The nucleotide sequence of pR72H cloned from Vibrio parahaemolyticus 93 was determined. We examined all V. parahaemolyticus gene sequences published in the GenBank-EMBL databases for homology and found that no other DNA sequence of V. parahaemolyticus was highly homologous to the sequence reported in this study. A pair of primers, VP33-VP32, derived from a pR72H fragment were selected to detect V. parahaemolyticus. The sensitivity of PCR detection for a pure culture of V. parahaemolyticus was 10 cells from crude bacterial lysates. Furthermore, a detection level of 2.6 fg, equivalent to 1 cell, was obtained by using purified chromosomal DNA as the template. The expected PCR products were obtained from all V. parahaemolyticus strains tested (n ؍ 124), while no PCR amplicons were found in other vibrios or related genera (n ؍ 50). High levels (10 6 to 10 10 CFU/ml) of Escherichia coli cells did not affect the PCR assay sensitivity. The presence of 10 8 V. parahaemolyticus cells or 10 9 E. coli cells in the PCR mixtures completely inhibited the PCR. When oyster samples were inoculated with V. parahaemolyticus 93 and cultured in tryptic soy broth containing 3% NaCl for 3 h at 35؇C, an initial sample inoculum level of 9.3 CFU/g was detected in a PCR assay with crude bacterial lysates. The PCR assay with enrichment culturing in salt polymyxin broth was compared with the conventional method for naturally contaminated shellfish and fish samples. We conclude that this PCR assay with enrichment culturing is a good alternative method for the detection of V. parahaemolyticus.
A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800 m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200 m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substance-producing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.
Oxidative stress is one of the major causes of cell death. Using time-lapse confocal recording of live cardiomyocytes, we showed that H2O2 (OH*) caused a marked increase in Na+ and Ca2+ levels in both the cytosol ([Na]cyt, [Ca]cyt) and mitochondria ([Na]m, [Ca]m). The H2O2-induced intracellular Na+ ([Na]i) overload contributed to the H2O2-induced [Ca]cyt/[Ca]m overload via activation of the reverse mode of the Na-Ca exchanger. When myocytes were treated for 40 min with 100 microM H2O2 in normal medium, then returned to H2O2-free medium, the percentage of apoptotic cells increased from 4% at 0 h to 55 and 85% at 4.5 and 16 h, respectively. H2O2-induced apoptosis was completely prevented by using Na-free, but not Ca-free, medium. When a Na+ ionophore cocktail in Ca-free medium was used instead of H2O2 to increase the [Na]i by more than 30 mM without any change in the [Ca]i, cytochrome c release and caspase 3-dependent apoptosis occurred, showing that [Na]i overload per se induced apoptosis. We also showed that the increase in the mitochondrial, but not the cytosolic, Na+ levels resulted in the opening of the permeation transition pore, followed by cytochrome c release. Our findings therefore suggest that H2O2-induced [Na]m overload is an important upstream signal for the apoptotic machinery, and the prevention of [Na]m overload thus represents a particularly attractive target for strategies aimed at preventing oxidative stress-induced cell death.
Lactococcus formosensis sp. nov., a lactic acid bacterium isolated from yan-tsai-shin (fermented broccoli stems) The Institute of Enology and Viticulture, University of Yamanashi, 1-13-1, Kitashin, Kofu, Yamanashi 400-0005, Japan A coccal-shaped organism, designated 516 T , was isolated from yan-tsai-shin (fermented broccoli stems), a traditional fermented food in Taiwan. 16S rRNA gene sequencing results showed that strain 516 T had 98.9 % sequence similarity to that of the type strain Lactococcus garvieae NBRC 100934 T . Comparison of three housekeeping genes, rpoA, rpoB and pheS, revealed that strain 516 T was well separated from Lactococcus garvieae NBRC 100934 T . DNA-DNA hybridization studies indicated that strain 516 T had low DNA relatedness with Lactococcus garvieae NBRC 100934 T (46.1 %). The DNA G+C content of strain 516 T was 38.1 mol% and the major fatty acids were C 16 : 0 (22.7 %), C 19 : 0 cyclo v8c (17.9 %) and summed feature 7 (29.0 %). Based on the evidence, strain 516 T represents a novel species of the genus Lactococcus, for which the name Lactococcus formosensis sp. nov. is proposed. The type strain is 516 T (5NBRC 109475 T 5BCRC 80576 T ).
Synthetic oligonucleotide primers derived from a sequence of the thermostable direct haemolysin (tdh) gene were used in a polymerase chain reaction (PCR) amplification technique to detect this gene in strains of Vibvio pavahaemolyticus. A total of 36 TDH-producing, and 89 TDH-negative Vibvio pavahaemolyticus strains and 46 other vibrios and enteric pathogens were studied. In all, 36 strains of Vibrio pavahaemolyticus from which the tdh gene could be successfully amplified by PCR were found to be TDH-positive in TDH haemolysin assay. No amplification products were obtained from Vibvio pavahaemolyticus strains that were TDH-negative in the haemolysin assay or from other vibrios and enteric pathogens, with the exception of two strains. The PCR results were consistent with DNA hybridization tests. The detection limit for the tdh gene by PCR amplification was 40 pg of total DNA, or broth culture containing 1000 viable cells. Amplification products were confirmed by restriction enzyme digestion and Southern blot hybridization. The PCR method could detect the tdh sequences in stool samples from patients with gastroenteritis caused by V. pavahaemolyticus. This PCR protocol clearly identified TDHproducing strains of V. pavahaemolyticus and provides an alternative to conventional methods for TDH detection by research laboratories, clinical laboratories, regulatory agencies, and the seafood industry.
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