The production of coagulation factor VII by adipocytes is enhanced by tumor necrosis factor-α or isoproterenol.

BACKGROUND: A relationship has been reported between blood concentrations of coagulation factor VII (FVII) and obesity. In addition to its role in coagulation, FVII has been shown to inhibit insulin signals in adipocytes. However, the production of FVII by adipocytes remains unclear. OBJECTIVE: We herein investigated the production and secretion of FVII by adipocytes, especially in relation to obesity-related conditions including adipose inflammation and sympathetic nerve activation. METHODS: C57Bl/6J mice were fed a low- or high-fat diet and the expression of FVII messenger RNA (mRNA) was then examined in adipose tissue. 3T3-L1 cells were used as an adipocyte model for in vitro experiments in which these cells were treated with tumor necrosis factor-α (TNF-α) or isoproterenol. The expression and secretion of FVII were assessed by quantitative real-time PCR, Western blotting and enzyme-linked immunosorbent assays. RESULTS: The expression of FVII mRNA in the adipose tissue of mice fed with high-fat diet was significantly higher than that in mice fed with low-fat diet. Expression of the FVII gene and protein was induced during adipogenesis and maintained in mature adipocytes. The expression and secretion of FVII mRNA were increased in the culture medium of 3T3-L1 adipocytes treated with TNF-α, and these effects were blocked when these cells were exposed to inhibitors of mitogen-activated kinases or NF-κB activation. The ß-adrenoceptor agonist isoproterenol stimulated the secretion of FVII from mature adipocytes via the cyclic AMP/protein kinase A pathway. Blockade of secreted FVII with the anti-FVII antibody did not affect the phosphorylation of Akt in the isoproterenol-stimulated adipocytes. CONCLUSION: Obese adipose tissue produced FVII. The production and secretion of FVII by adipocytes was enhanced by TNF-α or isoproterenol via different mechanisms. These results indicate that FVII is an adipokine that plays an important role in the pathogenesis of obesity.

Epstein-Barr Virus latent membrane protein 1 induces Snail and epithelial-mesenchymal transition in metastatic nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is distinctive among head-and-neck cancers in its undifferentiated histopathology and highly metastatic character. We have recently investigated the involvement of epithelial-mesenchymal transition (EMT) in NPC. In a previous study, we found a close association of expression of LMP1, the principal EBV oncoprotein, with expression of Twist and induction of EMT. METHODS: We analysed expression of Snail in 41 NPC tissues by immunohistochemistry. The role of Twist as well as Snail in EMT of NPC was investigated by using NP69SV40T human nasopharyngeal cells. RESULTS: In NPC tissues, overexpression of Snail is associated with expression of LMP1 in carcinomatous cells. In addition, expression of Snail positively correlated with metastasis and independently correlated inversely with expression of E-cadherin. Expression of Twist had no association with expression of E-cadherin. Further, in a human nasopharyngeal cell line, LMP1 induces EMT and its associated cellular motility and invasiveness. Expression of Snail is induced by LMP1 in these cells, and small hairpin RNA (shRNA) to Snail reversed the cellular changes. By contrast, Twist did not produce EMT in these nasopharyngeal cells. CONCLUSIONS: This study strengthens the association of EMT with the metastatic behaviour of NPC. These results suggest that induction of Snail by the EBV oncoprotein LMP1 has a pivotal role in EMT in NPC.

Transcription factor AP-2beta inhibits expression and secretion of leptin, an insulin-sensitizing hormone, in 3T3-L1 adipocytes.

BACKGROUND: We have previously reported an association between the activator protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with a disease-susceptible allele of AP-2beta showed stronger AP-2beta expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta led to lipid accumulation and induced insulin resistance in 3T3-L1 adipocytes. RESULT: We found that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the promoter activity of leptin, and subsequently decreased both messenger RNA (mRNA) and protein expression and secretion. Furthermore, knockdown of endogenous AP-2beta by RNA-interference increased mRNA and protein expression of leptin. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed specific binding of AP-2beta to leptin promoter regions in vitro and in vivo. In addition, site-directed mutagenesis of the AP-2-binding site located between position +34 and +42 relative to the transcription start site abolished the inhibitory effect of AP-2beta. Our results clearly showed that AP-2beta directly inhibited insulin-sensitizing hormone leptin expression by binding to its promoter. CONCLUSION: AP-2beta modulated the expression of leptin through direct interaction with its promoter region.

