MiR-20a suppresses proliferation and facilitates apoptosis of breast cancer cells via the MTOR signaling pathway.

OBJECTIVE: The paper aimed to explore the role of micro ribonucleic acid (miR)-20a in regulating the proliferation and apoptosis of breast cancer cells. MATERIALS AND METHODS: The expression of miR-20a in breast cancer cells was analyzed via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. Cell Counting Kit-8 (CCK-8) assay, colony formation assay, and flow cytometry were employed to analyze the proliferation and apoptosis of cells. Thereafter, the target proteins of miR-20a were predicted using TargetScan, a website for miRNA target gene prediction, and the interaction between miR-20a and the target genes was detected through the Luciferase reporter gene assay, qRT-PCR assay, and Western blotting. Finally, the miR-20a inhibitor and target gene expression plasmids were co-transfected for rescue experiment to study whether the target genes participate in the inhibitory effect of miR-20a on the proliferation of breast cancer cells. RESULTS: It was found that the expression of miR-20a was upregulated in breast cancer cell lines. Silencing miR-20a expression inhibited the proliferation and promoted the apoptosis of breast cancer cell. Besides, it was demonstrated that late endosomal/lysosomal adaptor, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) activator 3 (LAMTOR3) were a direct target of miR-20a. The knockdown of LAMTOR3 expression repressed the influence of miR-20a on the proliferation of breast cancer cells. CONCLUSIONS: MiR-20a targets LAMTOR3 gene to regulate the mTOR signaling pathway, thereby suppressing the proliferation and facilitating the apoptosis of breast cancer cells. It suggests that miR-20a exerts a carcinogenic effect in breast cancer, which may be a potential target for the treatment of breast cancer.

Activation of AMPK-SIRT3 signaling is chondroprotective by preserving mitochondrial DNA integrity and function.

OBJECTIVE: In osteoarthritis (OA), articular chondrocytes manifest mitochondrial damage, including mitochondrial DNA 4977-bp (mtDNA4977) deletion that impairs mitochondrial function. OA chondrocytes have decreased activity of AMPK, an energy biosensor that promotes mitochondrial biogenesis. Here, we tested if pharmacologic AMPK activation, via downstream activation of predominately mitochondrially localized sirtuin 3 (SIRT3), reverses existing decreases in mitochondrial DNA (mtDNA) integrity and function in human OA chondrocytes and limits mouse knee OA development. DESIGN: We assessed mtDNA integrity and function including the common mtDNA4977 deletion and mtDNA content, mitochondrial reactive oxygen species (mtROS) generation, oxygen consumption and intracellular ATP levels. Phosphorylation of AMPKα, expression and activity of SIRT3, acetylation and expression of the mitochondrial antioxidant enzyme SOD2 and DNA repair enzyme 8-oxoguanine glycosylase (OGG1), and expression of subunits of mitochondrial respiratory complexes were examined. We assessed effect of pharmacologic activation of AMPK on age-related spontaneous mouse knee OA. RESULTS: The mtDNA4977 deletion was detected in both OA chondrocytes and menadione-treated normal chondrocytes, associated with increased mtROS, decreased SIRT3, and increased acetylation of SOD2 and OGG1. AMPKα1 deficient chondrocytes exhibited significantly reduced SIRT3 activity. AMPK pharmacologic activation attenuated existing mtDNA4977 deletion and improved mitochondrial functions in OA chondrocytes via SIRT3 by reducing acetylation and increasing expression of SOD2 and OGG1, and limited aging-associated mouse knee OA development and progression. CONCLUSIONS: AMPK activation, via SIRT3, limits oxidative stress and improves mtDNA integrity and function in OA chondrocytes. These effects likely contribute to chondroprotective effects of AMPK activity.

Image-guided percutaneous liver biopsy in hepatosplenic gamma-delta T-cell lymphoma: a single centre experience.

OBJECTIVE: This study was to determine whether Image-Guided Percutaneous Liver Biopsy (IGPLB) is a safe and accurate procedure in patients with Hepatosplenic Gamma-Delta T-Cell Lymphoma (HSTCL) characterized by hepatosplenomegaly, and provide a rapid and safe diagnostic approach in this rare group of patients with a generally poor outcome. PATIENTS AND METHODS: We report five patients who underwent IGPLB for an initial diagnosis of HSTCL, in whom diagnosis by bone marrow biopsy and blood smear had failed. The patients presented with fever of undetermined origin, fatigue, night sweats and abdominal pain. Percutaneous liver biopsies were performed following location of the puncture point under the guidance of computed tomography (CT) or ultrasonography (US). RESULTS: CT and US can detect prominent hepatosplenomegaly without significant lymphadenopathy. IGPLB, assessed by immunohistochemical and molecular pathological analyses, was then performed without complications, which resulted in the diagnosis of HSTCL and facilitated treatment in a timely manner. CONCLUSIONS: IGPLB is a safe and accurate procedure in patients with suspected underlying malignant lymphoma characterized by hepatosplenomegaly, especially in those without readily accessible tissue amenable to biopsy.

