Analysis of the mechanism of the tight-junctional permeability increase by capsaicin treatment on the intestinal Caco-2 cells

In a previous experiment (Isoda et al., 2001), we showed that the tight-junctional (TJ) permeability increase in Caco-2 cells during capsaicin exposure was through binding of the capsaicin molecule to a capsaicin receptor-like protein. In the present study, we examined how actin, which modulates TJ permeability, is influenced by capsaicin. We showed that after treatment of the Caco-2 cells with capsaicin, the volume of F-actin decreased. Moreover, we also examined protein kinase C (PKC) and heat shock protein 47 (HSP47), which act as probable second messengers in causing TJ permeability increase. We showed that after capsaicin treatment, HSP47 was activated. However, PKC activity was the same in both control and treatment setups. These results suggest that, while PKC is not involved, it is highly possible that HSP47plays a role in TJ permeability increase in intestinal Caco-2 cells exposed to capsaicin. This revised version was published online in August 2006 with corrections to the Cover Date.     

Omega-3 fatty acids and neurological injury

Studies with omega-3 polyunsaturated fatty acids (PUFA) have shown that these compounds have therapeutic potential in several indications in neurology and psychiatry. Acute spinal cord injury (SCI) is an event with devastating consequences, and no satisfactory treatment is available at present. The pathogenetic mechanisms associated with SCI include excitotoxicity, increased oxidation and inflammation. We review here our recent studies, which suggest that omega-3 PUFA have significant neuroprotective potential in spinal cord trauma. In a first study, we administered an intravenous bolus of alpha-linolenic acid (LNA) or docosahexaenoic acid (DHA) 30 min after spinal cord hemisection injury in adult rats. The omega-3 PUFA led to increased neuronal and glial survival, and a significantly improved neurological outcome. In subsequent studies, we tested DHA in a more severe compression model of SCI. We also explored a combined acute and chronic treatment regime using DHA. Saline or DHA was administered intravenously 30 min after compression of the spinal cord. After injury, the saline group received a standard control diet, whereas DHA-injected animals received either a control or a DHA-enriched diet for 6 weeks following injury. We assessed locomotor recovery and analysed markers for cell survival and axonal damage, and we also investigated the effects of the treatment on the inflammatory reaction and the oxidative stress that follow SCI. We showed that the acute DHA treatment is neuroprotective after compression SCI, even if the treatment is delayed up to an hour after injury. The DHA injection led to an increased neuronal and glial cell survival, and the effect of the DHA injection was amplified by addition of DHA to the diet. Rats treated with a DHA injection and a DHA-enriched diet performed significantly better at 6 weeks in terms of neurological outcome. The analysis of the tissue after DHA administration showed that the fatty acid significantly reduced lipid peroxidation, protein oxidation and RNA/DNA oxidation, and the induction of COX-2. Parallel studies in a facial nerve injury model in mice also showed pro-regenerative effects of chronic dietary administration of DHA after nerve lesion. These observations suggest that treatment with omega-3 PUFA could represent a promising therapeutic approach in the management of neurological injury.     

Pro-apoptotic effect of aurothiomalate in prostate cancer cells

It has been recently demonstrated that small gold compounds could have a potential anti-tumoral activity. Here, we report that aurothiomalate (ATM), a gold compound already used in clinical therapy for the treatment of rheumatoid arthritis, has a pro-apoptotic effect in aggressive prostate cancer (PC3U) cells. In contrast, treatment of human primary epithelial prostate cells (PrEC) with ATM did not cause apoptosis. We demonstrated that ATM is able to disrupt the PKCι-Par6 complex in PC3U cells and that this disruption leads to the activation of ERK in a dose-dependent manner. Interestingly, we also showed that ERK acts upstream of the activation of caspase 3, leading to apoptosis. ATM treatment also causes activation of p38 and JNK MAP kinases. Moreover we could link ATM treatment to activation of the mitochondrial or so called intrinsic pathway, as we observed release of cytochrome c from mitochondria to cytoplasm, suggesting that the mitochondrial pathway is involved in the pro-apoptotic effect mediated by ATM. Taken together our data suggest that ATM could be a new promising drug for the treatment of advanced prostate cancer.     

