Cibenzoline, an ATP-sensitive K(+) channel blocker, binds to the K(+)-binding site from the cytoplasmic side of gastric H(+),K(+)-ATPase.

1. Cibenzoline, (+/-)-2-(2,2-diphenylcyclopropyl-2-imidazoline succinate, has been clinically used as one of the Class I type antiarrhythmic agents and also reported to block ATP-sensitive K(+) channels in excised membranes from heart and pancreatic beta cells. In the present study, we investigated if this drug inhibited gastric H(+),K(+)-ATPase activity in vitro. 2. Cibenzoline inhibited H(+),K(+)-ATPase activity of permeabilized leaky hog gastric vesicles in a concentration-dependent manner (IC(50): 201 microM), whereas no effect was shown on Na(+),K(+)-ATPase activity of dog kidney (IC(50): >1000 microM). Similarly, cibenzoline inhibited H(+),K(+)-ATPase activity of HEK-293 cells (human embryonic kidney cell line) co-transfected with rabbit gastric H(+),K(+)-ATPase alpha- and beta-subunit cDNAs (IC(50): 183 microM). 3. In leaky gastric vesicles, inhibition of H(+),K(+)-ATPase activity by cibenzoline was attenuated by the addition of K(+) (0.5 - 5 mM) in a concentration-dependent manner. The Lineweaver-Burk plot of the H(+),K(+)-ATPase activity shows that cibenzoline increases K(m) value for K(+) without affecting V(max), indicating that this drug inhibits H(+),K(+)-ATPase activity competitively with respect to K(+). 4. The inhibitory effect of H(+),K(+)-ATPase activity by cibenzoline with normal tight gastric vesicles did not significantly differ from that with permeabilized leaky gastric vesicles, indicating that this drug reacted to the ATPase from the cytoplasmic side of the membrane. 5. These findings suggest that cibenzoline is an inhibitor of gastric H(+),K(+)-ATPase with a novel inhibition mechanism, which inhibits gastric H(+),K(+)-ATPase by binding its K(+)-recognition site from the cytoplasmic side.

Staphylococcus aureus in inflammatory bowel disease.

BACKGROUND: The potential role of superantigens in inflammatory bowel disease (IBD), particularly Crohn disease, has been broached in studies of the functions of T cell receptors. Staphylococcal cells have been found in intestinal lymph follicles of IBDs. To clarify a role of staphylococcal superantigens in IBD, we attempted to determine whether Staphylococcus aureus could be detected in intestinal mucosa, including surgical specimens and lymph follicles of initial cases. METHODS: One-hundred-and-six colonic and ileal specimens were obtained from 38 Crohn disease, 25 ulcerative colitis and 36 non-IBD patients through therapeutic surgery or endoscopic biopsy. In Crohn disease, 23 surgical specimens and 11 biopsy specimens from initial cases were included. DNA was extracted with phenol-chloroform after homogenization and proteinase K treatment in 73 mucosal specimens. Using an inverted microscope, lymph follicle tissue was microdissected from the remaining 33, mostly biopsy, specimens. DNA was then extracted by freeze-thawing. A coagulase gene characteristic of S. aureus was sought. A nested polymerase chain reaction was performed utilizing primers that amplify a region of the coagulase gene. Polymerase chain reaction products were analyzed with polyacrylamide gel electrophoresis. RESULTS: Only one surgically resected colonic specimen, from a 42-year-old male ulcerative colitis patient, registered positive staphylocoagulase amplification. CONCLUSIONS: Staphylococcal superantigens are not involved in either the early lesions or the established lesions of Crohn disease. However, S. aureus infection occasionally may occur during the course of IBD.

[New evidence for detecting Helicobacter pylori in clinics].

A part of diagnostic tests for Helicobacter pylori was approved as National Health Insurance system in Ministry of Health and Welfare Japan on last November 2000. The gold standard for the presence of most infectious diseases is successful culture of the organism. H. pylori is fastidious and time consuming for growth in media. Instead of bacterial culture of H. pylori from biopsy sample, we need to effort to develop rapid methods to identify gene segment of H. pylori and its antibiotic resistance. Even the genetic methods would be developed, circular antigen product of H. pylori must be important for diagnosis of its clinical pathology. Here we listed the available diagnostic tests for histology, bacterial culture, rapid urease test, urea breath testing, and serological testing for H. pylori in Japan.