Impact of Malnutrition on Physical, Cognitive Function and Mortality among Older Men Living in Veteran Homes by Minimum Data Set: A Prospective Cohort Study in Taiwan.

OBJECTIVES: To evaluate the prevalence of malnutrition and its impact on mortality, functional decline and cognitive impairment among elder residents in long-term care settings. DESIGNS: A prospective cohort study. SETTINGS: Two veteran homes in Taiwan. PARTICIPANTS: A total of 1,248 male residents aged equal or more than 65 years. MEASUREMENTS: Charlson's comorbidity index (CCI), Minimum data set (MDS), resident assessment protocols (RAP), Activity of daily living-Hierarchy scale, Cognitive Performance Scale, MDS Social engagement scale. RESULTS: The mean age of participants is 83.1 ± 5.1 years, and the prevalence of malnutrition was 6.1%. Inadequate dietary content (57.9%) and unintentional weight loss (31.6%) account for the majority of malnutrition identified by MDS tool. Higher 18-month mortality rate (25% vs. 14.2%), higher baseline CCI (median 1 vs. 0), and higher baseline sum of RAP triggers (median 8.5 vs. 5) were noted among residents with malnutrition. Furthermore, malnutrition was shown predictive for functional decline (OR: 3.096, 95% CI: 1.715-5.587) and potential cognitive improvement (OR: 2.469, 95% CI: 1.188-5.128) among survivors after adjustment for age, body mass index and CCI. CONCLUSION: Malnutrition among elder men residing in veteran homes was associated with multimorbidities and higher care complexity, and was predictive for mortality and functional decline.

Factor VII promotes hepatocellular carcinoma progression through ERK-TSC signaling.

We previously demonstrated PAR2 starts upstreamed with tissue factor (TF) and factor VII (FVII), inhibited autophagy via mTOR signaling in HCC. However, the mechanism underlying for merging functions of PAR2 with the coagulation system in HCC progression remained unclear. The present study aimed to investigate the role of TF, FVII and PAR2 in tumor progression of HCC. The expressions of TF, FVII and PAR2 from HCC specimens were evaluated by immunohistochemical stains and western blotting. We found that the expression of FVII, but not TF and PAR2, directly related to the vascular invasion and the clinical staging. Importantly, a lower level of FVII expression was significantly associated with the longer disease-free survival. The addition of FVII but not TF induced the expression of PAR2 and phosphorylation of ERK1/2, whereas knockdown of FVII decreased PAR2 expression and ERK1/2 phosphorylation in HCC cell lines. Furthermore, levels of phosphor-TSC2 (Ser664) were increased after treatment with FVII and PAR2 agonist whereas these were significantly abolished in the presence of a potent and specific MEK/ERK inhibitor U0126. Moreover, mTOR knockdown highly reduced Hep3B migration, which could be reverted by FVII but not TF and PAR2. These results indicated that FVII/PAR2 signaling through MEK/ERK and TSC2 axis for mTOR activation has potent effects on the migration of HCC cells. In addition, FVII/PAR2 signaling elicits an mTOR-independent signaling, which promotes hepatoma cell migration in consistent with the clinical observations. Our study indicates that levels of FVII, but not TF, are associated with tumor migration and invasiveness in HCC, and provides clues that evaluation of FVII expression in HCC may be useful as a prognostic indicator in patients with HCC and may form an alternative target for further therapy.

Interconnections between autophagy and the coagulation cascade in hepatocellular carcinoma.

Autophagy has an important role in tumor biology of hepatocellular carcinoma (HCC). Recent studies demonstrated that tissue factor (TF) combined with coagulation factor VII (FVII) has a pathological role by activating a G-protein-coupled receptor called protease-activated receptor 2 (PAR2) for tumor growth. The present study aimed to investigate the interactions of autophagy and the coagulation cascade in HCC. Seventy HCC patients who underwent curative liver resection were recruited. Immunohistochemical staining and western blotting were performed to determine TF, FVII, PAR2 and light chain 3 (LC3A/B) expressions in tumors and their contiguous normal regions. We found that the levels of autophagic marker LC3A/B-II and coagulation proteins (TF, FVII and PAR2) were inversely correlated in human HCC tissues. Treatments with TF, FVII or PAR2 agonist downregulated LC3A/B-II with an increased level of mTOR in Hep3B cells; in contrast, knockdown of TF, FVII or PAR2 increased LC3A/B. Furthermore, mTOR silencing restored the impaired expression of LC3A/B-II in TF-, FVII- or PAR2-treated Hep3B cells and activated autophagy. Last, as an in vivo correlate, we administered TF, FVII or PAR2 agonist in a NOD/severe combined immunodeficiency xenograft model and showed decreased LC3A/B protein levels in HepG2 tumors with treatments. Overall, our present study demonstrated that TF, FVII and PAR2 regulated autophagy mainly via mTOR signaling. The interaction of coagulation and autophagic pathways may provide potential targets for further therapeutic application in HCC.

