Search

Hormonal induction triggers a cascade leading to the expression of CCAAT/enhancer-binding protein(C/EBP)alpha and peroxisome proliferator-activated receptor (PPAR) gamma, C/EBPalpha, and PPARgamma turns on series of adipocyte genes that give rise to the adipocyte phenotype. Previous findings indicate that C/EBPbeta, a transcriptional activator of the C/EBPalpha and PPARgamma genes, is rapidly expressed after induction, but lacks DNA-binding activity and therefore cannot activate transcription of the C/EBPalpha and PPARgamma genes early in the differentiation program. Acquisition of DNA-binding activity of C/EBPbeta occurs when CHOP-10, a dominant-negative form of C/EBP family members, is down-regulated and becomes hyperphosphorylated as preadipocytes traverse the G1-S checkpoint of mitotic clonal expansion. Evidences are presented in this report that lactacystin, a proteasome inhibitor, up-regulated the CHOP-10 expression, blocked the DNA-binding activity of C/EBPbeta, and subsequently inhibited MCE as well as adipocyte differentiation.

PAI-1 is expressed and secreted by adipose tissue which may mediate the pathogenesis of obesity-associated cardiovascular complications. Evidence is presented in this report that PAI-1 is not expressed by preadipocyte, but significantly induced during 3T3-L1 adipocyte differentiation and the PAI-1 expression correlates with the induction of peroxisome proliferator-activated receptor gamma (PPARgamma). A peroxisome proliferator responsive element (PPRE)-like cis-element (-206TCCCCCATGCCCT-194) is identified in the mouse PAI-1 gene promoter by electrophoretic mobility shift assay (EMSA) combined with transient transfection experiments; the PPRE-like cis-element forms a specific DNA-protein complex only with adipocyte nuclear extracts, not with preadipocyte nuclear extracts; the DNA-protein complex can be totally competed away by non-labeled consensus PPRE, and can be supershifted with PPARgamma antibody. Mutation of this PPRE-like cis-element can abolish the transactivation of mouse PAI-1 promoter mediated by PPARgamma. Specific PPARgamma ligand Pioglitazone can significantly induce the PAI-1 expression, and stimulate the secretion of PAI-1 into medium.

Background: Up to now there have been no satisfactory drugs to treat psychiatric disorders, and now bioactive compound from entomagenous fungi (BCEF0083) is a new type of bioactive compound from entomopathogenic fungi. Our previous investigations have shown that BCEF has an inhibition effect on monoamine oxidase. So, BCEF may be a latent antidepressant. This study aimed at observing the antidepressant effects and its mechanism of BCEF in the chronic unpredictable stress models in mice., Methods: The antidepressant effects of BCEF were examined on the chronic unpredictable stress models in mice. Sixty mice were randomly divided to six groups. Animals were housed and isolated except saline group. Mice were exposed to different stressors per day randomly from day 1 to day 21. Body weight were weighed on day 1, day 10 and on day 21 during the 21-day stress procedure. Awarding response was detected by using method of calculating the 24-hour consumption of saccharum water. Step through test was used to evaluate the behavioral response. AVP contents in plasma were also detected by using radioimmunoassays., Results: Chronic unpredictable stress resulted in a significant decrease of the body weight and could apparently cause escape behavior disturbance and gradual reduction of sensitivity to reward in animal models. Drug treatment (BCEF 25, 50, 100 mg/kg) could significantly ameliorate the decreased body weight and effectively reverse the escape behavior disturbance. The gradual reduction of sensitivity to reward, the anhedonic state, was also effectively reversed by BCEF. BCEF (50, 100 mg/kg) could also effectively restore the AVP content in the plasma., Conclusions: This evidence suggests that BCEF can effectively inhibit the depression behavior and show strong antidepressant effect. BCEF can effectively restore the plasma AVP release and this may be an important mechanism of its antidepressant effect.