Association of Dietary Variety and Appetite with Sleep Quality in Urban-Dwelling Older Japanese Adults.

OBJECTIVES: Although it has been shown that specific foods and nutrients are associated with sleep quality, few studies have examined the association of dietary variety and appetite with sleep quality in older adults. DESIGN AND SETTING: A cross-sectional study was conducted that examined the association of dietary variety and appetite with sleep quality in Japanese adults aged ≥70 years who resided in the metropolitan area of Tokyo, Japan. PARTICIPANTS: Data were collected in two steps: a mailed interview survey and an on-site survey. Those who responded to the surveys and met the inclusion criteria were included. MEASUREMENTS: Dietary variety, appetite, and sleep quality were assessed using a Dietary Variety Score (DVS), Council on Nutrition Appetite Questionnaire (CNAQ) score, and sleep efficiency, respectively. The sleep efficiency is the ratio of sleep duration to total time in bed (retiring time-awakening time). We defined the individuals with a sleep efficiency less than 75% as having poor sleep quality. RESULTS: Mean DVS and CNAQ score were 3.8 and 29.6 points, respectively. The rate of individuals with poor sleep quality was 11.7%. In the fully adjusted model, the odds ratios (OR) for low sleep efficiency in the middle and highest group categories of the DVS were 0.83 (95% confidence interval [CI], 0.54-1.29) and 0.50 (95% CI, 0.28-0.90), respectively, in reference to the lowest group category (p for trend = 0.023). The OR for low sleep efficiency in the middle and highest group categories of the CNAQ score were 0.73 (95% CI, 0.47-1.14) and 0.54 (95% CI, 0.30-0.96), respectively, in reference to the lowest group category (p for trend = 0.031). CONCLUSIONS: The higher DVS and CNAQ scores were significantly associated with higher sleep efficiency. Thus, dietary variety and good appetite might help maintain good sleep quality in urban-dwelling older Japanese adults.

Up-regulation of CC chemokine receptor 6 on tonsillar T cells and its induction by in vitro stimulation with alpha-streptococci in patients with pustulosis palmaris et plantaris.

Pustulosis palmaris et plantaris (PPP) is a tonsil-related disease; tonsillectomy is somewhat effective in treating the condition. However, the aetiological association between the tonsils and PPP has not yet been elucidated fully. Recently, some chemokines and chemokine receptors, including CC chemokine receptor (CCR) 4, CCR6 and CX chemokine receptor (CXCR) 3, have been reported to play important roles in the development of psoriasis, a disease related closely to PPP. In this study, we found that CCR6 expression on both tonsillar and peripheral blood T cells was up-regulated more intensively in PPP patients than in non-PPP patients (P < 0.001 for both), but CCR4 and CXCR3 expressions were not. In vitro stimulation with alpha-streptococcal antigen enhanced CCR6 expression significantly on tonsillar T cells in PPP patients (P < 0.05), but this was not observed in non-PPP patients. The chemotactic response of tonsillar T cells to the CCR6 ligand CC chemokine ligand (CCL) 20 was significantly higher in PPP patients than in non-PPP patients (P < 0.05). The percentage of CCR6-positive peripheral blood T cells decreased after tonsillectomy in PPP patients (P < 0.01); this decrease correlated with an improvement of skin lesions (P < 0.05, r = -0.63). The numbers of CCR6-positive cells and the expression of CCL20 were increased significantly in pathological lesions compared with non-pathological lesions in PPP skin (P < 0.01, P < 0.05 respectively). These results suggest that a novel immune response to alpha-streptococci may enhance CCR6 expression on T cells in tonsils and that CCR6-positive T cells may move to peripheral blood circulation, resulting in recruitment to target skin lesions expressing CCL20 in PPP patients. This may be one of the key roles in pathogenesis of the tonsil-related disease PPP.

