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The recently identified human herpesvirus 8 (HHV-8, or Kaposi's sarcoma-associated herpesvirus) has been implicated in the etiology of both Kaposi's sarcoma (KS) and primary effusion (body cavity-based) lymphoma (PEL) (Y. Chang et al., Science 266:1865-1869, 1994; P. S. Moore et al., J. Virol. 70:549-558, 1996). An important feature of the association of HHV-8 with these malignancies is the expression of an abundant, latency-associated 0.7-kb transcript, T0. 7 (W. Zhong et al., Proc. Natl. Acad. Sci. USA 93:6641-6646, 1996). T0.7 is found in all stages in nearly all KS tumors of different epidemiologic origin, including AIDS-associated, African endemic, and classical KS (K. A. Staskus et al., J. Virol. 71:715-719, 1997), as well as in a body cavity-based lymphoma-derived cell line, BCBL-1, that is latently infected with HHV-8 (R. Renne et al., Nat. Med. 2:342-346, 1996). T0.7 encodes a unique HHV-8 open reading frame, K12, also known as kaposin. In this study, we report that the kaposin gene induced tumorigenic transformation. Constructs with kaposin expressed either from its endogenous promoter or from a heterologous promoter induced focal transformation upon transfection into Rat-3 cells. All transformed Rat-3 cell lines containing kaposin sequences produced high-grade, highly vascular, undifferentiated sarcomas upon subcutaneous injection of athymic nu/nu mice. Tumor-derived cell lines expressed kaposin mRNA, suggesting a role in the maintenance of the transformed phenotype. Furthermore, kaposin protein was detected in transformed and tumor-derived cells by immunofluorescence and localized to the cytoplasm. More importantly, expression of kaposin protein was also detected in the PEL cell lines BCBL-1 and KS-1. These findings demonstrate the oncogenic potential of kaposin and suggest its possible role in the development of KS and other HHV-8-associated malignancies.

Since failure of differentiation has been suggested to be involved in the neoplastic process and progression of tumors, we evaluated whether the transcript levels of differentiation markers of proximal renal tubular cells, from which renal cell carcinoma (RCC) arises, could be used as prognostic markers. We used Northern blot analysis to study the expression of aquaporin 1 (aqp1) and carbonic anhydrase IV (ca4) genes in 66 paired samples of primary RCC and non-tumorous kidney tissues. Poor differentiation of tumor cells and non-clear cell-subtype RCC were significantly associated with low levels of aqp1 transcripts. When patients were divided into 2 groups according to level of aqpI transcript in RCC, a low level of aqp1 was significantly associated with unfavorable outcome. Among 18 patients with metastatic RCC and 40 patients with moderately differentiated RCC, those with RCC expressing low levels of aqpl mRNA demonstrated poorer survival than those with RCC expressing relatively high levels of aqp1. Similarly, decreased expression of ca4 mRNA in RCC was associated with poor survival. On multivariate analysis, transcript levels of aqpI and stage of the tumor were the independent factors predicting disease-specific survival. Transcript levels of aqp1 may serve as a new molecular prognostic marker in patients with RCC following nephrectomy.

In response to low ambient temperature, mammalian cells as well as microorganisms change various physiological functions, but the molecular mechanisms underlying these adaptations are just beginning to be understood. We report here the isolation of a mouse cold-inducible RNA-binding protein (cirp) cDNA and investigation of its role in cold-stress response of mammalian cells. The cirp cDNA encoded an 18-kD protein consisting of an amino-terminal RNAbinding domain and a carboxyl-terminal glycine-rich domain and exhibited structural similarity to a class of stress-induced RNA-binding proteins found in plants. Immunofluorescence microscopy showed that CIRP was localized in the nucleoplasm of BALB/3T3 mouse fibroblasts. When the culture temperature was lowered from 37 to 32 degrees C, expression of CIRP was induced and growth of BALB/3T3 cells was impaired as compared with that at 37 degrees C. By suppressing the induction of CIRP with antisense oligodeoxynucleotides, this impairment was alleviated, while overexpression of CIRP resulted in impaired growth at 37 degrees C with prolongation of G1 phase of the cell cycle. These results indicate that CIRP plays an essential role in cold-induced growth suppression of mouse fibroblasts. Identification of CIRP may provide a clue to the regulatory mechanisms of cold responses in mammalian cells.