Tea catechins inhibit cholesterol oxidation accompanying oxidation of low density lipoprotein in vitro.

Endogenous oxidized cholesterols are potent atherogenic agents. Therefore, the antioxidative effects of green tea catechins (GTC) against cholesterol oxidation were examined in an in vitro lipoprotein oxidation system. The antioxidative potency of GTC against copper catalyzed LDL oxidation was in the decreasing order (-)-epigalocatechin gallate (EGCG)=(-)-epicatechin gallate (ECG)>(-)-epicatechin (EC)=(+)-catechin (C)>(-)-epigallocatechin (EGC). Reflecting these activities, both EGCG (74%) and ECG (70%) inhibited the formation of oxidized cholesterol, as well as the decrease of linoleic and arachidonic acids, in copper catalyzed LDL oxidation. The formation of oxidized cholesterol in 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH)-mediated oxidation of rat plasma was also inhibited when the rats were given diets containing 0.5% ECG or EGCG. In addition, EGCG and ECG highly inhibited oxygen consumption and formation of conjugated dienes in AAPH-mediated linoleic acid peroxidative reaction. These two species of catechin also markedly lowered the generation of hydroxyl radical and superoxide anion. Thus, GTC, especially ECG and EGCG, seem to inhibit cholesterol oxidation in LDL by combination of interference with PUFA oxidation, the reduction and scavenging of copper ion, hydroxyl radical generated from peroxidation of PUFA and superoxide anion.

Detection of PCR products of Escherichia coli O157:H7 in human stool samples using surface plasmon resonance (SPR).

A method for the rapid detection of verotoxin-producing Escherichia coli O157:H7 in stools was evaluated. Strains possessing Shiga toxin-2 (stx-2) genes were isolated from stool samples and amplified using oligonucleotide primers. Stools spiked with cultured E. coli O157:H7 (strain 298 or strain 1646) were detected to be polymerase chain reaction (PCR) positive at 10(2) cfu per 0.1 g of stool. Stool samples from patients and healthy carriers showed a high correlation between positive results for a PCR and the presence of verotoxin-producing E. coli O157:H7, confirmed by isolation of serotype O157:H7 on sorbitol MacConkey medium (10 of 10 stool samples). These PCR products could be detected using a BIAcore 2000 surface plasmon resonance device using peptide nucleic acid as a sensor probe. In this report we use this method for the rapid detection of DNA from significant pathogenic organisms.

Presence of bacterial 16S ribosomal RNA gene segments in human intestinal lymph follicles.

BACKGROUND: There is currently no information regarding microbial agents inside the intestinal lymph follicles. METHODS: Biopsy or resected specimens, mostly from macroscopically normal areas, were sectioned with a cryostat. DNA was extracted from microdissected samples, exclusively from the lymph follicle. Amplification of DNA was performed using universal primers designed from conserved regions of bacterial 16S ribosomal RNA (rRNA). Several clones with inserts of around 400 base pairs were subjected to DNA sequence analysis followed by a database homology search. RESULTS: Bacterial 16S rRNA gene segments were detected in the lymph follicle in 2 of 14 (14%) non-inflammatory bowel disease (IBD) cases, 4 of 14 (28%) Crohn disease cases, and in 2 of 5 (40%) ulcerative colitis cases. Nineteen 16S rRNA gene segments were recognized in the eight positive cases. Five segments showed 100% identity to known bacterial 16S rRNAs, namely staphylococcus species, Streptococcus sanguis, and Paracoccus marcusii. However, the other 14 segments showed below 100% identity, indicating either the presence of unknown bacteria or of bacteria without known DNA data. No single identified or unidentified bacterium, characteristic of IBD, including Mycobacterium paratuberculosis and Listeria monocytogenes, was detected. CONCLUSIONS: The present study confirms the presence of bacterial 16S rRNA gene segments in human intestinal lymph follicles and paves the way for new investigations into the microbiology of the lymph follicle. Whether or not bacteria inside the lymph follicle is a primary stimulus in IBD has yet to be clarified.