Effects of Antidepressant Drug Imipramine on Gene Expression in Rat Prefrontal Cortex

We have investigated gene expression changes produced by acute and chronic daily treatment with a prototypical antidepressant, imipramine, using DNA microarrays. The analysis of similarities in gene expression patterns among functionally related genes revealed four expression profile cluster areas that showed a highly significant overrepresentation of several functional classes. Genes encoding for proteins involved in cAMP metabolism, postsynaptic membrane proteins, and proto-oncogenes were overrepresented in different cluster areas. Furthermore, we found that serine proteases as a group were similarly regulated by chronic antidepressant treatment. Our data suggest that cAMP metabolism, synaptic function, and protein processing by serine proteases may be important targets of antidepressant treatment and potential objects for antidepressant drug development.     

Neutral sphingomyelinase 2 induces dopamine uptake through regulation of intracellular calcium

Ceramide serves as a second messenger produced from sphingomyelin by the activation of sphingomyelinase (SMase). Here, we suggest that neutral SMase 2 (nSMase2) may regulate dopamine (DA) uptake. nSMase2 siRNA-transfected PC12 cells showed lower levels of nSMase activity and ceramide than scramble siRNA-transfected and control cells. Interestingly, transfection of nSMase2 siRNA or pretreatment with the nSMase2-specific inhibitor GW4869 resulted in decreased DA uptake. Reciprocally, exposure of PC12 cells to cell-permeable C₆-ceramide induced a concentration-dependent increase in DA uptake. Removal of extracellular calcium by EGTA increased DA uptake in scramble-transfected and control cells, but not in nSMase2 siRNA-transfected or GW4869-pretreated cells. Moreover, siRNA-transfected cells showed higher levels of intracellular calcium than scramble cells, while C₆-ceramide treatment resulted in decreased intracellular calcium compared to vehicle treatment alone. Taken together, these data suggest that nSMase2 may increase DA uptake through inducing ceramide production and thereby decreasing intracellular calcium levels.     

Evaluation of the use of the tobacco PR-1a promoter to monitor defense gene expression by the luciferase bioluminescence reporter system

Because of their marked responsiveness to induction signals, genes encoding pathogenesis-related proteins are used as markers to monitor defense gene expression in plants. To develop a non-invasive bioluminescence reporter assay system, we tested acidic PR-1 gene promoters from tobacco and Arabidopsis. These two promoters share common regulatory elements and are believed to show similar responsiveness to various stimuli but the results of transient expression assays by microprojectile bombardment of various plant cells and npr1 mutant Arabidopsis suggest that the tobacco PR-1a promoter is superior to its Arabidopsis counterpart in terms of responsiveness to salicylic acid treatment. Transgenic Arabidopsis seedlings harboring the tobacco PR-1a promoter fused to firefly luciferase showed marked induction in response to treatment with chemicals that induce defense gene expression in plants. These results suggest that the tobacco PR-1a promoter is applicable in monitoring defense-gene expression in various plant species.     

Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells

Adipocyte size is closely related to the occurrence of diabetes, metabolic syndrome, and insulin resistance. Thus, researchers are searching for active substances that function to reduce adipocyte size. In the present study, we focused on sugar cane vinegar, Kibizu, and evaluated the function of Kibizu to reduce adipocyte size by using an in vitro model system, because people in Amami Oshima famous for longevity regularly consume Kibizu. Results showed that Kibizu treatment significantly reduced the size and number of lipid droplets in 3T3-L1 cells, relative to treatment with Kurozu, another traditional vinegar. Results of an extraction experiment suggest that the active components in Kibizu are lipophilic and hydrophobic. In addition, an in vivo experiment on rats treated with Kibizu showed that the active components were contained in large vein blood. Results of an additional in vivo experiment suggest that metabolites generated by Kibizu-treated rats are primarily contained or modified specifically in the large vein blood.     

Vanillin Inhibits Translation and Induces Messenger Ribonucleoprotein (mRNP) Granule Formation in Saccharomyces cerevisiae: Application and Validation of High-Content,Image-Based Profiling

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells.     

The immunosuppressive agent 15-deoxyspergualin functions by inhibiting cell cycle progression and cytokine production following naive T cell activation

Immunosuppressive agents are commonly used in the prevention of graft rejection following transplantation and in the treatment of autoimmunity. In this study, we examined the immunosuppressive mechanism of the drug 15-deoxyspergualin (DSG), which has shown efficacy in the enhancement of graft survival and in the treatment of autoimmunity. Using a murine model of chronic relapsing and remitting experimental autoimmune encephalomyelitis, we were able to demonstrate that DSG both delayed and reduced the severity of experimental autoimmune encephalomyelitis. Subsequent in vitro studies to examine the mechanism of immune suppression showed that DSG was not able to inhibit early activation of naive CD4 T cells, but DSG did effectively inhibit the growth of naive CD4 T cells after activation. An analysis of cell proliferation and cell cycle showed that DSG treatment led to a block in cell cycle progression 2-3 days following Ag stimulation. In addition, DSG treatment inhibited the production of IFN-gamma by Th1 effector T cells. These studies suggest that CD4 T cells are a predominant target for DSG and the immunosuppressive effects of the drug may result from reduced CD4 T cell expansion and decreased polarization into IFN-gamma-secreting Th1 effector T cells in the induction of certain autoimmune disorders.     