Quantitative PCR methods are potentially useful for determining levels of specific gene expression and gene dosage. Previously we developed a non-radioisotopic quantitative RT-PCR method by utilizing the PCR amplification kinetics and CCD image analyzer. Recently, based on the principle of fluorescence energy transfer, the AmpliSensor system that can quantitate the PCR products concurrent to amplification in a single reaction vessel has been described. Herein, we compared the results obtained by both methods. cDNAs were synthesized from 10 human endometrial biopsy specimens. Aliquots of cDNA were used for quantitation by gel/image analyzer or AmpliSensor assay system. For estimation of the initial amount of the template(I), regression equations of the form:y = I x Ex, where y is the amount of PCR products and x is the number of cycles, were fitted to the data in the linear portion of the semi-logarithmic graphs. The relative levels of beta-actin cDNA estimated by AmpliSensor assay system was in good agreement (r = 0.91) with those of the gel/image analyzer assay system, which is cumbersome, time-consuming and needs post-amplification procedures. The AmpliSensor assay is suitable for a quantitative PCR assay based on PCR kinetics, and is applicable for diagnostic testings.

Background: Collateral growth is induced by chemical signals from the ischemic myocardium. We hypothesized that angiogenic growth factors are produced by cardiac tissue; they are diffusible, more concentrated in pericardial fluids, and are increased by myocardial ischemia., Methods and Results: With the use of an enzyme-linked immunosorbent assay, we measured the concentrations of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in pericardial fluids of 12 patients with unstable angina (group 1) and of 8 patients with nonischemic heart diseases (group 2). The levels of protein in pericardial fluids were quite comparable between the two groups (34 +/- 2 versus 32 +/- 4 mg/mL). The concentration of bFGF in pericardial fluids in group 1 was 2036 +/- 357 pg/mL, significantly (P < .001) higher than the 289 +/- 72 pg/mL in group 2. The amount of bFGF per milligram of protein was also significantly (P < .05) higher in group 1 than in group 2 (67 +/- 15 versus 12 +/- 4 pg/mg). The concentration of VEGF in pericandial fluids tended to be higher in group 1, but the difference was statistically insignificant (39 +/- 7 versus 22 +/- 6 pg/mL). The amount of VEGF per milligram of protein was 1.2 +/- 0.3 pg/mg in group 1, similar to the 0.8 +/- 0.4 pg/mg in group 2., Conclusions: This finding provides new evidence that bFGF plays an important role in mediating collateral growth in humans.

Vav is a signal transducing molecule containing SH2 and SH3 domains and a guanine nucleotide releasing factor-like domain. Its expression is thought to be highly specific for hematopoietic cells. Here we describe the expression of vav transcripts in human nonhematopoietic trophoblasts. By northern blotting, expression of 2.8-kb vav mRNA was detected in human decidual, placental, and chorionic villous tissues and in a choriocarcinoma cell line BeWo. By in situ hybridization, vav mRNA was found to be expressed in the cytotrophoblast shell and columns and in the extravillous trophoblasts in the maternal decidua from the first through third trimesters. Vav mRNA was also detected in villous syncytiotrophoblasts during the second and third, but not the first, trimesters. When 1 microM oligodeoxynucleotide antisense to the vav mRNA was added to the medium, growth of BeWo cells was significantly inhibited. These results suggest that vav plays an important role for successful implantation and placental development by regulating development of trophoblasts.

The distribution of the genes of streptococcal pyrogenic exotoxin (speA, B and C) among the 400 clinical isolates of Streptococcus pyogenes from Japanese pharnyx was detected by the PCR method (Nihonrinsho 50: 326, 1992). Sixty, 399 and 303 isolates were positive for the speA, B and C genes, respectively. However, only one isolate had none of these genes. Several isolates possessed two or three genes, i.e., both speB & C, speA & B or speA, B & C were found in 258, 30 and 30 isolates, respectively. No isolate possessing both speA & C without speB was found in this study. We concluded that the PCR method is much more useful for the epidemiological study on streptococcal pyrogenic exotoxins (SPE) of S. pyogenes isolates because of specificity, rapidity and sensitivity of this method compared with the conventional SPE identification method.