Cholesterol oxidation in meat products and its regulation by supplementation of sodium nitrite and apple polyphenol before processing.

The levels of cholesterol oxidation derivatives (OxChol) in eight commercial species of meat products were examined. These products contained more than 1 mg/100 g of OxChol, and 7beta-hydroxycholesterol + 5beta-epoxycholesterol (111-1092 microg/100 g), 5alpha-epoxycholesterol (80-712 microg/100 g), cholestanetriol (0-368 microg/100 g), and 7-ketocholesterol (708-1204 microg/100 g) were detected. To know the interaction of sodium nitrite supplementation against cholesterol oxidation in meat products, sausage was produced with or without varying levels of sodium nitrite and stored in the refrigerator for 15 days. As a result, cholesterol oxidation in sausage was inhibited by addition of sodium nitrite in a dose-dependent manner. This observation may be associated with inactivation of O(2)(-) radical and stabilization of polyunsaturated fatty acids (PUFAs). In fact, the levels of OxChol in sausage increased, accompanying the decrease of coexisting linoleic acid when sodium nitrite was not added to sausage meat. Thus, cholesterol oxidation in meat products seems to be considarably promoted by the oxidation of coexisting PUFAs. On the other hand, additive apple polyphenol also inhibited linoleic acid oxidation in sausage and then suppressed cholesterol oxidation through its radical scavenging effects. Therefore, apple polyphenol, having a large amount of an oligomer of catechin, may interfere with cholesterol oxidation in meat processing or storage of meat products through its antioxidative action and be useful as a new antioxitant for meat products when it is added to the original meat before processing.

[Measures for the disposal of non-regulated alternative medical wastes--cloned DNA of amplified DNA as waste materials].

Cloned DNA of amplified DNA, synthesized oligomer DNA and peptide nucleic acid is a candidate for hazardous medical waste material. The numerous identical base sequence of DNA has a risk associated with its handling in a laboratory and medical waste. To avoid the risk SD box, NaOCl, filtered chip of micropipette and a clean-bench are recommended for waste management.

Measuring respiration of cultured cell with oxygen electrode as a metabolic indicator for drug screening.

New trend in methods for assessing pharmacological action to bacteria and cell is to measure their metabolic activities induced, while the conventional methods used population growth. We focused on respiration volume as an indicator of cell metabolism, and developed inexpensive disposable oxygen electrode sensor and multi-channel dissolved oxygen meters (DOX-10 and DOX-96KB). Using these instruments, cytotoxicity was measured for 48 hrs and the method showed superior features to conventional methods in its handiness of one step assay, and excellent adaptability to automated systems. Total usability of this oxygen electrode method is being evaluated in bacterial drug susceptibility test, anticancer drug susceptibility test, and alternatives to animal experiment.

A chimeric gastric H+,K+-ATPase inhibitable with both ouabain and SCH 28080.

2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward that of Na+,K+-ATPase.

Rapid detection of the gene of Legionella pneumophila using the fluorescence polarization with the asymmetric PCR.

We attempted the rapid detection method of Legionella pneumophila by the asymmetric PCR and the fluorescence polarization. Eleven extracted DNAs from L. pneumophila serogroup 1 to approximately 6, L. bozemanii, L. dumoffii, L. gormanii, L. micdadei, and Pseudomonas aeruginosa were amplified by asymmetric PCR, and the polarization of those products were measured. Only the polarization of L. pneumophila serogroup 1 to approximately 6 rose within a few minutes after the beginning of measurement. The sensitivity to L. pneumophila using this method was 10(3) cells.

Microbiology of the intestinal lymph follicle: a clue to elucidate causative microbial agent(s) in Crohn's disease.