厚藤ASR基因克隆及功能初步分析

通过对厚藤(Ipomoea pes-caprae(Linn.)Sweet.)cDNA文库的筛选,获得了一个编码厚藤ASR(ABA-stress-ripening)基因的全长cDNA,命名为IpASR。研究结果显示,IpASR编码区全长648 bp,共编码215个氨基酸;蛋白质等电点为5.42,分子量为24.57 k D。通过在酵母中表达,发现IpASR能够提高转基因酵母的耐盐性及抗氧化能力。进一步以厚藤成年植株及幼苗为材料进行实时荧光定量PCR分析,结果表明,IpASR基因在厚藤成年植株各组织中广泛表达;高盐、甘露醇胁迫和ABA处理可诱导该基因在厚藤幼苗中的表达。结合GFP融合蛋白的亚细胞定位和生物信息学分析,发现IpASR蛋白为核蛋白,推测IpASR基因参与了厚藤生长发育的调控,并可响应ABA和非生物胁迫的诱导。     

The role of intracellular glutathione in the progression of Chlamydia trachomatis infection

The productive internalization in the host cell of Chlamydia trachomatis elementary bodies and their infectivity depends on the degree of reduction of disulfide bonds in the outer envelope of the elementary body. We have hypothesized that the reducing agent may be intracellular glutathione (GSH). Three approaches were used to modulate the intracellular GSH concentration: (1) treatment of cells with buthionine sulfoximine, which causes irreversible inhibition of GSH biosynthesis; (2) hydrogen peroxide-induced oxidation of GSH by intracellular glutathione peroxidases; and (3) treatment of cells with N-acetyl-l-cysteine (NAC), a precursor of glutathione. In the first two cases, we observed a four- to sixfold inhibition of C. trachomatis infection, whereas in NAC-treated cells we detected an increase in the size of chlamydial inclusions. Using a proteomics approach, we showed that the inhibition of chlamydial infection does not combine with alterations in protein expression patterns after cell treatment. These results suggest that GSH plays a key role in the reduction of disulfide bonds in the C. trachomatis outer envelope at an initial stage of the infection.     

Peroxisome proliferator perfluorodecanoic acid alters glutathione and related enzymes

Previously we have shown that treatment with the peroxisome proliferator perfluorodecanoic acid (PFDA) significantly increased hepatic reduced glutathione (GSH) content without altering the activity of selenium-glutathione peroxidase. In this study we examined some potential mechanisms by which PFDA treatment increases GSH levels. Male Sprague-Dawley rats were given a single injection of 0, 8.8, 17.5, and 35 mg PFDA in corn oil per kg body weight. Twelve days later the effects of PFDA on the activities of enzymes associated with GSH synthesis, utilization, and regeneration were assessed. The results showed that in a dose-dependent manner, PFDA treatment significantly decreased the activity of gamma-glutamylcysteine synthetase, while the activities of NADPH-generating enzymes, malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase were increased. PFDA treatment also dose dependently decreased cytosolic, but not microsomal, glutathione S-transferase activity, and the activity of glutathione reductase was decreased by the highest dose of PFDA. The data obtained suggest that increased hepatic GSH levels following PFDA treatment may result from increased regeneration and/or decreased utilization.     

Pomegranate juice reduces oxidized low-density lipoprotein downregulation of endothelial nitric oxide synthase in human coronary endothelial cells.

We examined the hypothesis that pomegranate juice (PJ) can revert the potent downregulation of the expression of endothelial nitric-oxide synthase (NOSIII) induced by oxidized low-density liporotein (oxLDL) in human coronary endothelial cells. Western blot and Northern blot analyses showed a significant decrease of NOSIII expression after a 24-h treatment with oxLDL. Accordingly, we observed a significant dose-dependent reduction in nitric oxide bioactivity represented by both basal and bradykinin-stimulated cellular cGMP accumulation. These phenomena were corrected significantly by the concomitant treatment with PJ. Our data suggest that PJ can exert beneficial effects on the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in humans by enhancing the NOSIII bioactivity.     

