• Journal of Microbiology and Biotechnology
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• v.21 no.11
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• pp.1193-1198
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• 2011

Encapsulation of biological material in the permiselective membrane allows to construct a system separating cells from their products, which may find biotechnological as well as biomedical applications in biological processes regulation. Application of a permiselective membrane allows avoiding an attack of the implanted microorganisms on the host. Our aim was to evaluate the performance of Bacillus subtilis encapsulated in an elaborate membrane system producing listeriolysin O, a cytolysin from Listeria monocytogenes, with chosen eukaryotic cells for future application in anticancer treatment. The system of encapsulating in membrane live Bacillus subtilis BR1-S secreting listeriolysin O was proven to exert the effective cytotoxic activity on eukaryotic cells. Interestingly, listeriolysin O showed selective cytotoxic activity on eukaryotic cells: more human leukemia Jurkat T cells were killed than human chronic lymphocytic B cells leukemia at similar conditions in vitro. This system of encapsulated B. subtilis, continuously releasing bacterial products, may affect selectively different types of cells and may have future application in local anticancer treatment.

• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.153-160
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• 2010

The present study was carried out for the isolation of bacteriocin-producing enterococci from indigenous sources. Gram-positive enterococci are known for having the ability to produce enterocins with good antimicrobial potential. A total of 34 strains were isolated from processed dairy products of Pakistan and seven out of them were found to be member of genus Enterococcus on selective enumeration. Biochemical and molecular characterization revealed that four of these isolates (IJ-03, IJ-07, IJ-11, and IJ-12) were Enterococcus faecalis and three (IJ-06, IJ-21, and IJ-31) were Enterococcus faecium. Local processed cheese was the source of all enterococcal isolates, except E. faecium IJ-21 and IJ-31, which were isolated from indigenous yoghurt and butter samples, respectively. Bacterial isolates were sensitive to commonly used antibiotics except methicillin and kanamycin. They also lacked critical virulence determinants, mainly cytolysin (cyl), gelatinase (gel), enterococcal surface protein (esp), and vancomycin resistance (vanA and vanB). Polymerase chain reaction amplification identified that enterocin A and P genes were present in the genome of E. faecium IJ-06 and IJ-21, whereas the E. faecium IJ-31 genome showed only enterocin P genes. No amplification was observed for genes that corresponded with the enterocins 31, AS-48, L50A, and L50B, and ent 1071A and 1071B. There were no signals of amplification found for E. faecalis IJ-11, indicating that the antimicrobial activity was because of an enterocin different from those checked by PCR. Hence, the indigenous bacterial isolates have great potential for bacteriocin production and they had antibacterial activity against a variety of closely related species.

• Journal of Microbiology
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• v.44 no.2
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• pp.226-232
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• 2006

Swarming has proven to be a good in vitro model for bacterial surface adherence and colonization, and the swarming differentiation of a bacterium has been shown to be coupled with changes in the expression of virulence factors associated with its invasiveness, particularly in the early stages of infection. In this study, we attempted to determine whether the expression of vvhA, which encodes for hemolysin/cytolysin (VvhA), is either upregulated or downregulated during the swarming differentiation of V. vulnificus. The insertional inactivation of vvhA itself exerted no detectable effect on the expression of V. vulnificus swarming motility. However, in our lacZ-fused vvhA transcriptional reporter assay, vvhA expression decreased in swarming V. vulnificus as compared to non-swarming or planktonic V. vulnificus. The reduced expression of vvhA in swarming V. vulnificus increased as a result of the deletional inactivation of luxS, a gene associated with quorum sensing. These results show that vvhA expression in swarming V. vulnificus is downregulated via the activity of the LuxS quorum-sensing system, suggesting that VvhA performs no essential role in the invasiveness of V. vulnificus via the adherence to and colonization on the body surfaces required in the early stages of the infection. However, VvhA may playa significant role in the pathophysiological deterioration occurring after swarming V. vulnificus is differentiated into planktonic V. vulnificus.

• Journal of Microbiology and Biotechnology
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• v.26 no.6
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• pp.1026-1034
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• 2016

In the current study, a total of 135 enterococci strains from different sources were screened for the presence of the enterocin-encoding genes entA, entP, entB, entL50A, and entL50B. The enterocin genes were present at different frequencies, with entA occurring the most frequently, followed by entP and entB; entL50A and L50B were not detected. The occurrence of single enterocin genes was higher than the occurrence of multiple enterocin gene combinations. The 80 isolates that harbor at least one enterocin-encoding gene (denoted "Gene⁺ strains") were screened for antimicrobial activity. A total of 82.5% of the Gene⁺ strains inhibited at least one of the indicator strains, and the isolates harboring multiple enterocin-encoding genes inhibited a larger number of indicator strains than isolates harboring a single gene. The indicator strains that exhibited growth inhibition included Listeria innocua strain CLIP 12612 (ATCC BAA-680), Listeria monocytogenes strain CDC 4555, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Salmonella typhimurium strain UK-1 (ATCC 68169), and Escherichia coli BAC 49LT ETEC. Inhibition due to either bacteriophage lysis or cytolysin activity was excluded. The growth inhibition of antilisterial Gene⁺ strains was further tested under different culture conditions. Among the culture media formulations, the MRS agar medium supplemented with 2% (w/v) yeast extract was the best solidified medium for enterocin production. Our findings extend the current knowledge of enterocin-producing enterococci, which may have potential applications as biopreservatives in the food industry due to their capability of controlling food spoilage pathogens.