It has been suggested that microbial agent(s) are involved in the onset of Crohn's disease. None of the candidates, however, has been unequivocally demonstrated to be a causative agent. The macroscopically earliest lesion takes place in the lymph follicle, irrespective of the initial attack or relapse in Crohn's disease. Human leucocyte antigen-DR (HLA-DR) antigens are expressed on the epithelium around the lymph follicle even in areas endoscopically uninvolved in Crohn's disease. These observations make the lymph follicle critical in the onset of Crohn's disease. The lymph follicle is a port of entry of a variety of microbial agent(s), leading to the speculation that microbial agent(s) exist in the lymph follicle. Polymerase chain reaction (PCR) using universal primers designed from conserved regions of bacterial ribosomal RNA or techniques such as representational difference analysis, may well identify microbial agent(s) in the lymph follicle that are specific to Crohn's disease. The existence of bacteria in the lymph follicle is here indicated by preliminary studies.

Functional expression of putative H+-K+-ATPase from guinea pig distal colon.

A guinea pig cDNA encoding the putative colonic H+-K+-ATPase alpha-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or Torpedo Na+-K+-ATPase beta-subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 microM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the beta-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (alpha-subunit) of the colonic H+-K+-ATPase. Although the beta-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase beta-subunits were found to act as a surrogate for the colonic beta-subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC50 = 0.4-1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).

Hepatitis B virus variants in patients with acute hepatitis in whom various clinical forms develop.

Ten patients who suffered from acute hepatitis with various clinical forms due to hepatitis B virus (HBV) were studied. HBV variants with a mutation in the precore region were dominant in two patients with fulminant hepatitis and in a patient with the most severe acute hepatitis. However, these mutant viruses were not detected in a patient who had the fulminant form of acute HBV infection on chronic liver damage or in most patients who had severe acute hepatitis. Furthermore, mutant viruses were also not detected in a patient with complicating myopathy and in one who had an atypical clinical course with three transaminase peaks. These results suggest that precore mutants may be involved in the pathogenesis of some cases of severe acute hepatitis, the same as for fulminant hepatitis, but not in other clinical forms of acute hepatitis.

Simultaneous detection of Streptococcus pneumoniae and Haemophilus influenzae by nested PCR amplification from cerebrospinal fluid samples.

Haemophilus influenzae and Streptococcus pneumoniae are often the cause of serious diseases such as meningitis. We designed a nested PCR assay to identify these pathogens from cerebrospinal fluid samples. The first-step PCR was able to detect eubacterial rRNA genes with a unified set of universal primers. In the second-step PCR, the identification primers, HI I and II and SP I and II, could detect H. influenzae and S. pneumoniae respectively through amplification of the rRNA spacer between the 16S and 23S rRNA genes. We suggest that the two-step PCR assay can be used as a novel method for the immediate and retrospective diagnosis of bacterial meningitis caused by H. influenzae and S. pneumoniae.

Rapid identification and typing of Staphylococcus aureus by nested PCR amplified ribosomal DNA spacer region.

We designed a polymerase chain reaction (PCR) assay for rapid detection of prokaryotic 16S-23S spacer regions. This PCR assay consisted of nested DNA amplifications. The first-step PCR was able to detect the general presence of eubacteriales with a unified set of universal primers. The universal primers were selected from highly conserved regions in 16S and 23S ribosomal RNA (rRNA) genes and amplified DNAs from all 62 different species of bacteria tested. In the second-step PCR, the identification primers could detect four important bacterial species through amplification of the rRNA spacer regions between the 16S-23S rRNA genes. For Staphylococcus aureus, intraspecies variation in spacer amplification products was observed with S. aureus specific primers. We suggest that the nested PCR assay could be used as a novel method for the identification and typing in epidemiological studies of S. aureus.

[Diagnostic microbiology of DNA in septicemia].

Bacterial species encode rRNAs that are functionally and evolutionarily conserved. The nucleotide sequences of bacterial 16S ribosomal RNAs (16S rRNAs) diverge in regions of around 1,400 bases. Bacterial 16S rRNAs possess nine or more conserved "universal" sequences. Between each conserved region there are species specific sequences. These regions provide targets for clinical detection and diagnoses based on molecular hybridization for the polymerase chain reaction. We designed one base discriminating primer in 16S rRNA for the specific amplification of these regions and the primers in 16S-23S spacer regions of the rrn operon. These DNA-amplification based diagnosis of the septicemia showed multiple bacterial infection and indicated uncommon species of bacteria in the blood.