Altered expression of retrovirus-like sequences and cellular oncogenes in mice fed methyl-deficient diets.

Methyl-deficient (lipotrope-deficient) diets enhance liver carcinogenesis in rodents. Although the mechanisms responsible for the cancer-promoting activity of such diets have not been identified, they have been observed to cause impaired immune response, alterations in methylation of liver RNA and DNA, and enhanced susceptibility to oxidative damage. Since alterations in gene expression may also play a critical role, the present studies examined the expression of the c-myc, c-H-ras, epidermal growth factor receptor, and ornithine decarboxylase genes, as well as endogenous retrovirus-like sequences, in C57BL/6J x C3H/HeJ F1 mouse liver during the first 2 weeks of feeding of a methyl-deficient diet. The kinetics of liver cell proliferation was investigated in parallel. Increased [3H]thymidine incorporation into liver DNA was found at day 4 and reached a maximum at days 7-11 after commencement of the methyl-deficient diet, when compared to age-matched mice fed a complete diet. Northern blot analysis of polyadenylated liver RNA samples indicated an increase in the levels of RNA homologous to Moloney murine leukemia virus and intracisternal A particle sequences but no significant change in the level of VL30 retrovirus-related RNA in the samples from mice fed methyl-deficient diets. A marked increase in the levels of c-myc and a slight increase in the levels of ornithine decarboxylase and c-H-ras transcripts were seen in the liver RNA samples from the treated mice. Of particular interest was a decrease in the abundance of epidermal growth factor receptor transcripts in the liver RNA samples from the treated mice. These changes in cellular levels of specific RNA resemble, in several respects, those we have previously described in rodent liver during regeneration and tumor promotion and also those seen in rodent hepatomas. They may reflect, therefore, a common profile of gene expression relevant to cell proliferation.

Zinc as modulator of oxygenation function and stabilizer of quaternary structure in earthworm hemoglobin.

Blood of the earthworm Pheretima hilgendorfi contains 10.7 mM Ca, 2.0 mM Mg, 75.5 mM Na, 5.9 mM K, 0.9 mM Zn and 0.3 mM Mn. Some of these cations cannot be removed completely from the blood by dialysis, and 36 atoms of Ca, 1-3 atoms of Mg and 1-2 atoms of Zn per 164 atoms of Fe were retained in purified preparation of the hemoglobin (Hb). At physiological pH, oxygen affinity and the Hill coefficient at half saturation (n1/2 value) of the Hb increased in the presence of 100 mM of Ca, Mg or Na. These effects were in the order of Ca > Mg > Na. At physiological concentration, however, the effect of each of these three cations on oxygenation was rather small. On the other hand, Zn gave a remarkable effect at less than 1 mM. This suggests a possibility that Zn acts as a primal modulator for the oxygenation function of the Hb in vivo. Oxygenation data at various pH values in the presence of each cation strongly suggest that Zn binds to a site different from those for the other three cations. Zn at a concentration of only 1 mM protected considerably the dissociation of the whole molecule to smaller components at alkaline pH and Zn thus contributes to the stabilization of the quaternary structure of the Hb.

Vascular endothelial growth factor and platelet-derived endothelial cell growth factor are frequently coexpressed in highly vascularized human breast cancer.

Vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) are known to be angiogenic growth factors in vitro and in vivo. In this study, we have investigated the relationship between VEGF expression and PD-ECGF expression in human breast cancer tissues using immunocytochemical methods. Of 152 primary breast cancers, 84 (55.3%) and 71 (46.7%) were positive for VEGF and PD-ECGF, respectively. Fifty-three (63. 1%) of 84 VEGF-positive tumors had a PD-ECGF-positive phenotype, whereas only 18 (26.5%) of 68 VEGF-negative tumors had a PD-ECGF-positive phenotype. There was a significant correlation between the VEGF expression and PD-ECGF expression (P < 0.01). As a single factor, VEGF expression and PD-ECGF expression were significantly associated with an increase in the microvessel density assessed by the immunocytochemical analysis using antifactor VIII-related antigen mAb. Interestingly, in addition, of 53 tumors with more than 100 microvessel counts/1 mm2, 40 (75.5%) had both VEGF- and PD-ECGF-positive phenotypes. It was found that VEGF and PD-ECGF were frequently coexpressed in highly vascularized tumors with high microvessel counts. It is suggested that VEGF and PD-ECGF might cooperatively function in the neovascularization of human breast cancer.