Score-Based and Nutrient-Derived Dietary Patterns Are Associated with Depressive Symptoms in Community-Dwelling Older Japanese: A Cross-Sectional Study.

OBJECTIVES: This study evaluated associations of score-based and nutrient-derived dietary patterns with depressive symptoms in community-dwelling older Japanese. DESIGN: Cross-sectional study. SETTING: Community-based. PARTICIPANTS: 982 community-dwelling adults aged 65 years or older. MEASUREMENTS: Score-based pattern was assessed by using dietary variety score (DVS), which covers 10 food group items in Japanese meals. Nutrient-derived dietary patterns were identified by using reduced rank regression (RRR), with folate, vitamin C, magnesium, calcium, iron, and zinc intakes as response variables. Depressive symptoms were assessed with the Geriatric Depression Scale. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated for these dietary patterns in multivariate logistic regression analyses with potential confounders. The lowest consumption category was used as the reference group. RESULTS: The prevalence of depressive symptoms was 13.5%. Higher DVS was associated with fewer depressive symptoms (OR=0.52, 95% CI=0.27-1.03 for the highest vs the lowest DVS; P for trend=0.031). The first RRR dietary pattern score was characterized by high intakes of fish, soybean products, potatoes, most vegetables, mushrooms, seaweeds, fruits, and green tea and a low intake of rice and was inversely associated with the prevalence of depressive symptoms (OR=0.53, 95% CI=0.30-0.92; P for trend=0.030). CONCLUSION: Greater dietary variety and a dietary pattern characterized by high intakes of fish, soybean products, potatoes, most vegetables, mushrooms, seaweeds, fruit, and green tea and a low intake of rice were consistently associated with lower prevalence of depressive symptoms in community-dwelling older Japanese. Therefore, both patterns identified the components of dietary habits essential to depression prevention.

Selective expansion of T cell receptor (TCR) V beta 6 in tonsillar and peripheral blood T cells and its induction by in vitro stimulation with Haemophilus parainfluenzae in patients with IgA nephropathy.

IgA nephropathy (IgAN), the most common form of primary glomerulonephritis, is recognized as a disease that often becomes worse during acute tonsillitis. Although many reports have shown that tonsillectomy is an effective treatment for IgAN patients, the immunological evidence has not yet been investigated fully. In this study, we compared the expression of T cell receptor (TCR) V beta families in tonsillar T cells of IgAN patients to those of non-IgAN patients. The reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analyses showed that the TCR V beta 6 was used more frequently in tonsillar T cells of IgAN patients than in those of non-IgAN patients (P < 0.01 each). Similarly, the proportions of TCR V beta 6-positive cells in peripheral blood T cells were significantly higher in IgAN patients than in non-IgAN patients (P < 0.05). After tonsillectomy, the proportions decreased in IgAN patients (P < 0.05), but did not in non-IgAN patients. Furthermore, in vitro stimulation with Haemophilus parainfluenzae antigen, which is reported to deposit in the glomerular mesangium of IgAN, enhanced expression of TCR V beta 6 in tonsillar T cells from both IgAN and non-IgAN patients. These results suggest that TCR V beta 6-positive tonsillar T cells might be activated by H. parainfluenzae, move into the kidney through blood circulation and induce glomerulonephritis.

C-Terminal Farnesylation of UCH-L1 Plays a Role in Transport of Epstein-Barr Virus Primary Oncoprotein LMP1 to Exosomes.