Tumor angiogenesis plays a pivotal role in the progress of tumor. Among the various endogenous angiogenic inhibitors discovered, the human plasminogen kringle 5 (K5) has been demonstrated to be a potential inhibitor of the proliferation and migration of vascular endothelial cells in vitro. The replication-incompetent adenovirus (Ad) vector Adeno-X-CMV-K5 (Ad-K5) (where CMV is cytomegalovirus) was constructed and its antiangiogenic effect was tested on vascular endothelial cell and tumor cell. For the construction, the K5 cDNA was fused in-frame with human plasminogen signal sequence and inserted into the eukaryotic expression vector pcDNA3 to form pcDNA3K5. The recombinant plasmid was subcloned into the shuttle plasmid pShuttle under the control of the constitutive CMV immediate-early promoter. The plasmid carrying the cDNA for K5 (pShuttleKS) was then recombined with the Adeno-X viral DNA and transformed into E. coli DH5alpha. The resultant recombinant plasmid pAd-K5 was transfected into human embryonic kidney (HEK) 293 cells with liposome. The adenovirus expressing human plasminogen kringle 5 (Ad-K5) was successfully packaged and propagated in 293 cells, as detected by the cytopathic effect (CPE) on the cells, and the viral titer in the supernatant was 5 x 10(8) pfu/mL by plaque assay. Both human umbilical vein endothelial cell line ECV304 and human breast carcinoma cell line MDA-MB-231 were infected with Ad-K5 and Ad-LacZ, which was used the negative control, and assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared with uninfected control and Ad-LacZ infected control, Ad-K5 infected group at 80 MOI (multiplicity of infection) significantly inhibited ECV304 proliferation; the difference between uninfected control and Ad-LacZ infected control was not significant. In contrast, there was no significant difference in the proliferation of MDA-MB-231 among all the treatments. In addition, the Ad-K5 at 100 MOI inhibited the differentiation and tube formation of ECV304 on ECMatrix gel. These results suggested that the recombinant replication-defective Adenovirus expressing human plasminogen kringle 5 inhibited the proliferation, differentiation and tube formation of ECV304 and had no effect on the proliferation of MDA-MB-231. Adenovirus mediated human plasminogen kringle 5 gene therapy may be a potential treatment of cancer through angiogenesis inhibition.

Aim: To study the protective effects of Ginkgo biloba extract (GbE) on gastric mucosa., Methods: By means of restaint-cold stress (RCS) in rats and 100% ethanol gavage in mice, the index of gastric mucosal injury was evaluated. The gastric juice was collected using pyloric ligation, and the volume and acidity of juice, and activity of pepsin were determined. The content of malondialdehyde (MDA) was measured by thiobarbituric acid (TBA) method., Results: GbE (25, 50, and 100 mg/kg, bid x 5 d, ig) inhibited dose-dependently the gastric mucosal injury induced by RCS and 100% ethanol gavage. The index of gastric mucosal injury after RCS in groups pretreated with GbE was 58%, 43%, and 31% of control group respectively. The index of gastric mucosal injury induced by ethanol in groups pretreated with GbE was 62%, 36%, and 26% of the control group, respectively. And GbE enhanced the protective effects of cimetidine (Cim) on gastric mucosa. But it did not obviously influence the volume and acidity of gastric juice as well as the activity of pepsin. One hour after the administration of ig 100% ethanol, the contents of MDA in gastric mucosa and serum in mice increased (P < 0.01) vs the control group. But pretreatment with GbE (25, 50, and 100 mg/kg, ig) could inhibit this increase of MDA both in gastric mucosa and in serum., Conclusion: GbE had protective effects on gastric mucosa and GbE plus Cim possessed the synergism in the treatment of acute gastric mucosal lesions.

Glucose and insulin stimulate leptin gene expression in vitro and in vivo. To identify cis-elements that are responsible for the glucose and insulin effects, mouse 3T3-L1 adipocytes were transiently transfected with reporter constructs with serial deletions in mouse ob gene promoter. The cis-elements were identified with Gel mobility shift assays (GMSA), DNase I footprint assays and PCR mediated site-directed mutation assays. Transient transfections detected a negative cis-acting element, a glucose-responsive element (GLRE), and an insulin-responsive element (IRE) in the region from -1 719 bp to -1 452 bp of mouse ob gene. This region does not contain any known GLRE or IRE. GMSA identified a DNA binding protein which specifically binds a native probe prepared from mouse ob gene promoter (-1 719 bp/-1 452 bp), and the binding was repressed by glucose or insulin. DNase I footprint assays and PCR mediated site-directed mutations assays identified that the binding motif AGCAAAA, spanning -1 698 bp to -1 692 bp of the mouse ob gene promoter, was responsible for the effects of glucose and insulin on ob gene expression. These studies suggest that a negative cis-acting element is located between -1 719 bp and -1 452 bp of the mouse ob gene promoter, and glucose and insulin simulate mouse ob gene expression by repressing the binding of a transcription factor to this element. This element, AGCAAAA, spanning -1 698 bp to -1 692 bp is a novel GLRE and IRE.

Relative quantitation RT-PCR was used to investigate the regulation of leptin expression in 3T3-F442A adipocytes by glucose and insulin. The results showed that glucose and insulin stimulated the expression of leptin in 3T3-F442A adipocytes, but they did not act synergically. Over-high concentration of glucose suppressed the expression of leptin and the effect of insulin. The elevation of the expression of leptin was characterized with saturation. The concentration of glucose is very important for the regulation of leptin expression.

Aim: To study the effects of hyperin (Hyp) on free intracellular calcium concentration ([Ca2+]i) of brain cells., Methods: The neonatal rat brain cells were dissociated. [Ca2+]i in presence and absence of extracellular high K+, L-glutamic acid (Glu), 5-hydroxytryptamine (5-HT), and norepinephrine (NE) were assayed with Fura 2-AM., Results: The resting [Ca2+]i in Hanks' solution (CaCl2 1.3 mmol.L-1) was (208 +/- 12) nmol.L-1 (n = 17). Hyp had no significant effects on the resting [Ca2+]i. Hyp 1.0, 4.0, and 16.0 mumol.L-1 markedly inhibited the increase of [Ca2+]i evoked by K+ 50 mmol.L-1 in a concentration-dependent manner. Hyp 16.0 mumol.L-1 inhibited the increases of [Ca2+]i induced by NE 1, 2, 4, and 8 mumol.L-1. Hyp (16.0 mumol.L-1) also markedly attenuated 5-HT and Glu-induced increase of [Ca2+]i., Conclusion: Hyp possessed inhibitory effects on influx of Ca2+ in the neonatal rat brain cells.

Tolerance to experimental autoimmune myasthenia gravis by nasal administration of microgram amounts of acetylcholine receptor (AChR) has been reported. To elucidate the mechanisms behind tolerance induction via the respiratory tract and the involvement of CD4(+) T cells, we established AChR-specific CD4(+)CD8(-) T cell clones from nasally tolerized rats. Nasal tolerance decreased leukocyte function-associated antigen-1 (LFA-1) expression in CD4(+) T cells from tolerized rats. There was no difference between nasally tolerized and control rats in expression of intercellular adhesion molecule-1. The levels of transforming growth factor-beta (TGF-beta) mRNA-expressing cells were upregulated in CD4(+) T cell clones after tolerance induction. These findings suggest that decreased LFA-1 expression in CD4(+) T cells contributes to reduction of the infiltration of inflammatory CD4(+) T cells, while upregulated TGF-beta may inhibit lymphocyte functions., (Copyright 1998 Academic Press.)

Various mouse and rat strains show different susceptibilities to experimental autoimmune myasthenia gravis (EAMG) that can be induced by immunization with acetylcholine receptor (AChR) and Freund's complete adjuvant, and represents a model for the antibody-mediated myasthenia gravis in humans. We examined AChR-induced B and T cell responses and cytokine mRNA expression to study the mechanisms behind susceptibility to EAMG in Lewis rats and resistance in Wistar Furth (WF) rats. Both strains had similarly elevated concentrations and affinities of serum anti-AChR antibodies, and no difference between the two strains for frequencies of cells in lymphoid organs expressing mRNA of the B cell stimulating cytokine interleukin-4 was found. In contrast, T cell responses to AChR measured by proliferation and by enumeration of interferon-gamma-expressing cells at both mRNA and protein level were lower in the resistant WF rats. This strain showed, instead, an up-regulation of the anti-inflammatory transforming growth factor-beta. Strain-related differences in the susceptibility to actively induced EAMG are thus related to quantitative differences in distribution between pro-inflammatory and anti-inflammatory cytokines.

Nitric oxide (NO) may be involved in myelin and oligodendrocyte injury associated with multiple sclerosis (MS), a demyelinating disease of unknown etiology. The cerebrospinal fluid (CSF) from MS patients may provide an important signal inducing a pathologic process within the central nervous system (CNS). To investigate this question, CSF-induced NO production by glial cells was studied in 38 patients with multiple sclerosis (MS), 30 patients with other CNS inflammatory diseases (ID) and 20 with tension headache (TH) as control. Neuron damage was estimated by release of lactate dehydrogenase (LDH), whereas oligodendrocyte damage was estimated by a percentage of viable cells in primary oligodendrocyte cultures. Here we show that CSFs from 13/38 (34%) patients with MS stimulate glial cells to produce NO, compared to 2/20 (10%) of patients with ID and 1/30 (3%) with tension headache. The levels of NO production correlated positively with the amounts of LDH released and negatively with percentage of viable oligodendrocytes, suggesting that NO may represent a mechanism for oligodendrocyte losses in affected tissues and play a role in lesion formation in MS and its animal model experimental allergic encephalomyelitis (EAE).

Aim: To study the protective and antiperoxidative effects of hyperin (hyperoside; quercetin-3-O-galactoside; Hyp) on myocardial ischemia/reperfusion., Methods: The rabbit anterior descenging branch of left coronary artery was occluded for 60 min and then released to allow reperfusion for 20 min. Hemodynamics (LVP, LV +/- dp/dt) and electrocardiogram (ECG, lead II) were monitored continuously with polygraph. After reperfusion, the blood sample and myocardium were taken to assay plasma creatine phosphokinase (CPK), lactate dehydrogenase (LDH), and cations in myocardium. Using a Langendorff system, the isolated heart of rat was initiated by ischemia for 40 min followed by 30 min of reperfusion. Malondialdehyde (MDA) contents of cardiac effluent and myocardium were measured with fluorescence spectrophotometer., Results: Hyp 10 mg.kg-1 i.v. depressed changes in LVP, LV +/- dp/dtmax, ECG, plasma CPK, LDH, and cations (Ca2+, Mg2+, Na+) in myocardium induced by ischemia/reperfusion in rabbits. Hyp 10 and 100 mumol.L-1 markedly reduced the increase in MDA production in isolated rat hearts after ischemia/reperfusion., Conclusion: Hyp possesses a protective effect against myocardial ischemia/reperfusion injury via attenuating lipid peroxidation.

Nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR plus Freund's complete adjuvant (FCA) resulted in prevention or marked decrease of the severity of EAMG, suppression of AChR-specific B cell responses and of AChR-reactive T cell functions. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radio-labelled synthetic oligonucleotide probes was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine IFN-gamma, the B cell stimulating IL-4 and the immune response-down-regulating TGF-beta. Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4 and TGF-beta mRNA-expressing cells compared with control rats receiving PBS nasally and injected with FCA only. Nasal tolerance to EAMG was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells, and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs compared with non-tolerized EAMG control rats. The relative affinity of anti-AChR antibodies was lower, but muscle AChR amounts were higher in nasally tolerized rats compared with non-tolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG, and that TGF-beta plays an important role in tolerance induction to EAMG.

We utilized a model of myelin basic protein (MBP) activation in vivo and MBP-stimulated cultures in vitro to study the influence of TGF-beta1 on glial cell proliferation and ICAM-1/leucocyte function-associated antigen-1 (LFA-1) expression, and to observe the antagonistic effects of TGF-beta1 and IFN-gamma. TGF-beta1 inhibited MBP-stimulated and MBP-activated glial cell proliferation, especially in MBP-stimulated separated microglia and astrocytes, and down-regulated the expression of ICAM-1 on MBP-stimulated glial cells and separated microglia. ICAM-1 expression on MBP-activated glial cells was intensely suppressed, whereas its expression on MBP-stimulated astrocytes was not influenced. TGF-beta1 had no effect on LFA-1 expression. In contrast, IFN-gamma up-regulated ICAM-1 expression, but inhibited proliferative response on MBP-stimulated glial cells when cultured without TGF-beta1. Examination of TGF-beta1 and IFN-gamma interactions revealed that TGF-beta1-mediated inhibition of proliferation and down-regulation of ICAM-1 on glial cells were prevented by IFN-gamma. The suppressive effect was re-established with high doses of TGF-beta1 in cultures, indicating that biological effects of TGF-beta1 vary depending on nitric oxide (NO) production, its concentration in the microenvironment and regulation of the cytokine network.

Oral and nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR and complete Freund's adjuvant (CFA) resulted in prevention or marked decrease of the severity of experimental autoimmune myasthenia gravis (EAMG) and suppression of AChR-specific B-cell responses and of AChR-reactive T-cell function. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabeled cDNA oligonucleotide proves was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine interferon-gamma (IFN-gamma), the B cell-stimulating interleukin-4 (IL-4), and the immunosuppressive transforming growth factor-beta (TGF-beta). Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-gamma, IL-4, and TGF-beta mRNA-expressing cells, compared to control rats receiving PBS orally or nasally and injected with CFA only. Oral and nasal tolerance was accompanied by decreased numbers of AChR-reactive IFN-gamma and IL-4 mRNA-expressing cells and strong up-regulation of TGF-beta mRNA-positive cells in lymphoid organs when compared to nontolerized EAMG control rats. The results suggest that IFN-gamma and IL-4 are central effector molecules in the development of EAMG and that TGF-beta plays an important role in tolerance induction to EAMG.

CD8+ T cells can perform both Th1- and Th2-like functions by producing cytokines such as interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), as well as the immune response down-regulating transforming growth factor-beta (TGF-beta), which are all involved in the development of experimental autoimmune myasthenia gravis (EAMG), a model for human MG. We have reported that depletion of CD8+ T cells results in the suppression of EAMG accompanied by the down-regulation of AChR-specific B cell responses and AChR-reactive IFN-gamma secreting Th1-like cells. To identify the involvement of IFN-gamma, IL-4 and TGF-beta in the development of EAMG after CD8+ T cell depletion, the expression of mRNA for these cytokines was studied in mononuclear cells from popliteal, inguinal and mesenteric lymph nodes, spleen and thymus by adopting in situ hybridization with complementary DNA oligonucleotide probes. Depletion of CD8+ T cells resulted in decreased levels of IFN-gamma and IL-4 mRNA expressing cells in different lymphoid organs except thymus, but no change in the numbers of TGF-beta mRNA expressing cells. The results imply that the suppression of EAMG after depletion of CD8+ T cells is caused by decreasing the effector factors but not by increasing the suppressor factor(s).

To understand the role of CD8+ T cells in experimental autoimmune myasthenia gravis (EAMG), CD8+ T cells were depleted by injecting a monoclonal anti-rat CD8 antibody (OX8) into Lewis rats immunized with Torpedo acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). CD8-depleted EAMG rats showed strikingly less muscle weakness and lower anti-AChR IgG antibody levels compared to Hy2-15-injected control EAMG rats. Moreover, the numbers of AChR-specific IgG antibody-secreting cells, AChR-reactive interferon-gamma-secreting T helper type 1-like cells and lymphocyte proliferation to AChR were lower in the CD8-depleted group than in control EAMG rats. These differences were significant among mononuclear cells from inguinal and popliteal lymph nodes, mesenteric lymph nodes and spleen, but not from thymus when examined 3, 5 and 7 weeks post-immunization. We suggest that CD8+ T cells are involved in the induction and persistance of EAMG by directly or indirectly affecting AChR-reactive T cells and anti-AChR IgG antibody-secreting cells.

Experimental autoimmune myasthenia gravis (EAMG) is a well established animal model, which can be induced in various animal species and strains with acetylcholine receptor (AChR) and represents an experimental counterpart of human myasthenia gravis (MG). Current immunotherapies of both EAMG and MG are non-specific and limited by their toxicity. Tolerance to EAMG has been achieved by oral administration of milligram quantities of Torpedo AChR. In the present report we demonstrate that nasal administration of microgram doses of Torpedo AChR to female Lewis rats prior to immunization with Torpedo AChR and complete Freund's adjuvant resulted in the prevention of subsequently induced EAMG, the suppression of serum anti-AChR antibody levels, the decrease of delayed-type hypersensitivity responses to AChR, as well as the suppression of AChR-specific immunoglobulin G-secreting cells, AChR-reactive interferon-gamma-secreting cells and T cell proliferation in peripheral lymphoid organs, particularly in popliteal and inguinal lymph nodes regional to immunization. We conclude that clinical signs of EAMG can be efficiently prevented by nasal administration of AChR in parallel with the downregulation of both B and T cell responses specific to AChR.

Hyperin(Hyp) was shown to inhibit the positive inotropic action of calcium ion on the isolated papillary muscles and shift the dose response curve of calcium ion to the right. As a plot lg(x-1) vs -lg(B) resulted in straight lines with slopes of -0.625. On the action potentials of guinea pig myocardial cells, Hyp markedly decreased the duration of plateau of action potentials (APD20%) but did not influence the APD100%, RP, APA, OS and Vmax. In the atrium preparations of mice, Hyp significantly inhibited influx of 45Ca induced by high K+. All findings indicate that Hyp can inhibit the inward flow of calcium ion.

In previous works, we reported that Hyp (hyperin) showed strong analgesic effect, and inhibited discharges induced by multianalgesics, but without anesthetic action. In this study, the analgesic mechanism of Hyp was further investigated. In the tail-flick test in mice, EGTA was shown to markedly enhance the analgesic effect of Hyp and shift the dose-effect curve to the upper-left. CaCl2 significantly antagonized the effect of Hyp and shifted the dose-effect curve to the lower-right. In the afferent discharges induced by analgesics, EGTA markly elevated while cacl2 lowered the effect of Hyp. It was also found that A23187, which promote influx of Ca2+, could antagonize the effect of Hyp. In the frog sciatic nerves, we found that Hyp could significantly inhibit influx of Ca2+. This finding suggests that the analgesic effect of Hyp may have close relationship with the reduction of calcium in afferent nerve endings.