Six hundred and seventy isolates from children with group A streptococcal infections from 1981 through 1990 were typed serologically and their antibiotic susceptibilities were determined. Productivity of streptococcal pyrogenic exotoxins was also investigated in some isolates. Four hundred and seventy-nine strains were isolated from patients with pharyngitis, 133 from those with scarlet fever, 35 from those with suppurative infection and 23 from those with non-suppurative disease. With immediate treatment (antibiotics were started at the same day throat swabs were taken) for 10 days, 5.3% of the patients with pharyngitis including scarlet fever had relapses and 13.4% of those patients had recurrences. Of the episodes of recurrences 15.7% were due to the same M serotype strains. Six patients had two episodes of scarlet fever. M type of isolate was different in the first and the second episode of each patient. Pyrogenic exotoxin type was unprecedented in the second episode of 4 out of 6 patients. M-typable and T-typable rates of isolates were 90.7% and 97.3%, respectively. Coincidence between M and T types was 73.3% (83.0% if including strains with the same and mixed T-type). Prevalent M-serotypes were 12 and 4, but M1, 3, or 28 was the most prevalent type of isolates in certain years. None of the 670 strains was resistant to penicillin G and cephalexin. Resistant rate of isolates to erythromycin and linecomycin was 26.5% in 1981 and 18.4% in 1982. But a marked decrease has noted since 1983 and only one has been resistant since 1986.(ABSTRACT TRUNCATED AT 250 WORDS)

During the screening of plant oils for their Epstein-Barr virus (EBV)-activating potency, we found that tung oil possesses an activity comparable to croton oil. Tung oil from various sources and the extracts from its parental plant Aleurites fordii (Chinese tung oil tree), when used in combination with n-butyrate, were shown to efficiently activate EBV persisting in human lymphoblastoid Raji cells (non-producer). The major diterpene ester in the plant extract, 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA), also exerted a similar activity. In producer P3HR-1 cells, both tung oil and HHPA increased the yield of infectious EBV by approximately five-fold. Since tung oil is used for the manufacture of oil paints, varnishes, waterproof substance, anticorrosives and other products, the implication of using such an agent with EBV-activating potency in our daily life is assessed and discussed.

Epstein-Barr virus (EBV)-associated early (EA) and virus capsid antigens (VCA) were efficiently induced in the viral genome-carrying human lymphoblastoid cells, P3HR-1 and Raji, by the culture fluids of Propionibacterium acnes, P. avidum, P. lymphophilum and Arachnia propionica, the anaerobes which are commonly seen among the normal flora of man. The active principle for EBV-induction in the 2 cell lines was the propionic acid produced by the microbes and such activity was shown to correlate with the fatty acid content of the culture media.

A monoclonal antibody designated L2 was produced against a late intracellular protein induced by Epstein-Barr virus (EBV). This protein was expressed in cells producing virus but not in EBV genome-positive nonproducer cell lines, EBV genome-negative cell lines, or producer cultures cultivated in the presence of phosphonoacetic acid as determined by immunofluorescence. In addition, the antibody did not react with the membranes of infected cells indicating that it was not directed against an EBV-induced membrane antigen component. The monoclonal antibody was shown to recognize a glycoprotein with a molecular weight of approximately 125K by SDS-polyacrylamide gel electrophoresis. This glycoprotein was consistently found to be slightly larger when isolated from the P3HR-1 cell line as opposed to the B-95-8 cell line. A similar difference was also noted by comparison of peptide maps of this protein isolated by immunoaffinity chromatography from the two cell lines. Serological studies indicated that this 125K glycoprotein was a major component of the viral capsid-antigen (VCA) complex as defined by immunofluorescence.

Epstein-Barr virus-associated early antigen and viral capsid antigen were efficiently induced in human lymphoblastoid P3HR-1 cells with culture fluid of Fusobacterium nucleatum, a member of the indigenous microbial flora of the human host. This finding may suggest a new approach to assess the possible role of the "cofactor(s)" in the etiology of Epstein-Barr virus-related diseases.