Increasing evidence shows that exosomes are key regulators in cancer cell-to-cell communication. Several reports on Epstein-Barr virus (EBV)-related malignancies demonstrate that latent membrane protein 1 (LMP1) secreted by exosomes derived from EBV- or LMP1-positive cells can promote cancer progression and metastasis. However, the mechanism by which LMP1 is loaded into exosomes is still poorly understood. Here, we examined whether the process of LMP1 loading into exosomes is linked to the multifunctional molecule of the ubiquitin system-ubiquitin C-terminal hydrolase-L1 (UCH-L1). For the first time, we demonstrate that LMP1 is physically associated with UCH-L1 and that directing of LMP1 to exosomes is mediated by C-terminal farnesylation of UCH-L1. Additionally, we found that the FTI-277 farnesyltransferase inhibitor reduces motility- and anchorage-independent growth of EBV-positive cells in functional assays. On the basis of our results, we conclude that C-terminal farnesylation of UCH-L1 is one of the key mechanisms by which LMP1 is sorted to exosomes. We hypothesize that inhibition of farnesylation with specific small-molecule inhibitors blocks exosome-mediated transfer of prometastatic molecules such as LMP1 during cancer cell-to-cell communications and thereby impedes the process of cancer invasion. IMPORTANCE Exosomes are small vesicles that cells secrete into the extracellular space, and there is increasing evidence that they have pivotal roles in cell-to-cell communication in malignancy. It is reported also that EBV-associated malignant cells, including those derived from nasopharyngeal carcinoma (NPC) and B-cell lymphoma, secrete exosomes. These EBV-related exosomes may contain viral products such as latent membrane protein 1 (LMP1) and may contribute to cancer progression. The aim of this study was to investigate the mechanism by which those viral products are loaded in exosomes. In this study, we show for the first time that ubiquitin C-terminal hydrolase-L1 (UCH-L1) and its C-terminal farnesylation, a posttranslational lipid modification, contribute to this mechanism. Our results also suggest that inhibition of UCH-L1 farnesylation is a potential therapeutic target against cancer metastasis and invasion.

APOBEC3A associates with human papillomavirus genome integration in oropharyngeal cancers.

The prevalence of human papillomavirus (HPV)-related oropharyngeal cancers has been increasing in developed countries. We recently demonstrated that members of the apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3, A3) family, which are antiviral factors, can induce hypermutation of HPV DNA in vitro. In the present study, we found numerous C-to-T and G-to-A hypermutations in the HPV16 genome in oropharyngeal cancer (OPC) biopsy samples using differential DNA denaturation PCR and next-generation sequencing. A3s were more abundantly expressed in HPV16-positive OPCs than in HPV-negative, as assessed using immunohistochemistry and reverse transcription quantitative PCR. In addition, interferons upregulated A3s in an HPV16-positive OPC cell line. Furthermore, quantitative PCR analysis of HPV DNA suggests that APOBEC3A (A3A) expression is strongly correlated with the integration of HPV DNA. These results suggest that HPV16 infection may upregulate A3A expression, thereby increasing the chance of viral DNA integration. The role of A3A in HPV-induced carcinogenesis is discussed.

Aspirin inhibits tumor cell invasiveness induced by Epstein-Barr virus latent membrane protein 1 through suppression of matrix metalloproteinase-9 expression.

Matrix metalloproteinases (MMPs) are thought to play crucial roles in tumor invasion and metastasis. Because we have shown that EBV latent membrane protein 1 (LMP1) enhances MMP-9 expression by activation of nuclear factor (NF)-kappaB and activator protein (AP)-1 (T. Yoshizaki, et al., Proc. Natl. Acad. Sci. USA, 95: 3621-3626, 1998), we therefore tested whether up-regulation of MMP-9 by LMP1 could be correlated with enhanced invasiveness of tumor cells in vitro. Whether aspirin and sodium salicylate could reduce invasiveness and whether LMP1 could enhance MMP-9 expression in tumors grown in nude mice were also tested. C33A cells stably expressing LMP1 had increased expression of MMP-9 and showed greater invasion through reconstituted basement membrane compared with vector-transfected C33A cells (P < 0.02). Treatment with aspirin or sodium salicylate inhibited invasiveness of the LMP1-expressing C33A cells (P < 0.03) and suppressed both the LMP1-induced MMP-9 expression in zymographic analyses and LMP1-induced MMP-9 promoter activity in CAT reporter assays (P < 0.01). Endogenous MMP-2 levels were unaffected by either drug. Both drugs repressed the CAT activity of the truncated MMP-9 promoter construct, which only contained a binding site for AP-1, to the basal level (P < 0.05). Moreover, EMSA indicated that the effects of the salicylates were through the inhibition of not only NF-kappaB but also AP-1 binding activity. Inhibitory effect of salicylates could be reversed by p50/p65 subunits of NF-kappaB or c-Jun overexpression. The inhibitory effect of aspirin on NF-kappaB activity was attributable to the inhibition of IkappaB kinase activity. Finally, tumors derived from C33A cells stably expressing LMP1 grown in nude mice showed enhanced MMP-9 levels compared with tumors derived from vector-transfected C33A cells. This enhancement was inhibited by treatment of the mice with aspirin. These results suggest that aspirin may be able to suppress invasion and metastasis of EBV-associated tumors that express LMP1 by suppression of MMP-9.

Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has a significant role in initiating EBV-associated lymphoproliferative disease and EBV-related malignancies. In view of clinical features related to the type of EBV latency, LMP1 may influence invasiveness of EBV associated tumors categorized as types II and III as represented on nasopharyngeal carcinoma (NPC). To screen for genes associated with invasion of epithelial cells transformed by LMP1, Madin-Darby canine kidney (MDCK) epithelial cells were transformed by LMP1. Stable transfection of a LMP1 gene into MDCK cells induced morphological change from cobblestone to a long spindle-shape, reduced cell-cell adhesion and caused high cell motility. Parental MDCK cells, which form spherical cysts in three-dimensional collagen gel matrix, form branching tubules following exposure to hepatocyte growth factor (HGF). MDCK cells transformed by LMP1 showed invasive growth to form branching tubules into collagen gel without HGF-treatment. mRNA differential display and Northern hybridization identified plasminogen activator inhibitor-1 (PAI-1), urokinase type plasminogen activator (uPA) and ets1 as genes upregulated during transformation by LMP1. Expression of a dominant negative type of Etsl in LMP1-transformed cells downregulated uPA expression and cell motility. Deletion of LMP1 cytoplasmic carboxy-terminal activating region 1 (CTAR1) domain abolished transformation, but a deletion mutant lacking CTAR2 domain still retained transforming and uPA-inducing ability. Expression of Ets1 was immunolocalized in tumor cells of NPC tissue which frequently express LMP1. Taken together, it is suggested that LMP1 induces expression of Ets1 which may contribute to invasion of NPC by stimulating cell motility and uPA expression.

Absence of bicarbonate abolishes the glycogenic effect of cortisol in cultured fetal rat hepatocytes.

The glycogenic action of cortisol in cultured fetal rat hepatocytes was completely abolished by the absence of NaHCO3 from the medium, while its presence stimulated the action in relation to its concentration. The absence of NaHCO3 slightly reduced glycogen storage by insulin but did not affect glucose-dependent glycogen deposition in the basal state. Also, the cortisol-induced increase in glycogen synthase a activity was reduced but that in total synthase activity was not affected. The absence of NaHCO3 did not reduce the cortisol-induced increase in tyrosine aminotransferase activity and the incorporation of [3H]dexamethasone into the nuclei. These results show that the absence of NaHCO3 specifically inhibits the glycogenic action of glucocorticoids in cultured fetal rat hepatocytes and indicate the need for further investigation into the role of HCO3- in universally used bicarbonate-buffered media.

Degradation of nucleic acids with ozone. II. Degradation of yeast RNA, yeast phenylalanine tRNA and tobacco mosaic virus RNA.

The degradation of a mixture of four 5'-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1-0.5 mg/l) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.

Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma.

PURPOSE: The EBV latent membrane protein-1 (LMP-1) is a multifunctional protein. Recently, the contribution of LMP-1 to the metastasis of nasopharyngeal carcinoma (NPC) has been suggested. Angiogenesis is a key step for metastasis. Thus, the association of LMP-1 to neovascularization of NPC was examined in this study. EXPERIMENTAL DESIGN: The association of LMP-1 to angiogenesis in 39 patients with NPC was evaluated by immunohistochemical study, and then induction of angiogenic factors by LMP-1 was examined by ELISA and luciferase reporter assay. RESULTS: In an immunohistochemical study, the expression of LMP-1 was significantly correlated to microvessel counts (P = 0.0003), suggesting that LMP-1 may induce some angiogenic factors. Therefore, we studied the relationship between LMP-1 expression and interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) expression by immunohistochemical analysis. IL-8, VEGF, and bFGF expression were correlated to microvessel counts, but only IL-8 expression was significantly correlated to LMP-1 expression (P < 0.0001). Transfection with LMP-1 expression plasmid induced IL-8 protein expression in C33A cells. The expression of LMP-1 transactivated IL-8 promoter, as demonstrated by IL-8 promoter luciferase reporter assay. Mutation of the nuclear factor kappaB responsive element in the IL-8 promoter region completely abolished transactivation by LMP-1, whereas mutation of the activator protein responsive element did not affect promoter activity. CONCLUSION: These results suggested that LMP-1 induces expression of IL-8 through the nuclear factor kappaB binding site, which may contribute in part to angiogenesis in NPC.