Characterization and N-terminal sequence of a 5 kDa polypeptide in the photosystem I core complex from spinach.

Photosystem I core complexes were isolated from spinach photosystem I particles after heat treatment in the presence of 50% (v/v) ethylene glycol (heat/EG treatment). The core complex from 58 degrees C/EG-treated particles was composed of polypeptides with apparent molecular masses of 63, 60 and 5 kDA; this complex contained the iron sulfur center Fx but lacked center FA and FB. The core complex obtained from the 70 degrees C/EG-treated preparation lacked FX and contained a lesser amount of the 5 kDa polypeptide. The N-terminal amino acid sequence of the 5 kDa polypeptide did not correspond to the sequence derived from any possible reading frame in the chloroplast DNA of liverwort or tobacco. Twelve of the first 29 N-terminal amino acids were hydrophobic, suggesting that this protein is intrinsic to the photosystem I reaction center.

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.

Direct detection and amplification of Helicobacter pylori ribosomal 16S gene segments from gastric endoscopic biopsies.

Helicobacter pylori is an organism thought to play an important causative role in gastritis and peptic ulcer diseases. We have designed an RNA dot blot assay for the detection of H. pylori, using as probe a synthetic oligonucleotide complementary to its 16S rRNA. We have also used oligonucleotide primers, complementary to conserved sequences within bacterial ribosomal 16S genes, to amplify a H. pylori ribosomal 16S DNA fragment via the polymerase chain reaction (PCR). After determining the DNA sequence of this amplified H. pylori fragment, primers were designed for specific PCR amplification of H. pylori ribosomal 16S DNA sequences. Samples from clinical endoscopic biopsies were PCR amplified with universal 16S ribosomal primers to detect the presence of bacteria and with H. pylori-specific primers to uniquely detect H. pylori. Finally, by comparing the H. pylori-specific PCR assay to commonly used diagnostic tests, we demonstrate that the molecular technique of PCR amplification shows promising applications for the clinical detection of H. pylori.

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.

Rapid identification of Streptococcus pneumoniae by PCR amplification of ribosomal DNA spacer region.

Streptococcus pneumoniae is one of the important human pathogens in clinical microbiology. A polymerase chain reaction assay was designed to detect and identify S. pneumoniae through amplification of the ribosomal DNA spacer regions between the pneumococcal 16S-23S ribosomal RNA genes. Thirty-two Streptococcus and non-Streptococcus strains were tested to verify the specificity of the assay, and only S. pneumoniae strains gave a positive reaction. This method is a powerful technique for the rapid identification of S. pneumoniae.

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.

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.

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.

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.

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.

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.

[Studies on phage type, beta-lactamase activity and sensitivity of multiple drug-resistant Staphylococcus aureus isolated from clinical specimens to cefmetazole].

The 868 strains of S. aureus were isolated at the Department of Pediatrics, Third Hospital and Aoto Hospital, The Jikei University, School of Medicine and the Kanagawa Prefectural Nursing and Hygienic School Hospital during 6 months from May to October in 1981. From them 66 strains not sensitive to CEZ were selected by a 3-concentration disk method. A total number of 54 strains except for 12 isolated from the same infant patient was examined for their MIC's for 6 drugs, CMZ, CEZ, CTM, CXM, MCIPC and GM. Moreover, phage typing and beta-lactamase activity determination were carried out in them. 1. Antibacterial activity Sixty-six (7.6) of 868 strains were not sensitive to CEZ. The MIC's of CMZ against these resistant strains were between 1.56 and 50 micrograms/ml with a peak between 3.13 and 6.25 micrograms/ml when the 10(5) cells/ml bacterial suspension were inoculated. CMZ was superior in antibacterial activity to CEZ and CXM by about 4 degrees and to CTM by about 3 degrees. MCIPC and GM has higher antibacterial activity against a few strains than CMZ. However, the number of strains with MIC higher than or equal to 50 micrograms/ml was 17 for MCIPC and 40 for GM, but only 2 for CMZ. Thus, the former 2 drugs were far inferior to the latter one. 2. Phage type (1) Nineteen strains (35.2%) had MIC's for CEZ greater than 50 micrograms/ml and CMZ less than 6.25 micrograms/ml. Seventeen of them belonged to the nontypable. (2) Fourteen (25.9%) had MIC's for CEZ greater than 100 micrograms/ml. Of them 9 were allocated to the group III, 3 to the mixed (I + II + III, I + III) group and 2 to the nontypable. (3) Of 20 strains (37.0%) which had MIC's for CEZ greater than 100 micrograms/ml and CMZ less than 6.25 micrograms/ml 5 belonged to the group I, 3 to the group III and 12 to the nontypable. (4) Five strains classified into the group I were all isolated at the Kanagawa Perfectural Nursing and Hygienic School Hospital. (5) Eleven of 15 strains belonging to the group III were isolated at the Third Hospital, The Jikei University, School of Medicine. (6) Seventeen of 21 strains isolated at the Aoto Hospital, The Jikei University, School of Medicine belonged to the nontypable. (7) Phage type was considered to be influenced by difference in areas, infection within hospitals, etc., 3. beta-Lactamase activity beta-Lactamase activity was demonstrated at levels between 0.07 and 3.26 mumol/min/mg in all 54 strains. There was no correlation between MIC for CEZ or CEZ and beta-lactamase activity. It was suggested that beta-lactamase might not contribute to mechanism of resistance of S. aureus to each drug examined.

[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.

[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.

[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.

[Significance of tumor angiogenesis as an independent prognostic factor in axillary node-negative breast cancer].

The value of tumor angiogenesis, EGFR and c-erbB-2 oncoprotein, a long with p 53 protein expression for predicting relapse-free survival was investigated in 110 node-negative breast cancer patients. The grade of neovascularization was assessed by the microvessel density which was obtained by an immunocytochemical staining by factor VIII-related antigen. EGFR, c-erbB-2 oncoprotein and p 53 oncoprotein were also determined by immunocytochemical assay. Univariate analysis showed no statistical significance of EGFR, c-erbB-2 and p53 status as a prognostic indicator. However, the microvessel density was a significant predictor of relapse-free survival. Patients with over 100 counts of factor VIII-RA positive cells per mm2 field in the most active areas of neovascularization showed significantly poorer prognosis compared to those with less than 100 counts (p < 0.005). Multivariate analysis demonstrated that microvessel density was an independent prognostic indicator in node-negative breast cancer patients (p < 0.0005). It was suggested that microvessel density might be of use in selecting the high-risk group in node-negative breast cancer patients needing adjuvant therapies.

[Significance of overexpression of p53 protein in human breast cancer].

Overexpression of p53 protein was examined by immunohistochemical assay. In 143 primary breast cancer patients, consisting of 18 patients with family history of breast cancer or past history of breast cancer and 125 patients with no such histories. In the later 125 patients, overexpression of p53 was shown in 42 patients (33.6%), however in the former 18 patients, p53 overexpression was found in 15 (83.3%). It was suggested that p53 protein is frequently overexpressed in patients with high risks like history of breast cancer and family history of breast. On the other hand, regarding the value of p53 overexpression as a prognostic indicator, an univariate analysis demonstrated that the relapse free survival of patients with over 50% p53 positive all rate was significantly worse compared to that of patients with less than 50% p53 positive all rate (p < 0.05). However multivariate analysis did not demonstrate the significant value (p < 0.1), suggesting that overexpression of p53 is marginal as a prognostic indicator.