Effect of the C-terminal domain of Vibrio proteolyticus chitinase A on the chitinolytic activity in association with pH changes.

AIMS: To reveal the cause of the difference in activity of chitinase A from Vibrio proteolyticus and chitinase A from a strain of Vibrio carchariae (a junior synonym of Vibrio harveyi), we investigated the pH-dependent activity of full-length V. proteolyticus chitinase A and a truncated recombinant corresponding to the V. harveyi form of chitinase A. METHODS AND RESULTS: After overexpression in Escherichia coli strain DH5α, the full-length and truncated recombinant chitinases were purified by ammonium sulphate precipitation and anion exchange column chromatography. Chitinase activity was measured at various pH values using α-crystal and colloidal chitins as the substrate. The pH-dependent patterns of the relative specific activities for α-crystal chitin differed between the full-length and truncated recombinant chitinases, whereas those for colloidal chitin were similar to each other. CONCLUSION: The difference in the activity of V. proteolyticus chitinase A and V. harveyi chitinase A might be partly due to a change in the pH dependence of the chitinase activities against α-crystal chitin, resulting from C-terminal processing. SIGNIFICANCE AND IMPACT OF STUDY: The present results are important findings for not only ecological studies on the genus Vibrio in association with survival strategies, but also phylogenetic studies.

Heterodisaccharide 4-O-(N-acetyl-ß-D-glucosaminyl)-D-glucosamine is an effective chemotactic attractant for Vibrio bacteria that produce chitin oligosaccharide deacetylase.

AIMS: To investigate the attractant effect of 4-O-(N-acetyl-ß-D-glucosaminyl)-D-glucosamine (GlcNAc-GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc-GlcN from N,N'-diacetylchitobiose (GlcNAc)(2). METHODS AND RESULTS: The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane-migration method. The results demonstrated that GlcNAc-GlcN functions as an effective chemoattractant in the CE family 4 COD-producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc-GlcN appears to stimulate polar flagellum rotation. CONCLUSIONS: GlcNAc-GlcN is a specific chemoattractant for the CE family 4 COD-producing vibrios, V. parahaemolyticus and V. alginolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: It was clarified for the first time that GlcNAc-GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc-GlcN from (GlcNAc)(2).

Purification and characterization of Vibrio parahaemolyticus extracellular chitinase and chitin oligosaccharide deacetylase involved in the production of heterodisaccharide from chitin.

A chitin-degrading bacterial strain, KN1699, isolated from Yatsu dry beach (Narashino, Chiba Prefecture, Japan), was identified as Vibrio parahaemolyticus. Treatment of powdered chitin with crude enzyme solution prepared from the supernatant of KN1699 cultures yielded a disaccharide, beta-D-N-acetylglucosaminyl-(1,4)-D-glucosamine (GlcNAc-GlcN), as the primary chitin degradation product. The extracellular enzymes involved in the production of this heterodisaccharide, chitinase (Pa-Chi; molecular mass, 92 kDa) and chitin oligosaccharide deacetylase (Pa-COD; molecular mass, 46 kDa), were isolated from the crude enzyme solution, and their hydrolysis specificities were elucidated. These studies confirmed that (1) Pa-Chi hydrolyzes chitin to produce (GlcNAc)(2) and (2) Pa-COD hydrolyzes the acetamide group of reducing end GlcNAc residue of (GlcNAc)(2). These findings indicate that GlcNAc-GlcN is produced from chitin by the cooperative hydrolytic reactions of both Pa-Chi and Pa-COD.

Structure of cytochrome c6 from the red alga Porphyra yezoensis at 1. 57 A resolution.

The crystal structure of cytochrome c(6) from the red alga Porphyra yezoensis has been determined at 1.57 A resolution. The crystal is tetragonal and belongs to space group P4(3)2(1)2, with unit-cell parameters a = b = 49.26 (3), c = 83.45 (4) A and one molecule per asymmetric unit. The structure was solved by the molecular-replacement method and refined with X-PLOR to an R factor of 19.9% and a free R factor of 25.4%. The overall structure of cytochrome c(6) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys14 and Cys17, and the iron has an octahedral coordination with His18 and Met58 as the axial ligands. The sequence and the structure of the eukaryotic red algal cytochrome c(6) are very similar to those of a prokaryotic cyanobacterial cytochrome c(6) rather than those of eukaryotic green algal c(6) cytochromes.

[Home hospice care at a clinic].

For the people who want to stay at home until their last day, the primary doctor and clinic where they were diagnosed will be the most reliable supports. We have been operating a 19 bed clinic since 1996. In these three years, we have established what we call a "combination palliative care system." A team composed of two doctors, 13 nurses, 3 care aids, a social worker, and a counselor provides home care services as well as outpatient and inpatient care. From April, 1998 to March, 1999, 59 patients died of cancer. Among them, 25 patients died at home. Their primary cancers were lung (7), colon (3), pancreatic (2), breast (2), ovarian (2), brain (1), stomach (1), hepatoma (1), neck (1) and others. First of all, sufficient consultation with patients and family makes this care successful. Through this, the patient can choose his style of care. The whole staff is involved in this care in turn, so that all of us become acquainted with each patient. Home care includes: 1) medical and nursing service available 24 hours a day, 2) activation of social resources for the support of the patient user, 3) constructive cooperation with relevant institutions, 4) relieving the patient's physical and mental suffering, 5) aroma therapy, oil massage, hair cuts and music therapy, and 6) support by volunteers. In this way, as a neighborhood clinic, the combination palliative care system is valuable.

[Home palliative care of brain tumor patients from our clinic].

This study investigated whether or not home palliative care of brain tumor patients was possible and various ideas for accomplishing this, over the 2 1/2 years since our clinic was opened. There were 22 patients, aged 42-72 years, comprising 7 cases of glioblastoma, 14 cases of metastatic brain tumor, and 1 case of unknown origin. The control of intracranial hypertension and attacks of convulsion was possible with oral medication or suppositories, but not injection. The prognosis of cancer patients which changed home care showed no remarkable changes, and the home care rate was 53%. Malignant brain tumor patients were able to enjoy time with their families at home during the terminal stage, and die with dignity.

Molecular cloning and expression patterns of Cu/Zn-superoxide dismutases in developing soybean seeds.

Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.

A class III acidic endochitinase is specifically expressed in the developing seeds of soybean (Glycine max [L.] Merr.).

A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase specific activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was purified from mature dry seeds. Based upon the selected part (the residue positions 10-17) of the determined N-terminal 38 amino acid sequence, a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The resulting 1340 bp cDNA was comprised of a 5'-untranslated region of 39 bases, a coding region corresponding to a 25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34,269, calculated pI 4.7), and a 235 nucleotide 3'-terminal untranslated region including 24 bases of the poly(A) tail. By comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than 50% identical to every class III acidic chitinase, but has no significant similarity to other families of chitinases. The comparison also showed that the C-terminal region of this chitinase is markedly extended, by at least 31 residues. Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean. Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed defense, not only in development but also in dormancy of soybean seed. This is the first reported isolation and cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose that this enzyme would be classified into a new class of chitinase PR-8 family, together with a Sesbania homologue.