Late-life metabolic syndrome prevents cognitive decline among older men aged 75 years and over: one-year prospective cohort study.

BACKGROUND: Although metabolic syndrome (MetS) has been reported to be associated with cognitive decline and dementia, little was known about late-life MetS and cognitive decline among older old population. The main purpose of this study was to evaluate the role of MetS and cognitive decline among men aged 75 and over in Taiwan. METHODS: This is a prospective cohort study which recruited men aged 75 years and older with intact cognitive function living in the Banciao Veterans Home, a retirement community for veterans in northern Taiwan. All participants received complete history taking, physical examinations, global cognitive tests and laboratory tests. Cognitive status was re-evaluated one year after enrollment to evaluate the role of MetS to cognitive decline in this study population. RESULTS: Overall, 338 people participated in the study and 62 of them were excluded due to low baseline MMSE score, and the remaining 276 people (mean age: 82.4±4.2 years) were enrolled for study. The prevalence of MetS and annual cognitive decline were 22.5% and 15.6%, respectively. During the follow-up period, 9 (3.3%) participants died, 229(83.0%) complete the study. Subjects with cognitive decline were older and had lower serum levels of serum total cholesterol. Multivariate logistic regression showed that older age (OR:1.13, 95% C.I.: 1.01-1.25, P=0.026) and central obesity (OR: 4.19, 95% CI: 1.26-13.91, P=0.019) were independent risk factors for cognitive decline; and MetS defined by Adult Treatment Panel III was a protective factor (OR: 0.20, 95% CI: 0.04-0.94, P=0.041). The protective effect of MetS remained the same when MetS was defined by the criteria of International Diabetes Federation. CONCLUSIONS: Age and central obesity were significant risk factors of cognitive decline, but late-life MetS, however defined, had protective effect on cognitive function. Further investigation is needed to clarify the possible mechanism of MetS and cognitive function in older adults.

Sex differences in U50,488H-induced phosphorylation of p44/42 mitogen-activated protein kinase in the guinea pig brain.

Recently there has been a widespread interest in the development of kappa opioid receptor (KOPR) ligands for treatment of pain, depression and anxiety, and prevention of stress-induced drug relapse. However, most of these preclinical studies have been conducted using male experimental animals. In the present study we examined if sex differences exist in neural activity induced by the KOPR agonist trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl) benzeneacetamide methanesulfonate (U50,488H). Here, we used immunohistochemistry to detect activation (phosphorylation) of p44/42 mitogen-activated protein kinase (MAPK) as an indicator of neural activity. Following habituation to injection for 3 days, adult guinea pigs received a single injection of U50,488H (5mg/kg, s.c.) and perfused 30-45 min later. U50,488H-induced an increase in the number of cells immuno-positive for phosphorylated p44/42 MAPK in subregions of the amygdala, thalamus, paraventricular nucleus of the hypothalamus, periaqueductal gray, and dorsal raphe nuclei. In contrast, U50,488H-induced a decrease in immuno-positive cells in the ventrolateral and lateral orbital cortex. Pretreatment with the KOPR antagonist norbinaltorphimine (10mg/kg, i.p.) 18 h prior to U50,488H significantly reversed the effects of U50,488H in most regions. In addition, we observed a notable sex difference in the basolateral amygdala; in males, U50,488H induced an increase in immuno-positive cell numbers but a decrease in females. However, across other brain regions males were generally more sensitive to U50,488H-induced alterations than females. These results suggest the need to include female subjects in studies examining emotional responses to KOPR ligands.

In vitro and in vivo therapeutics of ß-thujaplicin on LPS-induced inflammation in macrophages and septic shock in mice.