Metformin inhibits melanoma development through autophagy and apoptosis mechanisms

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.     

Chronic Electroconvulsive Treatment Augments Coupling of the GTP-Binding Protein Gs to the Catalytic Moiety of Adenylyl Cyclase in a Manner Similar to That Seen with Chronic Antidepressant Drugs

A significant increase of guanylylimidodiphosphate (GppNHp)-, fluoride-, and forskolin-stimulated adenylyl cyclase was observed in synaptic membrane preparations from rat cerebral cortex subsequent to chronic electroconvulsive shock (ECS) treatment. This effect required at least five treatments over a course of 10 days. The inhibition of adenylyl cyclase induced by GppNHp was not affected by these treatments. The dissociation constant (KD) and maximal binding for the photoaffinity GTP analog, [32P]P3-(4-azidoanilido)-P1-5'-GTP [( 32P]AAGTP), to each of the synaptic membrane G proteins also were unchanged after ECS treatment. Nonetheless, the transfer of [32P]AAGTP from Gi to Gs, which we suggest is indicative of the coupling between Gs and the adenylyl cyclase catalytic moiety, was accelerated by chronic ECS treatment but not by acute or sham treatment. Furthermore, chemical uncoupling of Gs from adenylyl cyclase rendered membranes from treated animals indistinguishable from controls. Finally, in all cases tested, membranes prepared from animals subjected to chronic treatment with amitriptyline or iprindole showed similar changes in the Gs-mediated activation of adenylyl cyclase. Acute treatments produced effects similar to controls, and liver and kidney membranes from animals receiving chronic treatment showed no changes in adenylyl cyclase despite the marked changes seen in brain. These results suggest that chronic administration of ECS enhances coupling between Gs and adenylyl cyclase enzyme and modifies interactions between Gs and Gi.     

Plant genotypic diversity and environmental stress interact to negatively affect arthropod community diversity

Many studies have found positive relationships between plant diversity and arthropod communities, but the interactive effects of plant genetic diversity and environmental stress on arthropods are not well documented. In this study, we investigated the consequences of plant genotypic diversity, watering treatment, and its interaction for the ground-dwelling arthropod community in an experimental common garden of quaking aspen (Populus tremuloides Michx.). We found that varying plant genotypic diversity and watering treatment altered multivariate arthropod community composition and structure. Arthropod biodiversity and richness showed a distinct response to the plant diversity × watering treatment interaction, declining sharply in water-limited genotypic mixtures. Abundance of arthropod functional groups did not show any response to diversity or the plant diversity × watering treatment interaction, but varied in their response to watering treatment, with predator and detritivore abundance increasing and parasitoid abundance decreasing in well-watered blocks. Our results conflict with most previous studies, and suggest that environmental stress can substantially change the nature of the plant-arthropod diversity relationship. Additionally, we suggest that the plant-arthropod diversity relationship is dependent on the type of plant and arthropod species sampled, and that the association between tree diversity and ground-dwelling arthropods may be much different than more commonly studied grassland species and herbivorous arthropods.     

Emergent multiple predator effects in an experimental microbial community

Studies on multiple predator effects have typically focused on metazoans. Here we examined the effects of multiple consumers on bacteria. Specifically, we investigated the separate and combined effects of two common bacterivorous ciliates, Colpidium striatum and Paramecium caudatum, on a simple bacterial assemblage. Non-additive multiple predator effects emerged for all bacterial species significantly affected by grazing, where bacterial responses in the two-consumer treatment cannot be predicted by summing the separate effects of the two consumers. Some species showed risk reduction (observed responses less than expected), whereas others showed risk enhancement (observed responses larger than expected). We attributed risk alteration to the interference between the two consumers. Contradictory to theoretical predictions, total bacterial abundance in the two-consumer treatment did not differ from single-consumer treatments and consumer-free controls, due largely to risk reduction and compensatory responses within the bacterial assemblage. Decomposition of particulate organic matter was greater in the two-consumer treatment than the Paramecium single-consumer treatment, but did not differ among other treatments. These results suggest that the presence of multiple consumers may have unexpected impacts on the structure and functioning of bacterial communities.     

Histone deacetylase inhibitors facilitate dihydroartemisinin-induced apoptosis in liver cancer in vitro and in vivo

Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and p-ERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy.