Aim: In Okinawa, a subtropical island located between the East China Sea and Pacific Ocean, 2000 km south of mainland Japan, the incidence of oral squamous cell carcinoma is 1.5 times higher than that seen in mainland Japan, and a large number of these patients have been reported to be infected with the Epstein-Barr virus (EBV). This study aimed to gain a better understanding of the pathogenesis of this malignancy in this area by carrying out genomic analysis of EBV. Methods: Fifty four patients with oral squamous cell carcinoma reported from 1997 to 1999 in Okinawa were compared with 21 and 20 patients from Kitakyushu and Kumamoto in Kyushu, mainland Japan, respectively. Diagnosis was confirmed by conventional histological examination of paraffin wax sections. EBV was detected by non-isotopic in situ hybridisation (NISH) and the polymerase chain reaction (PCR) (Bam HI-F, EBV nuclear antigen 2 (EBNA2), and latent membrane protein 1 (LMP-1) regions). Sequence analysis of the PCR products was also carried out. Results: In Okinawa, 25 patients were found to be infected with EBV type A by analysing the 3′ sequence divergence of the EBNA2 genes. Six patients were positive for EBV type B, and eight for both type A and B. Therefore, type A virus infection was demonstrated in 33 of 54 patients, and type B in 14 of 54. In total, 39 of 54 patients were infected with EBV. However, the "f" variant was shown in only one patient, who was also infected with type A virus. In contrast, 97.0% of EBV type A infected patients showed a 30 bp deletion of the LMP-1 gene, but those infected with EBV type B did not. Sequence analysis of the type A virus EBNA2 gene revealed slight variations of the sequence (mutations)-48991 G→T and 48998 C→A-in 18 of 33 cases compared with the B95-8 strain, and in 14 cases, in addition to these, a further mutation of 48917 T→C was demonstrated; in the single remaining case, only one mutation at 49137 A→G was detected. The mutations at 48991 (G→T), and 49137 (A→G) are associated with amino acid changes Arg→Met and Thr→Ala, respectively. In contrast, no mutation was seen in the EBNA2 DNA from the 14 cases of type B virus when compared with that of the Jijoye strain. In Kitakyushu and Kumamoto, only 10 of 41 patients (six in Kitakyushu and four in Kumamoto) were infected with EBV. Among them, nine patients were infected with type A virus, and only one patient from Kitakyushu was infected with type B virus. The 48991 G→T and 48998 C→A mutations of the EBNA2 region were demonstrated in type A virus, but the 48917 T→C and 49137 A→G mutations were not when compared with the B95-8 strain. In the case of type B virus no mutation was noted. A 30 bp deletion was found in these nine cases of type A, but not in type B. The sequence analysis of EBV type A in Okinawa, Kitakyushu, and Kumamoto showed slight variations when compared with B95-8, but EBV type B LMP-1 did not when compared with the Jijoye strains. Conclusion: In Okinawa, EBV infection was frequently demonstrated in oral squamous cell carcinoma (p < 0.001)....

Mycoplasma penetrans isolated from clinical specimens of AIDS patients showed potent activity in tumor necrosis factor alpha (TNFα) production in THP‐1, U937 and J22HL60 cell lines, and in the enhancement of HIV‐1 replication in a dormantly‐infected J22HL60 cell line as compared with the activities of other mycoplasmas. Both activities were found in the methanol layer but not in the chloroform layer of the membrane extracted by the Bligh‐Dyer method. TNFα production was observed in the peritoneal macrophages from both lipopolysaccharide‐responsive and ‐unresponsive mouse strains, and was not inhibited by polymyxin B. The induction of TNFα production and enhancement of HIV‐1 replication were strongly inhibited by Concanavalin A‐Sepharose. The inhibitory effect of Concanavalin A‐Sepharose was partially prevented by sugars in the order methyl‐α‐D‐mannopyranoside and methyl‐α‐D‐glucopyranoside but not methyl‐α‐D‐galactopyranoside. Anti‐human TNFα antibody, however, did not reduce the activity of the methanol layer to enhance HIV‐1 replication, suggesting that the methanol layer could enhance HIV‐1 replication directly. These results suggest that the carbohydrate derived from M. penetrans might be responsible for the progression of HIV‐1 infection.

Many individuals are chronically infected or parasitically colonized with mycoplasmas in their respiratory or urogenital tracts without apparent clinical significance. However, prolonged close interaction between prokaryotic agents and eukaryotic host cells may gradually and significantly alter normal biological or physiological properties of infected hosts. Steroid hormones are associated with rates of cancer formation in human. The purpose of this study is to establish a sensitive reporting system to examine whether mycoplasmal infections affect biological responses to steroid hormones in mammalian cells. We established pMTV-CAT stably transfected cell lines to test the effect of mycoplasmal lipid-associated membrane proteins (LAMPs). Results showed that LAMPs (1 microg/ml) from seven different species of human mycoplasmas-M. penetrans, M. fermentans, M. genitalium, M. salivarium, M. pneumoniae, M. orale, and M. hominis-had an inhibitory effect on androgen receptor (AR) response to 5alpha-dihydrotestosterone (DHT) in the E82 transfectants. The inhibitory effect of mycoplasmal LAMPs appeared to be dose dependent. LAMPs from M. penetrans, M. genitalium, M. salivarium, M. pneumoniae, and M. orale also had an inhibitory effect on glucocorticoid receptor (GR) response to hormone dexamethasone (Dex) in TSU transfectants. In contrast, LAMPs from M. fermentans and M. hominis showed a stimulatory effect on the GR response to Dex in these TSU cells. The results suggest that colonization or chronic infection by mycoplasmas may significantly affect the responses of mammalian host cells to various steroid hormones, potentially affecting rates of cancer formation.

Chloramphenicol acetyltransferase (CAT) is widely used as a reporter to determine the transcriptional specificity of promoters and for the quantification of transcriptional activity of transcription factors such as nuclear receptors. However, large-scale quantification of CAT activity in transfected mammalian cells is still heavily labor-intensive, time-consuming and expensive. Here, we describe a simplified method that combined using multiwell tissue culture plates in transfection and sample preparation and a modified single step method for quantitatively assaying CAT activity. By using multiwell plates, the tedious sample preparation procedure was dramatically simplified. The CAT assay is performed by mixing cell lysate, chloramphenicol, 3H-acetyl co-enzyme A and non-aqueous scintillation fluid in scintillation vials, followed by automatically continuously counting samples two or three cycles at fixed time intervals. The catalytic reaction and determination of CAT activity are carried out in the vials simultaneously. This simplified protocol is faster, less expensive and more accurate than other CAT assay procedures and the results can be normalized easily. The utility of the assay is demonstrated by the analysis of the transcriptional activity of the glucocorticoid and androgen receptors cotransfected into cells with a CAT reporter.

The activity of induce tumor necrosis factor alpha (TNF alpha) production of several mycoplasmas, including AIDS associated mycoplasmas was investigated. M. penetrans which was detected and isolated from urine and tissue of Kaposi's sarcoma of patients with AIDS markedly exhibited the induction of TNF alpha production of both THP-1 cells and murine peritoneal macrophages to compare to other mycoplasmas. Each amount of M. penetrans, M. fermentans, M. incognitus, Acholeplasma ladilawii, M. orale, M. salivarium, M. hominis required for induction of 50% cytotoxicity to L cells in the supernatants of mouse peritoneal macrophages cultured with those microorganisms was 0.65 micrograms/ml, 11.3 micrograms/ml, 19.6 micrograms/ml, 6.6 micrograms/ml, 7.7 micrograms/ml, 6.3 micrograms/ml, 5.7 micrograms/ml respectively. Next, the components of M. penetrans were extracted by Bligh-Dyer method, in order to investigate chemical component to induce TNF alpha-production. The activity of TNF alpha induction was mainly found in the methanol-phase, but not in the chloroform-phase, where lipid and glycolipid of the microorganisms were generally thought to be accumulated. The binding of the active component to concanavalin A-Sepharose was blocked in the presence of Methyl alpha-D-mannopyranoside and Methyl alpha-D-glucopyranoside. These results suggest that the component possess mannoside and glucoside active site.