BACKGROUND: Among the many Vibrio species that can cause infections in humans, several species can cause a fatal outcome. Therefore, accurate identification of Vibrio species is very important. Since some species show atypical phenotypic features, selecting an appropriate molecular method is necessary to avoid misdiagnosis. METHODS: Vibrio clinical isolates (N=53) and reference strains (N=8) were used in this study. We analyzed the following sequences for identification: dnaJ gene, 16S rDNA, gyrase B (gyrB) V. vulnificus-specific sequence, gyrB V. navarrensis-specific sequence, and V. vulnificus hemolysin gene PCR (Vvh PCR). We performed phylogenetic analysis of the 16S rDNA, dnaJ, and gyrB sequences. Final identification was based on the combined results of all tests described above. Concordance of the 16S rDNA and dnaJ sequence analysis was measured using the Chi-square test. RESULTS: The 61 Vibrio strains were identified as follows, in descending order: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), and Grimontia hollisae (1.64%). The accuracy rates of the dnaJ gene and 16S rDNA sequence for identification were 91.80% and 86.89%, respectively. The 16S rDNA and dnaJ sequences showed a concordance rate of 0.45, which indicates moderate agreement. CONCLUSIONS: Our results suggest that analysis of the dnaJ sequence may be a useful method for the identification of clinical isolates of Vibrio species, especially for distinguishing between closely related Vibrio species.
Cholera ; Diagnostic Errors ; DNA, Ribosomal ; Fatal Outcome ; Humans ; Methods ; Polymerase Chain Reaction ; Sequence Analysis ; Vibrio

BACKGROUND: Tigecycline resistance has emerged recently and has shown diverse mechanisms. The aim of this study was to assess the role of efflux activity in tigecycline resistance in 120 clinical isolates of A. baumannii using two methods: the H33342 accumulation assay and adeB real-time reverse transcriptase polymerase chain reaction. In addition, we analyzed the correlation between the expression level of adeB and H33342 accumulation level. METHODS: A. baumannii clinical isolates was divided into tigecycline-resistant (49 strains), intermediate (40 strains), and susceptible (31 strains) groups. The H33342 accumulation was measured in the absence or presence of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Real-time RT-PCR was performed to determine the relative expression of the adeB gene in A. baumannii clinical isolates. RESULTS: The level of H33342 accumulation in the resistant group was relatively lower than those in the other groups. The addition of CCCP caused a significantly increased fold change in H33342 accumulation in the tigecycline-resistant group. Significant difference in the fold change level in H33342 accumulation was found between tigecycline-susceptible and resistant isolates. Those findings support the role of efflux pumps of which substrates are H33342 in the resistance of tigecycline. Significant differences in the relative expression levels of adeB were shown between tigecycline-susceptible and resistant groups also. CONCLUSION: The results showed that several efflux pumps of which substrates were H33342 can contribute to tigecycline resistance. The adeB overexpression can also contribute to tigecycline resistance. It is possible that efflux pumps other than adeB efflux pumps contribute to tigecycline resistance because there was no correlation between fold change level in H33342 accumulation and adeB expression level.
Acinetobacter baumannii* ; Acinetobacter* ; Carbonyl Cyanide m-Chlorophenyl Hydrazone ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND: Cancer stem cells have been investigated as new targets for colorectal cancer (CRC) treatment. We recently reported that CD133+ colon cancer cells showed chemoresistance to 5-fluorouracil through increased survivin expression and proposed the survivin inhibitor YM155 as an effective therapy for colon cancer in an in vitro study. Here, we investigate the relationship between survivin and CD133 expression in surgically resected CRC to identify whether the results obtained in our in vitro study are applicable to clinical samples. METHODS: We performed immunohistochemical staining for survivin and CD133 in surgically resected tissue from 187 stage II or III CRC patients. We also comparatively analyzed apoptosis according to survivin and CD133 expression using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. RESULTS: The results of the Mantel-Haenszel test established a linear association between nuclear survivin and CD133 expression (p = .018), although neither had prognostic significance, according to immunohistochemical expression level. No correlation was found between survivin expression and the following pathological parameters: invasion depth, lymph node metastasis, or histologic differentiation (p > .05). The mean apoptotic index in survivin+ and CD133+ tumors was higher than that in negative tumors: 5.116 ± 4.894 in survivin+ versus 4.103 ± 3.691 in survivin– (p = .044); 5.165 ± 4.961 in CD133+ versus 4.231 ± 3.812 in CD133– (p = .034). CONCLUSIONS: As observed in our in vitro study, survivin expression is significantly related to CD133 expression. Survivin may be considered as a new therapeutic target for chemoresistant CRC.
Apoptosis ; Colonic Neoplasms ; Colorectal Neoplasms* ; Deoxyuridine ; Fluorouracil ; Humans ; In Vitro Techniques ; Lymph Nodes ; Neoplasm Metastasis ; Neoplastic Stem Cells

