Crystallization and preliminary X-ray analysis of plant class I chitinase from rice

We report here on crystallization and preliminary X-ray analysis of plant class I chitinase from rice (OsChia1b). Similar single crystals of full-length OsChia1b were obtained under two independent conditions. The crystals grown under these conditions diffracted up to 2.1 and 2.5 angstroms resolution, respectively, at a synchrotron beamline, and were found to belong to the tetragonal space group P4(3)2(1)2.     

Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function

Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD(ChiC)) differs from those of the plant enzymes. ChBD(ChiC) and the catalytic domain (CatD(ChiC)), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD(ChiC) binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD(ChiC) resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD(ChiC) itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD(ChiC) in trans.     

Percutaneous cryoablation of pulmonary metastases from colorectal cancer

Objective

To evaluate the safety and efficacy of cryoablation for metastatic lung tumors from colorectal cancer.

Methods

The procedures were performed on 24 patients (36–82 years of age, with a median age of 62; 17 male patients, 7 female patients) for 55 metastatic tumors in the lung, during 30 sessions. The procedural safety, local progression free interval, and overall survival were assessed by follow-up computed tomographic scanning performed every 3–4 months.

Results

The major complications were pneumothorax, 19 sessions (63%), pleural effusion, 21 sessions (70%), transient and self-limiting hemoptysis, 13 sessions (43%) and tract seeding, 1 session (3%). The 1- and 3-year local progression free intervals were 90.8% and 59%, respectively. The 3-years local progression free intervals of tumors ≤15 mm in diameter was 79.8% and that of tumors >15 mm was 28.6% (p = 0.001; log-rank test). The 1- and 3-year overall survival rates were 91% and 59.6%, respectively.

Conclusion

The results indicated that percutaneous cryoablation is a feasible treatment option. The local progression free interval was satisfactory at least for tumors that were ≤15 mm in diameter.     

Importance of Trp59 and Trp60 in chitin-binding, hydrolytic, and antifungal activities of Streptomyces griseus chitinase C

The chitin-binding domain of Streptomyces griseus chitinase C (ChBD_ChiC) belongs to CBM family 5. Only two exposed aromatic residues, W59 and W60, were observed in ChBD_ChiC, in contrast to three such residues on CBD_Cel5 in the same CBM family. To study importance of these residues in binding activity and other functions of ChBD_ChiC, site-directed mutagenesis was carried out. Single (W59A and W60A) and double (W59A/W60A) mutations abolished the binding activity of ChiC to colloidal chitin and decreased the hydrolytic activity toward not only colloidal chitin but also a soluble high Mr substrate, glycol chitin. Interaction of ChBD_ChiC with oligosaccharide was eliminated by these mutations. The hydrolytic activity toward oligosaccharide was increased by deletion of ChBD but not affected by these mutations, indicating that ChBD interferes with oligosaccharide hydrolysis but not through its binding activity. The antifungal activity was drastically decreased by all mutations and significant difference was observed between single and double mutants. Taken together with the structural information, these results suggest that ChBD_ChiC binds to chitin via a mechanism significantly different from CBD_Cel5, where two aromatic residues play major role, and contributes to various functions of ChiC. Sequence comparison indicated that ChBD_ChiC-type CBMs are dominant in CBM family 5.     

Chromosome numbers of European blackberries (Rubus subg.Rubus,Rosaceae)

Chromosome numbers are presented for 32 collections of 29 European blackberry species (Rubus subg.Rubus) from Germany. One species is triploid (2n = 21), 27 species are tetraploid, (2n = 28), and one species is pentaploid (2n = 35). Chromosome numbers are reported for the first time ofR. adspersus, R. amisiensis, R. calvus, R. conothyrsoides, R. contractipes, R. demissus, R. elegantispinosus, R. ferocior, R. foliosus, R. hypomalacus, R. leucandrus, R. nemorosus, R. platyacanthus, R. praecox, R. rhombifolius, andR. rhytidophyllus. Chromosome numbers forR. dasyphyllus, R. gelertii, R. glandithyrsos, R. lamprocaulos, R. lindebergii, R. macrophyllus, R. montanus, R. muenteri, R. pedemontanus, R. polyanthemus, R. senticosus, R. silvaticus, andR. vigorosus are confirmed.     

Cytotaxonomic study on two putative hybrids in the genusDuchesnea (Rosaceae)

The karyotypes and chromosome associations at meiosis in two types of natural hybrids, 7x and 8x, betweenDuchesnea chrysantha (2x) andD. indica (12x) were investigated. The 7x hybrid had a haploid chromosome set from each parent plant, whereas the 8x hybrid was considered to have a full set ofD. chrysantha and half a set ofD. indica. In the two hybrids, the chromosomes ofD. chrysantha andD. indica conjugated only slightly at meiosis. It is probable that no common genome set between the diploidD. chrysantha and the dodecaploidD. indica exists. The present evidence indicates thatD. chrysantha andD. indica should be considered to be distinct species, although they have sometimes been treated as a single species.     

Molecular cloning, expression and characterization of pyridoxamine-pyruvate aminotransferase

Pyridoxamine-pyruvate aminotransferase is a PLP (pyridoxal 5'-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine-pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the alpha family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429-432]. The K(d) value for pyridoxal determined by means of CD was 100-fold lower than the K(m) value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.     

A novel polymorphism in CDC6 is associated with the decline in lung function of ex-smokers in COPD

The effect of smoking cessation on the rate of decline in lung function in patients with advanced stages of chronic obstructive pulmonary disease (COPD) has not been clarified. Saccharomyces cerevisiae cell division cycle 6 homolog (CDC6) protein possesses the pro-apoptotic properties. We tested our hypothesis that the individual susceptibility to rapid decline in lung function despite smoking cessation in patients with advanced stages of COPD is attributed to the genetic variants in the CDC6 gene. We prospectively followed 82 patients (ex-smokers) during 30 months and evaluated the differences among the genotypes in the annual rate of decline in FEV_1.0 (%predicted) with ten single nucleotide polymorphisms (SNPs) in and around the CDC6 gene. We found significant differences in SNP5 (National Center for Biotechnology Information SNP reference: rs2077464), SNP6 (rs13706), SNP7 (rs7217852), and SNP8 (rs9904270) with a gene-dosage effect (ANOVA overall-P = 0.029-0.030). The individual allele of SNP5G, SNP6A, SNP7G, and SNP8T were associated with rapid decline in FEV_1.0 (%predicted) [odds ratio (95% confidence interval) = 2.35 (1.19-4.65), P = 0.014]. The SNP5G/SNP6A/SNP7G/SNP8T haplotype was associated with an increased risk of deterioration of FEV_1.0 (%predicted) (P = 0.017). Importantly, SNP6 caused a change in amino acids in CDC6 protein (Val441Ile), immediately upstream of the caspase-3-dependent cleavage site of CDC6 (Asp442) during apoptosis. These results suggest that CDC6 may be one of the susceptibility genes that contribute to rapid decline in lung function despite smoking cessation in these patients with COPD.