Presence of Streptococcus DNA sequence in surgical specimens of gastric cancer

In Southern blot analysis using Mycoplasma 16S ribosomal DNA (rDNA) as a probe, positive signals were detected in DNA samples from surgical specimens of gastric cancers. The DNA that hybridized to Mycoplasma 16S rDNA was eluted from the gel, cloned and sequenced. The cloned sequence was identical to 16S rDNA of Streptococcus anginosus. In Southern blot analysis with the S. anginosus 16S rDNA fragment as a new probe, positive signals were detected in 9 (20%) out of 43 cases of gastric cancer.     

Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?

The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.     

Type D retrovirus markers in healthy Africans from Guinea

Sixteen matching sera and DNA samples from healthy African blood donors living in rural areas of Guinea were analysed for the presence of type D retrovirus markers. Screening for the antibodies against structural proteins of Mason-Pfizer monkey virus (M-PMV) was carried out by Western blot with a purified M-PMV as an antigen. Eight out of 16 sera samples were found to contain antibodies against at least two gag gene-coded proteins, and three of these were weakly positive against env gene-coded protein. Using PCR amplification and Southern hybridization, we detected M-PMV-like gag sequences in 11 out of 16 samples and env-related sequences in 8 out of 16 samples. Six DNAs were found to contain both M-PMV gag- and env-related sequences. Restriction endonuclease analysis of the PCR-amplified gag sequences from two individuals and direct DNA sequencing analysis of the amplimers confirmed their M-PMV-like origin. Detection of antibodies and M-PMV-related sequences in blood donors from Guinea, but not in French or Algerian blood donors, indicated exogenous SRV infection in humans from certain geographic areas of Western Africa.     

Antioxidant systems in rat epididymal spermatozoa

Down-regulation of KAI1 mRNA expression has been shown to be associated with the formation of metastases or disease progression in pancreatic cancer. Whether KAI1 possesses similar characteristics in other malignancies of the gastrointestinal tract is not known. Here, we compared the patterns of KAI1 mRNA expression in 41 esophageal cancers and 35 stomach cancers to assess whether KAI1 might also be of biological relevance in the metastatic ability of these tumors. By Northern blot analysis, KAI1 mRNA levels ranged widely in both normal and cancerous esophageal and gastric tissue samples, with no statistical differences. No association between KAI1 mRNA expression and tumor stage or tumor differentiation was seen in these tumors. In addition, KAI1 mRNA expression was similar in primary esophageal and gastric cancer samples with or without metastases. By in situ hybridization, KAI1 mRNA expression was evident in the cytoplasm of most squamous epithelial cells in the normal esophagus and in nonmucosal epithelial cells of the normal stomach. The staining intensity in the esophageal and gastric cancer cells was similar to that in the normal controls. This differential pattern of KAI1 mRNA expression in esophageal and gastric cancers in comparison to pancreatic cancer indicates that KAI1 seems to influence the potential of gastrointestinal cancer cells to metastasize differently. In esophageal and gastric cancers, the formation of metastases is not dependent on the reduction of KAI1 in the cancer cells.     

Two cases of gastric involvement of esophageal cancer: intramural metastasis and intramural lymph node metastasis

Cases of esophageal cancer with intramural metastasis to the stomach and esophageal cancer with metastasis to an intramural lymph node of the stomach are herein reported. The former patient was a 52 year-old male. Squamous cell carcinoma (SCC) of the lower esophagus with an intramural metastasis located at the gastric cardia and a small leiomyoma at the fornix were resected. The latter patient was a 70 year-old female. SCC of the lower esophagus and an intramural lymph node metastasis located at the anterior wall of the gastric cardia were resected. The patient died nevertheless of multiple liver metastases. These gastric involvements were detectable by endoscopy before surgery. The clinicopathological features of these esophageal cancers were characterized; the sites were the lower part of the esophagus, and extreme lymphatic and vascular invasions were shown histologically. The gastric tumors were located in the upper third of the stomach, and the findings revealed submucosal tumors. It is therefore important to discriminate other gastric tumors, and to resect them simultaneously with esophageal cancer when a gastric tube has been used for reconstruction after esophagectomy.     

Isolation and identification of DNA fragments frequently deleted from tumor cell lines of human esophageal cancer

OBJECTIVE: To clone tumor suppressor genes associated with human esophageal cancer (EC). METHODS: DNA fragments deleted from cell lines of esophageal cancer EC8712 and EC8733 were isolated by using modified genomic substractive hybridization. Twenty EC tissues paired with adjacent normal mucosa were analysed by Southern blot and PCR with these fragments as probes. RESULTS: Seven DNA fragments deleted from EC cell lines obtained were named as 12H1, 12H2, 33H3, 33H4, 12B1, 33B2 and 33B3. Sequencing revealed their size of 512bp, 428bp, 509bp, 355bp, 680bp (partially sequencing), 519bp and 425bp, respectively. 12H1 and 33H3 showed high homology (98%) to a new repeated sequence in human genome. 33H4 displayed 87% nucleotide identity with human alphoid-like repetitive sequence located on chromosome 17. 12B1 was homologous (90%) to LINE 1 transoposon containing two open reading frames. 12H2, 33B2 and 33B3 were novel sequences. These DNA fragments were deleted in 20%-60% of EC tissues and in 10%-20% of adjacent mucosa. CONCLUSIONS: Loss of 33H4 may be accompanied by deletion of an adjacent gene. 12B1 as transposon may suppress the tumorigenesis by activating suppressor genes or/and by inactivating oncogenes. 12H1/33H3, 12H2, 33B2 and 33B3 may be candidates of tumor suppressor genes associated with the development and the progression of esophageal cancer.     

Potential mechanism(s) involved in the regulation of glycogen synthesis by insulin

The aim of this study was to elucidate the significance of aberrant DNA methylation in gastric carcinogenesis. The DNA methylation status at the D17S5 locus, at which a candidate tumor suppressor gene, HIC-1, was identified, of gastric cancers and non-cancerous gastric mucosae from 42 gastric cancer patients was examined by Southern blotting using a methylation-sensitive restriction enzyme. DNA hypermethylation was observed in 15, 13, 25 and 45% of the tissues showing no remarkable histological findings, chronic gastritis without intestinal metaplasia, intestinal metaplasia and gastric cancer, respectively. The incidence of DNA hypermethylation was significantly higher in gastric cancers than in non-cancerous gastric mucosae (P < 0.05). DNA hypermethylation was often accompanied by allelic loss at the same locus in gastric cancers. DNA hypermethylation at the D17S5 locus, which was even detected in precancerous conditions, including intestinal metaplasia, may play a role in gastric carcinogenesis.     

Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction

A polymerase chain reaction (PCR)-based test was developed for the detection of Salmonella typhi in the blood specimens from patients with typhoid fever. Two pairs of oligonucleotide primers were designed to amplify a 343-bp fragment of the flagellin gene of S. typhi. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization by using a 32P-labeled 40-base probe internal to the amplified DNA. The nested PCR with two pairs of primers could detect 10 organisms of S. typhi as determined by serial dilutions of DNA from S. typhi. The peripheral mononuclear cells from 11 of 12 patients with typhoid fever confirmed by blood culture were positive for DNA fragment of the flagellin gene of S. typhi, whereas 10 blood specimens of patients with other febrile diseases were negative. With the nested PCR, S. typhi DNAs were detected from blood specimens of four patients with suspected typhoid fever on the basis of clinical features but with negative cultures. We suggest that the PCR technique could be used as a novel diagnostic method of typhoid fever, particularly in culture-negative cases.     

Chromosome breakpoint distribution in nonmelanoma skin cancers

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330(233) primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood.     