This study was designed to examine the effects of histamine on gastric motility and its specific receptor in the circular smooth muscle of the human gastric corpus. Histamine mainly produced tonic relaxation in a concentration-dependent and reversible manner, although histamine enhanced contractility in a minor portion of tissues tested. Histamine-induced tonic relaxation was nerve-insensitive because pretreatment with nerve blockers cocktail (NBC) did not inhibit relaxation. Additionally, K+ channel blockers, such as tetraethylammonium (TEA), apamin (APA), and glibenclamide (Glib), had no effect. However, N(G)-nitro-L-arginine methyl ester (L-NAME) and 1H-(1,2,4)oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), did inhibit histamine-induced tonic relaxation. In particular, histamine-induced tonic relaxation was converted to tonic contraction by pretreatment with L-NAME. Ranitidine, the H2 receptor blocker, inhibited histamine-induced tonic relaxation. These findings suggest that histamine produced relaxation in circular smooth muscle of human gastric smooth muscle through H2 receptor and NO/sGC pathways.
Apamin ; Glyburide ; Guanylate Cyclase ; Histamine ; Humans ; Muscle, Smooth* ; Nerve Block ; NG-Nitroarginine Methyl Ester ; Nitric Oxide* ; Ranitidine ; Receptors, Histamine H2 ; Relaxation* ; Tetraethylammonium

OBJECTIVES: To examine the effect of extracts of Korean native Cimicifuge species on cell proliferation in vascular smooth muscle cells (VSMC). METHODS: VSMC were isolated from rat aorta. Cell proliferation was assessed by measure of bromodeoxyuridine incorporation into the cells. Differences in Reactive oxygen species (ROS) levels were examined after exposure to the extracts of Korean native Cimicifuge species using the detection reagents dichlorofluorecin diacetate. The rhizomes/roots were air-dried and milled with a commercial food mixer. Milled rhizomes/roots of each Cimicifuga species were separately extracted by 80% ethanol, absolute methanol, and 40% 2-propanol using homogenizer and evaporated under reduced pressure at low temperatures. Effects of extracts dissolved in phosphate-buffered saline (0.3 mg/mL) were examined. RESULTS: Ethanolic, methanolic or propanolic extracts of 4 Korean native Cimicifuge species (Cimicifuga [C] davurica, C. japonica, C. heracleifolia var. bifida Nakai, C. simplex) were screened. The addition of extracts of each Korean native Cimicifuge species to cells in the presence of 10% fetal bovine serum (FBS) potently inhibited cell proliferation. Significant decrease of 23%-30% was observed. Vitamin E, a potent antioxidant, inhibited 10% FBS-stimulated cell proliferation of VSMC. We also demonstrated that extracts of each Korean native Cimicifuge species decreased intracellular ROS generation induced with 10% FBS. The effect of Korean native Cimicifuge species was not species-specific and solvent-specific. CONCLUSION: TExtracts of Korean native Cimicifuge species inhibit VSMC proliferation via inhibition of intracellular ROS. These findings suggest that Cimicifuge species used for reducing menopause symptoms might be cardioprotective in women.
1-Propanol ; 2-Propanol ; Animals ; Aorta ; Bromodeoxyuridine ; Cardiovascular Diseases ; Cell Proliferation ; Cimicifuga ; Estrogens ; Ethanol ; Female ; Humans ; Indicators and Reagents ; Menopause ; Methanol ; Muscle, Smooth, Vascular ; Rats ; Reactive Oxygen Species ; Vitamin E ; Vitamins

The balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis. PAI-1 expression increases in atherosclerotic arteries and vascular smooth muscle cells (VSMCs) are one of major constituents of atheroma. We investigated the impact of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, on the plasminogen activator system of the rat VSMCs. The lysoPC stimulated the protein and gene expressions of PAI-1 but did not affect the protein expression of t-PA. Fibrin overlay zymography revealed that lysoPC increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA. Vitamin E inhibited the lysoPC-induced PAI-1 expression. Further, lysoPC increased the intracellular reactive oxygen species (ROS) formation. Caffeic acid phenethyl ester, an inhibitor of NF-kappaB, blocked this lysoPC effect. Indeed, lysoPC induced the NF-kappaB-mediated transcriptional activity as measured by luciferase reporter assay. In addition, genistein, an inhibitor of protein-tyrosine kinase (PTK), diminished the lysoPC effect, while 7,12-dimethylbenz[a]anthracene, a stimulator of PTK, stimulated PAI-1 production. In conclusion, lysoPC does not affect t-PA expression but induces PAI-1 expression in the VSMC by mediating NF-kappaB and the genistein-sensitive PTK signaling pathways via oxidative stress. Importantly, lysoPC stimulates the enzyme activity of PAI-1 and suppresses that of t-PA.
Animals ; Benz(a)Anthracenes/pharmacology ; Caffeic Acids/pharmacology ; Cells, Cultured ; Genistein/pharmacology ; Lipoproteins, LDL/metabolism ; Lysophosphatidylcholines/*pharmacology ; Muscle, Smooth, Vascular/cytology/*drug effects/metabolism ; NF-kappa B/antagonists & inhibitors/metabolism ; Oxidative Stress/drug effects ; Phenylethyl Alcohol/analogs & derivatives/pharmacology ; Plasminogen Activator Inhibitor 1/agonists/genetics/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Protein-Tyrosine Kinases/antagonists & inhibitors/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects ; Tissue Plasminogen Activator/*metabolism ; Transcription, Genetic/drug effects ; Up-Regulation/drug effects ; Vitamin E/pharmacology

OBJECTIVES: To investigate the cytotoxic effects of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoproteins (LDL), on vascular smooth muscle cells (VSMCs). METHODS: VSMCs were derived from rat aorta. Cell death was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, lactic dehydrogenase (LDH) assay, and DNA fragmentation assay. Apoptosis was quantified by propidium iodide staining and fluorescent activated cell sorting (FACS) analysis, and intracellular free radical production was determined using 2',7'-dichlorofluorescin diacetate (DCF-DA). In addition, the changes in caspases, bcl-2 and bax proteins were evaluated by western blot analysis. RESULTS: LysoPC over 25 microM induced more than 50% of the cell death at 10 hours on MTT assay with no change in the level of LDH. The DNA ladder pattern showed that cell death induced by lysoPC was caused by apoptosis, which was associated with increased free radical production. Vitamin E, a potent antioxidant and caffeic acid phenylethyl ester (CAPE), an inhibitor of nuclear factor-kappaB (NF-kappaB), blocked apoptosis. The casepase-3 precursor decreased and the active form of caspase-8 increased. Total bcl-2 and bax proteins did not change with lysoPC treatment, but translocation of bax from cytosole to the mitochondria membrane was observed. CONCLUSION: LysoPC induces apoptosis in VSMCs via an oxidant mechanism, dependent on NF-kappaB.
Animals ; Aorta ; Apoptosis ; Atherosclerosis ; bcl-2-Associated X Protein ; Blotting, Western ; Caffeic Acids ; Caspase 8 ; Caspases ; Cell Death ; Cytosol ; DNA ; DNA Fragmentation ; Fluoresceins ; Lipoproteins, LDL ; Lysophosphatidylcholines ; Membranes ; Mitochondria ; Muscle, Smooth, Vascular ; NF-kappa B ; Oxidoreductases ; Propidium ; Rats ; Vitamin E ; Vitamins