PMEA polymer-coated PVC tubing maintains anti-thrombogenic properties during in vitro whole blood circulation.

Poly(2-methoxyethylacrylate) (PMEA) is a new coating material that appears to reduce protein and platelet adsorption. However, the exact performance of PMEA coated circuit remains to be revealed in well-controlled experiments. Therefore, we compared its hemocompatibility with covalent-bound heparin-, and non-coated circuits during 6 hours of in vitro circulation, using donor blood from six volunteers. In our model, simple tubing circuits containing one-way ball valve were placed on the rotary table, which moved alternatively to generate pulsatile recirculation of heparinized human blood inside the tubing. Using this model, we expected fine assessment of the material surface, because we could reduce blood damage by avoiding air and a blood pump. Moreover, the small capacity of circuit allowed us to compare three kinds of circuits using a single unit of donor blood, eliminating effects by possible variations between blood donors. The anti-thrombin capacity of the PMEA-coated circuits was maintained even after six hours blood circulation, whereas surface thrombin generation increased markedly after use in non-coated circuits (P<0.05). Deposition of fibrin onto PMEA circuits was reduced more than 30% compared with heparin and non-coated circuits (P<0.05). However, the increase of plasma Factor XIIa was similar in all circuits. Increase of CD11b expression on circulating leukocytes and of plasma C3a was ameliorated in the heparin- and PMEA-coated circuits (P<0.05). PMEA-coated circuits appear to maintain their anti-thrombogenicity during use, otherwise PMEA-coated and heparin-coated circuits showed a similar character in hemocompatibility. This long-standing anti-thrombogenicity might be attributable to less adsorption of activated blood components onto the surface.

Matrix metalloproteinase 9 is induced by the Epstein-Barr virus BZLF1 transactivator.

Type IV collagenases, matrix metalloproteinase (MMP) 2 and MMP9 are implicated in tumor invasion and metastasis. In patients with nasopharyngeal carcinoma (NPC), poor prognosis due to development of local and distant metastasis has been reported to be predicted by antibody titers against the Z protein which is an AP-1 family transcription factor encoded by the EBV BZLF1 immediate-early gene. Here we report that in patients with NPC, expression of Z in tumor cells correlates with advanced cervical lymph node metastasis which may suggest that Z affects tumor invasion and metastasis. We therefore tested if Z would induce expression of type IV collagenases. Transfection of Z expression plasmid into the C33A epithelial cell line increased expression of MMP9, but MMP2 expression was unaltered. Mutational analysis of the Z protein revealed that, in addition to all three functional domains of Z (dimerization domain, DNA binding domain, and activation domain), the carboxyl terminal 17 amino acids which stabilize the Z protein were necessary for induction of MMP9 expression. Analysis of the MMP9 promoter demonstrated that only AP-1 site close to the transcriptional start-site was essential for transactivation by Z. Previously we reported that Epstein-Barr virus latent membrane protein 1 (LMP1) stimulates MMP9 expression (Yoshizaki et al. Proc. Natl. Acad. Sci. 1998; 95: 3621-6). Thus, Z together with LMP1 may contribute to invasion and metastasis of NPC by inducing expression of MMP9.

Depression of liver microsomal vitamin K epoxide reductase activity associated with antibiotic-induced coagulopathy.