• Korean Journal of Environment and Ecology
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• v.12 no.1
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• pp.58-77
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• 1998

Four species(25%) of Viperidae(Agkistrodon brevicaudus, Agkistrodon ussuriensis, Agkistrodon saxatilis) and Cloubridae(Rhabdophis tigrenus tigrenus) were Korean poisonous snake. Copulation season of these species was from July to August. Reproduction mode of genus Agkistrodon species was ovoviviparous but Rhabdophis tigrinus tigrinus was the other pattern of oviparous. Optimal movement temperature range was from 20$\circ $C to 29$\circ $C(March~September). Wjen atmosphere temperature was below 10$\circ $C, at that time they hibernate at the ground, rock bottom, stone wall and embankment around the end of a field. The venom of these snakes consist mainly Hematoxin, Cytolysin, Neurotoxin and Cardiotoxin of poisonous liquids. These material injection to animal cause systemic syndrome such as Dizziness(25.7%), Vomitting(23.1%), Fever(22%), Visual trouble(18%), Headace(17.7%), Dyspnoea(17.6%) and bring about other local syndrome such as Discoloration(54.2%), Bleeding(20.2%), Bullae(10.7%) and Skin ulcer(!0.8%). The annual distribution was appeared to decrease 1972 after 1992 and average snakebiting patients was 25.6 per year, but practically total estimated snakebiting was 2,700 per year. The seasonal distribution was most frequent in August(25%), and mortality was 1.8%(26 per 1,430). The sex ratio was 2:1 and according to age distribution, it was most prevalent at one's fifties(19%). The most frequent place where the accident happened was the field(48.2%) and most predilection site of the body for victim were hand(47.8%) and foot(39.5%), Commonly bite snake were Agkistrodon ussuriensis(27.1%), Agkistrodon brevicaudus(22.6%) and Agkistrodon saxatilis(9.6%) but 40.7% of species could not be identified. Treatment of antivenin patient was 75.9% (1,068/1,407).

• The Journal of the Korean Society for Microbiology
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• v.35 no.3
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• pp.251-261
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• 2000

V. vulnificus is an estuarine bacterium which causes septicemia and shock in susceptible patients. The organism produces a hemolytic cytolysin (VvH), which has a membrane damaging effect on erythrocytes. To clarify the mechanisms by which VvH might contribute to virulence, we examined its effect on macrophages. When mouse peritoneal macrophages were harvested and co-cultured with hemolysin-positive V. vulnificus strains (100 bacteria/cell), about 60% of the macrophages were killed; macrophages were not killed when co-cultured V. vulnificus strain CVD 707, a VvH-negative deletion mutant. Exposure of macrophages to filtered culture supernatants (2.5 HU/ml) and purified VvH (3 HU/ml) resulted in an increase in dead cells (80 and 90%, respectively), as determined by the trypan blue dye exclusion method and LDH release from macrophages was also increased (70 and 65.5%, respectively). The cytotoxic effect of VvH on macrophages was both the dose- and time-dependent. The VvH caused damage to the macrophage membrane and was blocked significantly by preincubation with cholesterol (p<0.01). Fetal bovine serum showed remarkable inhibition of VvH synthesis by V. vulnificus and inhibited VvH activity in culture supernatant. Cell viability was increased by 35% (p<0.01) and LDH release decreased by 28% (p<0.01) when macrophages were incubated with V. vulnificus (100 bacterial cell) in DMEM-10% FBS for 2 hr. Bacterial clearance activity of mice against V. vulnificus CVD 707 was decreased by pretreatment with 10 HU of VvH. This result suggests that the VvH can impair the membrane of macrophages and may playa role in the pathogenesis of V. vulnificus septicemia.

• Archives of Pharmacal Research
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• v.22 no.5
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• pp.437-447
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• 1999

Type I diabetes, also known as insulin-dependent diabetes mellitus (IDDM) results from the destruction of insulin-producing pancreatic $\beta$ cells by a progressive $\beta$ cell-specific autoimmune process. The pathogenesis of autoimmune IDDM has been extensively studied for the past two decades using animal models such as the non-obese diabetic (NOD) mouse and the Bio-Breeding (BB) rat. However, the initial events that trigger the immune responses leading to the selective destruction of the $\beta$ cells are poorly understood. It is thought that $\beta$ cell auto-antigens are involved in the triggering of $\beta$ cell-specific autoimmunity. Among a dozen putative $\beta$ cell autoantigens, glutamic acid decarboxylase (GAD) has bee proposed as perhaps the strongest candidate in both humans and the NOD mouse. In the NOD mouse, GAD, as compared with other $\beta$ cell autoantigens, provokes the earliest T cell proliferative response. The suppression of GAD expression in the $\beta$ cells results in the prevention of autoimmune diabetes in NOD mice. In addition, the major populations of cells infiltrating the iselts during the early stage of insulitis in BB rats and NOD mice are macrophages and dendritic cells. The inactivation of macrophages in NOD mice results in the prevention of T cell mediated autoimmune diabetes. Macrophages are primary contributors to the creation of the immune environment conducive to the development and activation of $\beta$cell-specific Th1-type CD4+ T cells and CD8+ cytotoxic T cells that cause autoimmune diabetes in NOD mice. CD4+ and CD8+ T cells are both believed to be important for the destruction of $\beta$ cells. These cells, as final effectors, can kill the insulin-producing $\beta$ cells by the induction of apoptosis. In addition, CD8+ cytotoxic T cells release granzyme and cytolysin (perforin), which are also toxic to $\beta$ cells. In this way, macrophages, CD4+ T cells and CD8+ T cells act synergistically to kill the $\beta$ cells in conjunction with $\beta$ cell autoantigens and MHC class I and II antigens, resulting in the onset of autoimmune type I diabetes.