Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2 kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50 °C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50 °C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

[A CASE OF REPEATED DRUG-INDUCED LUNG DISEASES DUE TO MULTIPLE HERBAL MEDICINES CONTAINING COMMON INGREDIENTS].

We present a rare case of repetitive lung disease caused by various herbal medicines containing common ingredients. In June 201X-2, an 81-year-old man with chronic sinusitis was treated with Shini-seihai-to. One month later, the patient experienced liver dysfunction, and pulmonary opacity was observed on a chest radiograph; this condition improved following the discontinuation of Shini-seihai-to. In October 201X-2, the patient developed fever and dyspnea after treatment with Saiko-keishi-to, which was administered to treat irritable bowel syndrome, and was diagnosed with pneumonia. His condition did not improve with antimicrobial treatment but did improve with systemic corticosteroids. Following discharge from the hospital, the patient took both Shini-seihai-to and Hochu-ekki-to. He developed a fever two days later, which improved after discontinuing the medicines. The patient developed a cough after taking Sairei-to in February 201X and was subsequently admitted to our hospital with respiratory failure; pulmonary opacity was observed on a chest computed tomography scan. On the basis of clinical course, lymphocytosis in bronchoalveolar lavage fluid, and drug-induced lymphocyte stimulation tests, we diagnosed the patient with Sairei-to-induced lung disease. The patient's condition improved after discontinuing Sairei-to. We conclude that common ingredients in different herbal medicines may cause drug-induced lung injury. Therefore, we recommend that scrupulous attention should be paid to Chinese herbal medicine use in patients with a history of lung injury induced by herbal medicines.

Molecular Identification, Characterization, and Expression Analysis of a Metallothionein Gene from Septifer virgatus.

This study provides a preliminary characterization of a metallothionein (MT) gene in Septifer virgatus and highlights its potential use in biomonitoring. The full-length SvMT cDNA and the complete sequence of the SvMT gene were identified using reverse transcriptase PCR coupled with the rapid amplification of cDNA ends and the primer walking method. The SvMT cDNA encodes a protein of 72 amino acids having nine classical Cys-X-Cys motifs. Moreover, the deduced amino acids contained the conserved motif (Cys-x-Cys-x(3)-Cys-Thr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys) of MT family 2. Its molecular mass and isoelectric point were estimated to be 7.01 kDa and 7.00, respectively. BLAST-based searching indicated that SvMT shared 81.0% amino acid sequence identity with Mytilus edulis MT-20-II. The SvMT gene has three coding exons and two introns. After exposure to 1 mg/L cadmium chloride, the expression of SvMT increased 15-fold by 3 days (d), with a maximum expression of 27-fold by 5 d compared with the pre-exposure level. After exposure to 2 mg/L zinc chloride, the expression of SvMT increased 2.5-fold by 3 d and 4.7-fold by 5 d compared with the pre-exposure level. A significant increase in the expression level of SvMT mRNA was observed after the exposure of S. virgatus to the combination of 0.003 mg/L cadmium chloride and 0.2 mg/L zinc chloride compared with the pre-exposure level. Our work indicates that the SvMT gene is associated with stress responses and could be a potential biomarker for marine pollution.

Oral administration of multispecies microbial supplements to sows influences the composition of gut microbiota and fecal organic acids in their post-weaned piglets.

The timings of the administration of microbial supplements to control the populations of gut microbiota of piglets have been poorly understood. Here the effects of temporal administering multispecies microbial supplements to sows on the composition of gut microbiota and on the bacteria-mediated fecal metabolites in their offsprings were investigated. During gestation and lactation, pregnant sows were fed either a normal diet (group A) or a diet with multispecies supplements comprised of nine microbial species such as Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium bifidum, Enterococcus faecium, Candida pintolopesii, and Aspergillus oryzae etc. (group B). All of the sows' piglets were temporarily fed with the same supplements around weaning in accordance with the guideline of the farm. This regimen was followed by a normal diet in both groups over one month thereafter. Under such conditions, the concentration of short-chain fatty acids (SCFAs) in fecal samples remarkably increased in group B compared to group A. When 16S rDNA sequences of the fecal bacteria were analyzed, the microbial structure of bacteria was different between both goups. Especially the Clostridium cluster IV and subcluster XIVa were particularly increased in group B, although the administered microbes were undetectable. Thus, temporal administration of multispecies-microbial supplements to pregnant sows changes the composition of SCFAs and gut microbiota in their offsprings.

Distribution of oil-degrading bacteria in coastal seawater, Toyama Bay, Japan.

Oil-degrading bacteria are considered to play an important role in the biodegradation of spilled or released oil in the sea. The distribution of indigenous oil-degrading bacteria in the coastal seawater of Toyama Bay, Japan, was examined. Surface seawater samples with or without oil film in fishing port were analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of bacterial 16S rDNA. Sequence analysis revealed that several DGGE bands clearly detected only in samples with oil film corresponded to Cyanobacteria. Moreover, we cultured surface seawater samples with oil film in two different liquid culture media, a marine broth and an NSW medium; each culture contained 0.5% (w/v) C-heavy oil. Emulsification of the oil was observed at day 6 in the marine broth and day 9 in the NSW medium. Time-dependent changes of bacterial communities in those culture media were analyzed by DGGE. Interestingly, we found that Alcanivorax sp. became one of the dominant bacteria in each culture medium when emulsification of the oil began. Alcanivorax sp. is one of the well-known oil-degrading bacteria in seawater and is associated with the production of biosurfactants. These results suggest that Cyanobacteria and Alcanivorax play important roles in the bioremediation of oil-contaminated areas in Toyama Bay.