This study was designed to elucidate high K(+)-induced relaxation in the human gastric fundus. Circular smooth muscle from the human gastric fundus greater curvature showed stretch-dependent high K+ (50 mM)-induced contractions. However, longitudinal smooth muscle produced stretch-dependent high K(+)-induced relaxation. We investigated several relaxation mechanisms to understand the reason for the discrepancy. Protein kinase inhibitors such as KT 5823 (1 microM) and KT 5720 (1 microM) which block protein kinases (PKG and PKA) had no effect on high K(+)-induced relaxation. K+ channel blockers except 4-aminopyridine (4-AP), a voltage-dependent K+ channel (KV) blocker, did not affect high K(+)-induced relaxation. However, N(G)-nitro-L-arginine and 1H-(1,2,4)oxadiazolo (4,3-A)quinoxalin-1-one, an inhibitors of soluble guanylate cyclase (sGC) and 4-AP inhibited relaxation and reversed relaxation to contraction. High K(+)-induced relaxation of the human gastric fundus was observed only in the longitudinal muscles from the greater curvature. These data suggest that the longitudinal muscle of the human gastric fundus greater curvature produced high K(+)-induced relaxation that was activated by the nitric oxide/sGC pathway through a KV channel-dependent mechanism.
4-Aminopyridine ; Carbazoles ; Contracts ; Gastric Fundus ; Guanylate Cyclase ; Humans ; Muscle, Smooth ; Muscles ; Nitric Oxide ; Protein Kinase Inhibitors ; Protein Kinases ; Pyrroles ; Relaxation

This study was designed to elucidate high-K+induced response of circular and longitudinal smooth muscle from human gastric corpus using isometric contraction. Contraction from circular and longitudinal muscle stripes of gastric corpus greater curvature and lesser curvature were compared. Circular smooth muscle from corpus greater curvature showed high K+ (50 mM)-induced tonic contraction. On the contrary, however, longitudinal smooth muscle strips showed high K+ (50 mM)-induced sustained relaxation. To find out the reason for the discrepancy we tested several relaxation mechanisms. Protein kinase blockers like KT5720, PKA inhibitor, and KT5823, PKG inhibitor, did not affect high K+-induced relaxation. K+ channel blockers like tetraethylammonium (TEA), apamin (APA), glibenclamide (Glib) and barium (Ba2+) also had no effect. However, N(G)-nitro-L-arginine (L-NNA) and 1H-(1,2,4) oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC) and 4-AP (4-aminopyridine), voltage-dependent K+ channel (KV) blocker, inhibited high K+-induced relaxation, hence reversing to tonic contraction. High K+-induced relaxation was observed in gastric corpus of human stomach, but only in the longitudinal muscles from greater curvature not lesser curvature. L-NNA, ODQ and KV channel blocker sensitive high K+-induced relaxation in longitudinal muscle of higher portion of corpus was also observed. These results suggest that longitudinal smooth muscle from greater curvature of gastric corpus produced high K+-induced relaxation which was activated by NO/sGC pathway and by KV channel dependent mechanism.
Apamin ; Barium ; Carbazoles ; Contracts ; Glyburide ; Guanylate Cyclase ; Humans ; Intracellular Signaling Peptides and Proteins ; Isometric Contraction ; Muscle, Smooth ; Muscles ; Protein Kinases ; Pyrroles ; Relaxation ; Stomach ; Tetraethylammonium

PURPOSE: The purpose of this study was not only to evaluate the relative mRNA expression of interleukin-1beta(IL-1beta), cyclooxygenase2 (COX-2) and prostaglandin E2 (PGE2) by RT-PCR analysis but to observe pattern of edema by light microscopic and electron microscope after topical apply of hyaluronic acid in inflammation-guided mouse. MATERIAL AND METHODS: Mice of this study were devided into 4 groups: Control group (no inflammation guided), Positive control (inflammation guided + vaselin apply), Protopic group (inflammation guided + protopic apply), Hyaluronic group (inflammation guided + hyaluronic acid apply). RESULTS: Hyaluronic group showed less expressions of IL-1beta, COX-2, PGE2 than those of positive control & protopic group. Hyaluronic group revealed a decreased inflammation than positive control & protopic group in Light Microscope. Hyaluronic group appeared decreased edema of ear compare to positive control & protopic group in Elecron Microscope. CONCLUSION: It was considered that hyaluronic acid has an antiinflammatory effect for intercepting the gene expression of cytokines related to inflammation.
Animals ; Cytokines ; Dinoprostone ; Ear ; Edema ; Electrons ; Gene Expression ; Hyaluronic Acid ; Inflammation ; Light ; Mice ; RNA, Messenger