Suppression of arachidonic acid cascade-mediated apoptosis in aflatoxin B1-induced rat hepatoma cells by glucocorticoids

We hypothesized that lower ovarian and gonadotropin hormone concentrations would be associated with lower levels of peak bone mineral density (BMD) in apparently normally menstruating women who did not exercise intensively and did not report anorexia or bulimia. This hypothesis was evaluated using a case-with-control study design (n = 65) which was nested within a population-based longitudinal study of peak bone mass (Michigan Bone Health Study) with annual assessment in women aged 25-45 years (n = 582). Cases were 31 premenopausal women with BMD of the lumbar spine, femoral neck, and total body less than the 10th percentile of the distribution, where controls were 34 premenopausal women with BMD between the 50th and 75th percentile. BMD was measured by dual-energy X-ray absorptiometry. In addition to their annual measurement, these 65 participants collected first-voided morning urine specimens daily through two consecutive menstrual cycles. The urine from alternating days of this collection was analyzed for estrone-3-glucuronide (E1G), pregnanediol glucuronide (PdG), testosterone, and follicle-stimulating hormone by radioimmunoassay and these values adjusted for daily creatinine excretion levels. Additionally, analyses of daily urine specimens for luteinizing hormone (uLH) was undertaken to better characterize the possible uLH surge. Cases had significantly lower amounts of E1G (p = 0.009) and PdG (p = 0.002) than did controls, whether amounts were characterized by a mean value, the highest value, or the area under the curve, and after statistically controlling for body size. Further, when B-splines were used to fit lines to the E1G and PdG data across the menstrual cycle, the 95% confidence intervals (CIs) about the line for the controls consistently excluded and excluded and exceeded the 95% confidence bands for the cases in the time frame associated with the luteal phase in ovulatory cycles. Likewise, 95% CIs for the LH surge in controls exceeded the fitted line for cases around the time associates with the LH surge. The cases and controls were not different according to dietary intake (energy, protein, calcium), family history of osteoporosis, reproductive characteristics (parity, age at menarche, age of first pregnancy), follicular phase serum hormone levels, calciotropic hormone levels, or by evidence of perimenopause. We conclude that these healthy, menstruating women with BMD at the lowest 10th percentile from a population-based study had significantly lower urinary sex steroid hormone levels during the luteal phase of menstrual cycles as compared with hormone levels in premenopausal women with BMD between the 50th and 75th percentile of the same population-based study, even after considering the role of body size. These data suggest that subclinical decreases in circulating gonadal steroids may impair the attainment and/or maintenance of bone mass in otherwise reproductively normal women.     

Central action of interleukin 1 beta on intestinal motility in rats: mediation by two mechanisms

BACKGROUND: Interleukin 1 (IL-1) can influence gut functions by inhibiting gastric acid secretion. This study was performed to investigate the effects of IL-1 on intestinal motility and the mechanisms involved. METHODS: The effects of IL-1 beta were determined by electromyography in conscious rats with implanted electrodes and a permanent catheter in a lateral brain ventricle. RESULTS: Intracerebroventricular IL-1 beta (15 ng) administered to fed rats immediately stimulated cecocolonic spike bursts and caused a migrating myoelectric complex pattern after a delay in the small intestine. Tenfold higher doses of peripherally administered IL-1 beta did not promote similar reactions. The IL-1 antagonist reduced the small intestinal effect of IL-1 beta and blocked the cecocolonic stimulation. Indomethacin and SC 19220 reduced the small intestinal effects but did not antagonize the increase in cecocolonic contractions. In contrast, alpha-helical CRF9-41 blocked the increase of cecocolonic contractions but did not antagonize the IL-1 beta-induced effects on the small intestine. CONCLUSION: IL-1 beta's effects on intestinal motility can be mainly ascribed to a central action. The cecocolonic stimulation may be mediated by brain corticotropin-releasing factor, whereas the small intestinal effects involve a prostaglandin mediation.     

Role of CD59 in experimental glomerulonephritis in rats

CD59 is a molecule which is present on the host cell membranes and inhibits formation of membrane attack complex. A monoclonal antibody, 6D1, recognizes a rat analogue of human CD59. 6D1 inhibits function of rat CD59 and can enhance complement-mediated hemolysis in vitro. To assess the role of CD59 in complement-mediated glomerular injury, 6D1 was tested in a model of experimental glomerulonephritis induced by a lectin and its antibodies. The left kidney of a rat was perfused either with 200 micrograms of Lens culinaris hemagglutinin (LCH) plus 1 mg of 6D1 (IgG1 fraction) (Group I and III) or with LCH only (Group II) through a cannula placed in the left renal artery. All the perfusate was discarded from a cannula in the renal vein. The holes in the artery and vein were repaired by microsurgery and the blood circulation was re-established. Rats were injected either with 0.125 ml of rabbit anti-LCH serum (Group I and II), or with normal rabbit serum (Group III) via tail vein one minute after the recirculation. Fifteen minutes after injection, significant C9 deposition in the glomeruli was observed only in Group I, whereas C3 deposition in Group I and II were comparable. At Day 4, total glomerular cells, proliferating cells, glomerular expression of intercellular adhesion molecule-1 and fibrin deposition in Group I were all significantly increased when compared with Group II. At Day 7, number of total glomerular cells and leukocytes in the glomeruli of Group I were significantly higher than in Group II. The glomeruli in Group III appeared normal throughout experiments. These data indicate that the functional inhibition of a rat analogue of human CD59 worsens complement-mediated glomerular injury in vivo.     

Suppression of mesangial proliferative glomerulonephritis development in rats by inhibitors of cAMP phosphodiesterase isozymes types III and IV

Excessive mesangial cell (MC) proliferation is a hallmark of many glomerulopathies. In our recent study on cultured rat MC (Matousovic, K., J.P. Grande, C.C.S. Chini, E.N. Chini, and T.P. Dousa. 1995. J. Clin. Invest. 96:401-410) we found that inhibition of isozyme cyclic-3',5'-nucleotide phosphodiesterase (PDE) type III (PDE-III) suppressed MC mitogenesis by activating cAMP-dependent protein kinase (PKA) and by decreasing activity of mitogen-activated protein kinase (MAPK). We also found that inhibition of another PDE isozyme, PDE-IV, suppresses superoxide generation in glomeruli (Chini, C.C.S., E.N. Chini, J.M. Williams, K. Matousovic, and T.P. Dousa. 1994. Kidney Int. 46:28-36). We thus explored whether administration in vivo of the selective PDE-III antagonist, lixazinone (LX), together with the specific PDE-IV antagonist, rolipram (RP), can attenuate development of mesangioproliferative glomerulonephritis (MSGN) induced in rats by anti-rat thymocyte serum (ATS). Unlike the vehicle-treated MSGN rats, rats with MSGN treated with LX and RP did not develop proteinuria and maintained normal renal function when examined 5 d after injection of ATS. In PAS-stained kidneys from PDE-antagonists-treated MSGN-rats the morphology of glomeruli showed a reduction in cellularity compared with control rats with ATS. Compared with MSGN rats receiving vehicle, the MSGN rats receiving PDE-antagonists had less glomerular cell proliferation (PCNA delta -65%), a significantly lesser macrophage infiltration (delta -36% ED-1) and a significant reduction of alpha-smooth muscle actin expression by activated MC; in contrast, immunostaining for platelet antigens and laminin were not different. The beneficial effect of PDE inhibitors was not due to a moderate decrease (approximately -20%) in systolic blood pressure (SBP); as a similar decrease in SBP due to administration of hydralazine, a drug devoid of PDE inhibitory effect, did not reduce severity of MSGN in ATS-injected rats. We conclude that antagonists of PDE-III and PDE-IV administered in submicromolar concentrations in vivo to ATS-injected rats can decrease the activation and proliferation of MC, inhibit the macrophage accumulation, and prevent proteinuria in the acute phase of MSGN. We propose that PDE isozyme inhibitors act to block (negative "crosstalk") the mitogen-stimulated intracellular signaling pathway which controls MC proliferation due to activating of the cAMP-PKA pathway. These results suggest that antagonists of PDE-111 and IV may have a suppressive effect in acute phases or relapses of glomerulopathies associated with MC proliferations.     

N-alpha-tosyl-L-lysine chloromethylketone prevents expression of iNOS in vascular smooth muscle by blocking activation of NF-kappa B

OBJECTIVE: To demonstrate that an agreement approach to applying equations on the basis of clinical and exercise test variables is an accurate, self-calibrating, and cost-efficient method for predicting severe coronary artery disease in clinical populations. DESIGN: Retrospective analysis of consecutive patients with complete data from exercise testing and coronary angiography referred for evaluation of possible coronary artery disease. After developing an equation in a training set, this equation and two other equations developed by other investigators were validated in a test set. The study was performed at two university-affiliated Veteran's Affairs medical centers. PATIENTS: 1080 consecutive men studied between 1985 and 1995 who had coronary angiography within 3 months of the treadmill test. The population was randomly divided into a training set of 701 patients and a test set of 379 patients. Patients with previous coronary artery bypass surgery, valvular heart disease, marked degrees of resting ST depression, and left bundle branch block were excluded. MEASUREMENTS: Recording of clinical and exercise test data along with visual interpretation of the electrocardiogram recordings on standardized forms and abstraction of visually interpreted angiographic data from clinical catheterization reports. RESULTS: Simple clinical and exercise test variables improved the standard application of exercise-induced ST criteria for predicting severe coronary artery disease. By setting probability thresholds for severe disease of <20% and >40% for the three prediction equations, the agreement approach divided the test set into three groups: low risk (patients with all three equations predicting <21% probability of severe coronary disease), no agreement, and high risk (all three equations with >39% probability) for severe coronary artery disease. Because the patients in the no agreement group would be sent for further testing and would eventually be correctly classified, the sensitivity of the agreement approach was 89% and the specificity was 96%. The agreement approach appeared to be unaffected by disease prevalence, missing data, variable definitions, or even angiographic criteria. CONCLUSIONS: Requiring diagnosis of severe coronary disease to be dependent on agreement between these three equations has made them likely to function in all clinical populations. The agreement approach should be an efficient method for the evaluation of populations with varying prevalence of coronary artery disease, limiting the use of more expensive noninvasive and invasive testing to patients with a higher probability of left main or triple-vessel coronary artery disease. This approach provides a strategy that can be applied by inputting the results of basic clinical assessment into a programmable calculator or a computer to assist the practitioner in deciding when further evaluation is appropriate, thus assuring patients access to subspecialty care.     

Role of unphosphorylated, newly synthesized I kappa B beta in persistent activation of NF-kappa B

Stimulation with inducers that cause persistent activation of NF-kappa B results in the degradation of the NF-kappa B inhibitors, I kappa B alpha and I kappa B beta. Despite the rapid resynthesis and accumulation of I kappa B alpha, NF-kappa B remains induced under these conditions. We now report that I kappa B beta is also resynthesized in stimulated cells and appears as an unphosphorylated protein. The unphosphorylated I kappa B beta forms a stable complex with NF-kappa B in the cytosol; however, this binding fails to mask the nuclear localization signal and DNA binding domain on NF-kappa B, and the I kappa B beta-NF-kappa B complex enters the nucleus. It appears therefore that during prolonged stimulation, I kappa B beta functions as a chaperone for NF-kappa B by protecting it from I kappa B alpha and allowing it to be transported to the nucleus.     

Enhanced AP-1 binding in brain induced by D1 and D2 agonists in methamphetamine-sensitized rats

The activator protein-1 (AP-1) binding activities induced by a separate challenge with SKF38393 and quinpirole after 1 weeks' abstinence from chronic methamphetamine (4 mg/kg/day, 14 days) were increased significantly in the striatum, nucleus accumbens and cingulate cortex compared with the saline-treated controls. Quinpirole-, but not SKF38393-induced AP-1 binding activities were still significantly higher after a 4-week abstinence period in the chronic methamphetamine group than in the chronic saline control group. Downward sniffing, which occurred following a quinpirole-challenge, was significantly intensified after both a 1 and 4 weeks' abstinence from chronic methamphetamine. These results indicate that chronic administration of methamphetamine induces alterations of the interactions of dopamine D1 and D2 receptors which are reflected as enhanced AP-1 binding activities.     

G-protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G-protein recognition

Asian students of seven Japanese language schools participated, and data of 292 Chinese, Taiwanese, and Korean students were analyzed in this study. They were asked about (1) attitudes toward their own and other cultures, (2) high regard for their country and culture, (3) self-efficacy and social skills at the moment and when they were in their country, (4) aspired level of social skills in this country, and (5) feeling of adjustment to life in Japan. Main findings were as follows: (1) psychological factors had stronger effects on the feeling than demographic factors. Self-efficacy in particular had a strong effect. (2) Attitudes to own and other cultures were related to self-efficacy and the feeling. (3) Structural analysis revealed a difference in the feeling between students from socialist and capitalist regions. Based on the analysis, a causal model was proposed of psychological and demographic factors leading to feeling of adjustment, and Asian students' adjustment to life in Japan was discussed in terms of adjustment to their inner, psychological environment.     

Effects of FK506 on experimental membranous glomerulonephritis induced by cationized bovine serum albumin in rats

BACKGROUND: There have been no reports on the effect of FK5 06, a new immunosuppressive agent, on experimental membranous glomerulonephritis (MN) induced by an exogenous antigen. Therefore we investigated the effects of FK506 on MN induced by cationized bovine serum albumin (C-BSA) in rats. METHODS: Two weeks after the rats were immunized with 1 mg of C-BSA and Freund's complete adjuvant, they received intravenous injections of 3 mg/kg of C-BSA 3 times weekly for 4 weeks. Rats were divided into four groups: group A (n = 5) received intramuscular injections of 3 mg/kg of FK506 daily for 5 days beginning 2 days before the first immunization; group B (n =4) received 1 mg/kg of FK506 daily for 2 weeks beginning 2 weeks after the first immunization; and group C (n =4) received 1 mg/kg of FK506 daily for 2 weeks beginning 4 weeks after the first immunization. Group D (n = 5) received no FK506 and served as the control group. Rats were sacrificed 8 weeks after the first immunization. RESULTS: Administration of FK506 in the preimmunization stage almost completely suppressed the development of MN in group A. Histological findings in groups B and C were similar to those in group D, the control group. However, urinary protein excretion was significantly lower in group B (24+/-46 mg/day) and C (220+/-44 mg/day) than in group D (330+/-61 mg/day). Expression of intracellular adhesion molecule-1 in glomeruli and the number of leukocyte functioning-associated molecules-1 were significantly decreased in groups A, B, and C compared with the control group. Administration of FK506 was not associated with any significant side-effects or histological abnormalities. The whole-blood trough levels of FK506 in groups B and C were 9.1 ng/ml and 9.2 ng/ml respectively. CONCLUSIONS: The efficacy of FK506 was significantly increased when the drug was administered in the early phase of immunization in this model. The present study suggests that FK506 may be useful in patients with intractable nephrotic syndrome such as MN.     

NF-kappaB and AP-1 activation by nitric oxide attenuated apoptotic cell death in RAW 264.7 macrophages

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 microM) or with lipopolysaccharide, interferon-gamma, and NG-monomethyl-L-arginine (LPS/IFN-gamma/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-gamma/NMMA prestimulation activated nuclear factor (NF)-kappaB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-kappaB and activator protein-1 (AP-1). NF-kappaB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-kappaB site derived from the murine Cox-2 promoter, confirmed NF-kappaB activation after NO addition. An NF-kappaB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-gamma/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-kappaB activation, a low-level NO-elicited Cox-2 response required activation of both NF-kappaB and AP-1.     

Transcriptional activation of NF-kappa B activity by Epstein-Barr virus (EBV) LMP1 as a selective therapeutic strategy for EBV-associated diseases

Epstein-Barr virus (EBV) has been known to be associated with many malignant tumors, including nasopharyngeal carcinoma (NPC). Previous studies have indicated that an EBV-encoded oncoprotein, latent membrane protein 1 (LMP1), is expressed in many NPC tissues. LMP1 has been shown to stimulate HIV LTR through the two NF-kappa B binding sites within this promoter. In this study, we examined the feasibility of using this property of LMP1 as a therapeutic strategy for the treatment of NPC. This therapy consists of the preferential killing of the LMP1-expressing cells by gene transfer using the NF-kappa B-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system. The 800-bp HIV-LTR, which contains two NF-kappa B binding sites, was used to drive the HSVtk gene. Stable C33A cell clones expressing the LMP1 and the HSVtk genes were subjected to the GCV sensitivity test. Results showed that cells expressing both the LMP1 and the HSVtk genes were highly sensitive to GCV treatment. These cells were introduced into nude mice subcutaneously and tumors became palpable within 2 weeks. GCV was then introduced intraperitoneally to these mice and the sizes of the tumors were measured daily. Results showed that the tumors regressed in the group of mice carrying cells that stably expressed both the LMP1 and the HSVtk genes, but not in mice carrying cells containing LMP1 or HSVtk alone. Our data indicate that the HSVtk gene expressed from a NF-kappa B-binding motif-containing promoter that is regulated by LMP1 may be used as an in vivo gene therapy strategy of EBV LMP1-expressing cancers such as NPC.