Hypoprothrombinemic changes in blood coagulation parameters, such as prolongation of prothrombin time, increase in the level of plasma protein induced by vitamin K absence, and decrease in plasma prothrombin level, were detected in rats fed a vitamin K-deficient diet. These changes were enhanced by the administration of beta-lactam antibiotics containing N-methyltetrazolethiol, thiadiazolethiol or methyl-thiadiazolethiol. Microsomal vitamin K epoxide reductase activity was suppressed with the maximum effect at 1-2 days after the treatment and with recovery, thereafter, gradually to the normal level after 5-7 days. Hypoprothrombinemic alterations in blood coagulation parameters following a single administration of antibiotic to vitamin K-deficient rats were somewhat delayed compared with the change in the epoxide reductase activity, but the effects of the antibiotic on both blood coagulation parameters and the enzyme activity disappeared completely 7 days after the antibiotic treatment. Antibiotic-induced depression of the epoxide reductase activity was observed even in the vitamin K sufficient rats, although the hypoprothrombinemic changes in the blood coagulation parameters did not develop. Vitamin K administration could normalize the blood coagulation parameters in the hypoprothrombinemic rats caused by treatment with the antibiotics but without recovery of the decreased epoxide reductase activity. These results suggest that some antibiotics inhibit liver microsomal vitamin K epoxide reductase, which causes hypoprothrombinemia to develop under vitamin K-deficient conditions.

In vivo effects of beta-lactam antibiotics and heterocyclic thiol compounds on vitamin K-dependent carboxylation activity and blood coagulation factors in vitamin K-deficient rats.

The in vivo effects of heterocyclic thiol compounds, corresponding to the 3'-position substituents of several beta-lactam antibiotics, on blood coagulation factors and on liver microsomal gamma-glutamylcarboxylation (gamma-carboxylation) activity were evaluated in rats maintained on a vitamin K-deficient diet. These rats, when compared to normal control animals, exhibited hypoprothrombinemic changes: prolongation of both prothrombin time and activated partial thromboplastin time, decreases in factor VII and plasma prothrombin, and increases in PIVKA II (descarboxyprothrombin) both in plasma and liver. They also displayed a marked increase in liver microsomal gamma-carboxylation activity. These blood coagulation variables could be altered markedly by administering various heterocyclic thiol compounds to the vitamin K-deficient rats, although these compounds did not inhibit gamma-carboxylation activity in an assay system using phylloquinone. A similar pattern of alteration was observed when some beta-lactam antibiotics were administered. Increased microsomal gamma-carboxylation activity in antibiotic-treated vitamin K-deficient rats was normalized by the administration of vitamin K, concomitant with the recovery of blood coagulation variables to the normal range. The results indicate that antibiotic-induced hypoprothrombinemia in vivo is not caused by inhibition of enzymes of the gamma-carboxylation system, such as vitamin K reductase and gamma-glutamylcarboxylase, but is related to the endogenous vitamin K level.

Elevation of antibody against Epstein-Barr virus genes BRLF1 and BZLF1 in nasopharyngeal carcinoma.

PURPOSE: Epstein-Barr virus immediate early genes BZLF1 and BRLF1 encode the Z and R proteins respectively. Elevation of the anti-Z immunoglobulin (Ig) antibody titer is a common feature of nasopharyngeal carcinoma (NPC) in areas where the disease endemic, whereas R levels have not been examined. This study was performed to analyze antibody titers against Z and R in Japan. METHODS: Sera from 14 patients with newly diagnosed NPC, 7 with NPC in complete remission, 15 with head and neck squamous cell carcinoma (HNSCC), and 31 controls were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Both anti-Z and anti-R Ig were significantly increased in sera from patients with NPC compared with those in remission, those with HNSCC, and controls (Z, P = 0.0016, = 0.0344, = 0.0002; R, P = 0.0015, = 0.0004, <0.0001 respectively). By immunoblot analysis, anti-Z and anti-R Ig were detected in 9 of 9 cases of NPC, 1 of 3 cases NPC in remission, and 2 of 13 cases of HNSCC. Anti-Z and anti-R antibody titers were nine times higher in NPC than in other groups. None of 15 control cases showed positive reactivity against either Z or R. CONCLUSION: Coupled evaluation of anti-Z and anti-R Ig by ELISA might be a useful system for screening NPC.