The total activity and Vmax of NAD(P)H:menadione reductase in the cytoplasm were greater than those in the periplasm. A transient increase in menadione-catalyzed LC was observed in the exponential phase and the LC decreased in the stationary phase during growth of E. coli. Menadione-catalyzed LC was sensitive to antibiotic action. A decrease in menadione-catalyzed LC by the impairment of membrane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that menadione-catalyzed luminol chemiluminescent assay is applicable to rapid antimicrobial assay because LC is sensitive to the change in growth and cytotoxic events caused by antimicrobial agents.     

Muscular adaptations to combinations of high- and low-intensity resistance exercises

Acute and long-term effects of resistance-training regimens with varied combinations of high- and low-intensity exercises were studied. Acute changes in the serum growth hormone (GH) concentration were initially measured after 3 types of regimens for knee extension exercise: a medium intensity (approximately 10 repetition maximum [RM]) short interset rest period (30 s) with progressively decreasing load ("hypertrophy type"); 5 sets of a high-intensity (90% of 1RM) and low-repetition exercise ("strength type"); and a single set of low-intensity and high-repetition exercise added immediately after the strength-type regimen ("combi-type"). Postexercise increases in serum GH concentration showed a significant regimen dependence: hypertrophy-type > combi-type > strength-type (p < 0.05, n = 8). Next, the long-term effects of periodized training protocols with the above regimens on muscular function were investigated. Male subjects (n = 16) were assigned to either hypertrophy/combi (HC) or hypertrophy/ strength (HS) groups and performed leg press and extension exercises twice a week for 10 weeks. During the first 6 weeks, both groups used the hypertrophy-type regimen to gain muscular size. During the subsequent 4 weeks, HC and HS groups performed combi-type and strength-type regimens, respectively. Muscular strength, endurance, and cross sectional area (CSA) were examined after 2, 6, and 10 weeks. After the initial 6 weeks, no significant difference was seen in the percentage changes of all variables between the groups. After the subsequent 4 weeks, however, 1RM of leg press, maximal isokinetic strength, and muscular endurance of leg extension showed significantly (p < 0.05) larger increases in the HC group than in the HS group. In addition, increases in CSA after this period also tended to be larger in the HC group than in the HS group (p = 0.08). The results suggest that a combination of high- and low-intensity regimens is effective for optimizing the strength adaptation of muscle in a periodized training program.     

Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.     

Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce beta-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.