Human papillomavirus genotype as a major determinant of the course of cervical cancer

PURPOSE: To determine whether the prognosis of invasive cancers of the uterine cervix is related to the type of human papillomavirus (HPV) associated with the tumor. PATIENTS AND METHODS: Two hundred ninety-seven patients with invasive cervical cancer were prospectively registered from 1986 to 1994. HPV typing was performed on DNA extracted from frozen tumor specimens by means of Southern blot hybridization (SBH) and polymerase chain reaction (PCR) techniques. The median follow-up was 38 months. RESULTS: HPV sequences were detected in 246 patients (83%): 150 patients had HPV16, 31 patients had HPV18, and 14 patients had one of the intermediate-oncogenic-risk HPV types (HPV31, 33, 35, 52, 58). In 51 patients, HPV type remained undetermined, and in 51 patients, no viral sequences were found. No significant associations were observed between virologic data and tumor stage or node status. The 5-year disease-free survival (DFS) rate was 100% for patients with intermediate-risk HPV-associated tumors, 58% for patients with HPV16-positive tumors, and 38% for patients with HPV18-positive tumors (P = .02). In multivariate analysis, patients with HPV18-associated tumors had a relative risk (RR) of death 2.4 times greater (95% confidence interval [CI], 1.29-4.59) than that for patients with HPV16, and 4.4 times greater (95% CI, 3.48-5.32) than that for patients with a tumor associated with a viral type different from HPV16/18. CONCLUSION: The prognosis for invasive cancers of the uterine cervix is dependent on the oncogenic potential of the associated HPV type. HPV typing may provide a prognostic indicator for individual patients and is of potential use in defining specific therapies against HPV-harboring tumor cells.     

Differentiation of Arthroderma spp. by random amplification of polymorphic DNA (RAPD) and Southern hybridization

To develop the molecular differentiation analysis of dermatophytes, we carried out RAPD and Southern hybridization analyses using genomic DNAs of six Arthroderma species, including A. fulvum, A. grubyi, A. gypseum, A. incurvatum, A. otae and A. racemosum. The RAPD analysis gave different band patterns specific to each of the six Arthroderma fungi. However, minor differences in the banding patterns were observed between the strains of plus (+) and minus (-) mating types of A. gypseum, A. fulvum and A. incurvatum. Southern blot analysis using a probe (1S) obtained from A. grubyi DNA gave specific bands only in the DNA samples of A. grubyi and A. incurvatum. On the other hand, Southern blot analysis using a probe (C3) obtained from A. otae DNA gave specific bands in all six Arthroderma species examined, and the size of the bands were specific to each species. These findings indicate that RAPD and Southern hybridization analyses are useful in the differentiation of these Arthroderma species.     

Lymph node dissection for T1 esophageal cancer

Proper mucosal cancer of esophagus of esophageal has no lymph node metastasis, and lymph node metastasis occurs when the tumor invades to muscularis mucosa. Submucosal cancer of esophagus has lymph node metastasis in the rate of 44.4% (40/90). The incidence and number of metastatic lymph node increase with the depth of invasion. Lymph node metastasis of esophageal cancer spreads widely to cervix, mediastinum and abdomen. It's same in submucosal cancer and first metastasis occurs also appears at everywhere from cervix to abdomen. There are high rate of lymph node metastasis in 101L, 105, 106rR, 106rL, 108, 110, 1, 2, 3, 7 lymph nodes. The cancer in upper thoracic esophagus has high rate of lymph node metastasis in cervix and upper mediastinum and lymph node metastasis of lower thoracic esophageal cancer is liable to appear in lower mediastinum and abdomen. Then the cancer in middle thoracic esophagus should be performed the lymph node dissection in cervix, mediastinum and abdomen, especially 101, 102m, 104, 105, 106r, 106t, 107, 108, 110, 1, 2, 3, 7 lymph nodes. On the other hand, cancers limited to proper mucosal layer should be treated with endoscopic mucosal resection. And its same as in the greater part of cancers invaded to muscularis mucosa and shallow layer of esophageal submucosa. The 5 year survival rate of T1 cancers of esophagus is 85.6%, which were performed surgical treatment.     

Characteristics and clinical outcome of proximal-third gastric cancer

BACKGROUND: It is generally accepted that the prognosis of patients with proximal gastric cancer (PGC) is worse than that of patients with more distal gastric cancer. STUDY DESIGN: The aim of this study was to compare the clinical features and outcomes of PGC with those of middle- and distal-third gastric cancers. A total of 646 primary gastric cancers was analyzed as a retrospective study. RESULTS: Proximal gastric cancer occurred in 21.8% of the 646 cancers analyzed, and approximately 21% of PGCs had esophageal invasion. The 5-year survival rate for patients with PGC was significantly lower than that of patients with more distal tumors. When the PGC group was divided into patients with esophageal invasion and without esophageal invasion, patients with esophageal invasion had significantly worse outcomes. When corrected for depth of invasion, lesions with esophageal invasion had significantly worse outcomes than those of other sites in T2 curative cancers. Proximal gastric cancer with esophageal invasion was characterized by a larger tumor, deeper penetration, and a higher incidence of lymph node metastasis compared with tumors in other sites, and in multivariate analysis of all curative cases, these variables were independent prognostic factors for survival. The frequency of positive proximal margins of PGC was higher than those of other sites. CONCLUSIONS: The relatively poor prognosis associated with PGC is mainly from advanced tumor stages of esophageal invasion. Early detection is the most important strategy to improve the survival of patients with PGC. In addition, aggressive lymph node dissection and chemotherapy for esophageal invasion should be considered even if the tumor invasion is moderate (T2 tumor), and a tumor-free margin is important.     

Risk of esophageal and gastric adenocarcinomas in relation to use of calcium channel blockers, asthma drugs, and other medications that promote gastroesophageal reflux

Incidence of adenocarcinomas of the esophagus and gastric cardia has risen dramatically over the past 2 decades in the U. S., for reasons that are not yet clear. A number of common medications (e.g., calcium channel blockers, tricyclic antidepressants, and certain asthma medications) promote gastroesophageal reflux by relaxing the lower esophageal sphincter (LES). Reflux is thought to increase cancer risk by promoting cellular proliferation, and by exposing the esophageal epithelium to potentially genotoxic gastric and intestinal contents. Recent studies have suggested that calcium channel blockers may also increase cancer risk by inhibiting apoptosis. Using personal interview data from a multicenter, population-based case-control study conducted between 1993 and 1995 in three areas of the U. S., we evaluated whether the use of LES-relaxing drugs was associated with increased risk of adenocarcinomas of the esophagus and gastric cardia. Cases of esophageal adenocarcinoma (n = 293) and gastric cardia adenocarcinoma (n = 261) were compared with general population controls (n = 695). Information on additional case groups of esophageal squamous cell carcinoma (n = 221) and noncardia gastric cancer (n = 368) were also available for comparison. Overall, 27.4% of controls had used one or more of these drugs for at least 6 months, compared with 30.2% of esophageal adenocarcinoma and 23.8% of gastric cardia adenocarcinoma cases. The adjusted odds ratios (ORs) for ever use were 1.0 [95% confidence interval (CI) = 0.7-1.5] and 0.8 (95% CI = 0.5-1.1), respectively. There was little evidence of increasing risk with increasing duration of use of all LES-relaxing drugs together. We found an increased risk of esophageal adenocarcinoma among persons reporting use of asthma drugs containing theophylline (OR = 2.5; 95% CI = 1.1-5.6) or beta agonists (OR = 1.7; 95% CI = 0.8-3.8). Risks were higher among long-term users (>5 years) of these drugs (OR = 3.1; 95% CI = 0.9-10.3 and OR = 2.3; 95% CI = 0.8-7.0, respectively). In contrast, there was no evidence that the use of calcium channel blockers or other specific groups of drugs increased the risk of any of the cancers studied. These results provide reassuring evidence that the increases in incidence of adenocarcinomas of the esophagus and gastric cardia are not likely to be related to the use of LES-relaxing drugs as a group, or calcium channel blockers in particular, but they do suggest that persons treated for long-standing asthma may be at increased risk of esophageal adenocarcinoma.     

No evidence of HTLV-I proviral integration in lymphoproliferative disorders associated with cutaneous T-cell lymphoma

Several recent studies have reported detection of HTLV-I genetic sequences in patients with cutaneous T-cell lymphoma (CTCL) including mycosis fungoides and Sezary syndrome. The purpose of this study was to determine whether HTLV-I was detectable in lesional tissues of patients suffering from diseases known to be associated with CTCL. Thirty-five cases were obtained from diverse geographical locations including Ohio, California, Switzerland, and Japan. Six of them had concurrent CTCL. Cases were analyzed using a combination of genomic polymerase chain reaction (PCR)/ Southern blot, dot blot, and Southern blot analyses. All assays were specific for HTLV-I provirus. Sensitivity ranged from approximately 10(-6) for PCR-based studies to 10(-2) for unamplified genomic blotting. Lesional DNA from patients with lymphomatoid papulosis (fourteen cases), Hodgkin's disease (twelve cases), and CD30+ large-cell lymphoma (nine cases) was tested for the HTLV-I proviral pX region using a genomic PCR assay followed by confirmatory Southern blot analysis with a nested oligonucleotide pX probe. All cases were uniformly negative. All of the Hodgkin's disease cases, eight of the large-cell lymphoma cases, and six of the lymphomatoid papulosis cases were then subjected to dot blot analysis of genomic DNA using a full-length HTLV-I proviral DNA probe that spans all regions of the HTLV-I genome. Again, all cases were negative. Finally, eleven of the Hodgkin's disease cases were also subjected to Southern blot analysis of EcoRI-digested genomic DNA using the same full-length HTLV-I probe. Once again, all cases were negative. These findings indicated that, despite utilization of a variety of sensitive and specific molecular biological methods, HTLV-I genetic sequences were not detectable in patients with CTCL-associated lymphoproliferative disorders. These results strongly suggest that the HTLV-I retrovirus is not involved in the pathogenesis of these diseases.     

28 K in squamous metaplasia of the bladder in patients with spinal cord injury

Actinobacillus actinomycetemcomitans has been strongly implicated in the etiology of localized juvenile periodontitis. Techniques used in the identification of this periodontal pathogen include cultural, biochemical, immunological and DNA hybridization analysis. In this study, we report the use of polymerase chain reaction (PCR) to amplify unique sequences of A. actinomycetemcomitans. Specific oligonucleotide primers LKT2 and LKT3 were designed to hybridize to the A. actinomycetemcomitans lktA gene, which encodes leukotoxin, a putative A. actinomycetemcomitans virulence factor. The LKT2 and LKT3 primers amplified lktA-specific sequences from all 12 A. actinomycetemcomitans strains tested. In another set of experiments, 13 other bacterial species, most of which are normal residents of the oral cavity, were tested with these primers. These PCR amplifications also contained 2 additional primers, RRN4 and RRN5, which served as positive controls; RRN4 and RRN5 were designed to amplify specific sequences of eubacterial 16S ribosomal DNA (rDNA). PCR amplifications of all bacterial species tested, including A. actinomycetemcomitans, yielded 16S rDNA-specific DNA fragments. Furthermore, each bacterial species tested, with the exception of A. actinomycetemcomitans, failed to amplify lktA sequences. The LKT and RRN primers were used in further PCR experiments to detect A. actinomycetemcomitans directly from gingival fluid samples. The results clearly demonstrate the simplicity, rapidity, specificity and accuracy of the LKT primers in the identification of A. actinomycetemcomitans.     

Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.