ß-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of ß-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-a gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of ß-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of ß thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-α were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-κB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, ß-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, ß-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-α production as well as iNOS, COX2, and NF- κB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, ß-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-α production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of ß-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-α and iNOS production. ß-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications. ß-thujaplicin, an active constituent from Chamaecyparis obtusa, has been shown to have acaricidal and antimicrobial effects. Very few studies have focused on the potential of the anti-inflammatory effect of ß-thujaplicin. Moreover, its capability of inhibiting inflammatory mediators e.g. TNF-alpha gene transcription, nitric oxide (NO) and prostaglandin E2, remains unknown. Besides those molecular mechanisms behind the anti-inflammatory effect of ß-thujaplicin, solid proof of its effectiveness in vivo has not yet been studied. In our study, in vitro effects of ß-thujaplicin were verified on RAW 264.7 macrophages which were stimulated by LPS. Indomethacin was used as a positive control. The inducible NO production after stimulation was measured by Griess reagent. PGE2, IL-6 and TNF-alpha were measured by ELISA methods. Protein expressions of iNOS, COX2, and NF-kB were evaluated by Western blotting. Septic ICR mice were administered 20 mg/kg of LPS and then the mortality rate was monitored. Within the concentration range which was devoid of cytotoxicty, ß-thujaplicin exhibited a clear dose-dependent inhibition on LPS-induced NO production. Furthermore, ß-thujaplicin inhibited LPS-induced PGE2, IL-6, and TNF-alpha production as well as iNOS, COX2, and NF-kB protein expression more substantially potent than indomethacin. In agreement with the in vitro study, ß-thujaplicin was shown to be effective in vivo for inhibiting LPS-induced NO and TNF-alpha production and a significant decrease in mortality rate of mice suffering from septic shock was observed. This study demonstrates the potential of ß-thujaplicin in treatment of inflammation and sepsis. These effects occur through an efficient blockage of TNF-alpha and iNOS production. ß-thujaplicin efficacy is comparable to that of indomethacin thus it can be a substitution but bear less depletion of PGE2, making this compound very promising in clinical applications.

CHMP6 and VPS4A mediate the recycling of Ras to the plasma membrane to promote growth factor signaling.

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III (Endosomal Sorting Complex Required for Transport-III), were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding loop, and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound to N-Ras but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked epidermal growth factor receptor (EGFR) recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling.

Reduced expression of the µ opioid receptor in some, but not all, brain regions in mice with OPRM1 A112G.

OPRM1 A118G is a common single nucleotide polymorphism (SNP) in the coding region of the human mu opioid receptor (MOPR) gene OPRM1. This SNP is associated with higher morphine doses required for postoperative analgesia as well as a variety of drug addiction phenotypes. A mouse model possessing the equivalent substitution (A112G) in the Oprm1 gene was generated to facilitate mechanistic studies. Mice homozygous for the G112 allele (G/G) displayed lower antinociception to morphine compared with those homozygous for A112 allele (A/A), similar to humans, suggesting that the mice are a good model to further characterize underlying factors contributing to phenotypes associated with this SNP. Here, we compared [³H]DAMGO binding to the MOPR in the brains of A/A and G/G mice using quantitative in vitro autoradiography. A/A mice exhibited higher [³H]DAMGO binding than G/G in the cingulate, motor, and insular cortices, nucleus accumbens core and shell, hypothalamus, thalamus, amygdala, periaqueductal gray, superficial gray of superior colliculus, and ventral tegmental area. No genotype differences were observed in somatosensory cortex, caudate putamen, and hippocampus. When males and females were examined separately, A/A mice showed higher [³H]DAMGO binding than G/G mice in more brain regions in males than in females. Radioligand binding using brain membranes also showed higher [³H]DAMGO binding in the cortex and thalamus in A/A mice than G/G mice but no genotype differences in the caudate putamen or hippocampus. Thus, the A112G SNP is associated with reduced MOPR expression in some, but not all, brain regions, and appears to have some sex differences. The elevated MOPR expression in periaqueductal gray and thalamus in A/A mice are consistent with their higher antinociceptive responses to morphine. The higher MOPR levels in nucleus accumbens and/or ventral tegmental area of A/A mice is consistent with the higher morphine-induced hyperactivity and locomotor sensitization observed in these mice. Thus, these results provide some insights into the observed decreased clinical opioid potency in humans with the A118G SNP.

A whole genome methylation analysis of systemic lupus erythematosus: hypomethylation of the IL10 and IL1R2 promoters is associated with disease activity.

The etiology of systemic lupus erythematosus (SLE) involves a complex interaction of genetic and environmental factors. Investigations have shown that environmentally driven epigenetic changes contribute to the etiology of SLE. Here, we hypothesize that aberrant DNA methylation may contribute to the activation of the immune machinery and trigger lupus disease activity. A whole genome methylation array was applied to investigate the DNA methylation changes between 12 pairs of active SLE patients and healthy controls. The results were further confirmed in 66 SLE patients, 102 healthy controls. The methylation statuses of the IL10 and IL1R2 genes were significantly reduced in the SLE patient samples relative to the healthy controls (age-adjusted odds ratios, 64.2 and 16.9, respectively, P<0.0001). There was a trend toward SLE patients having hypomethylated IL10 and IL1R2 genes accompanied by greater disease activity. We observed that the methylation degree of IL10 and IL1R2 genes were reduced in the rheumatoid arthritis (RA) patients as well but the hypomethylation change was more significant in IL1R2 genes than in the IL10 genes in RA patients. This study demonstrated that DNA hypomethylation might be associated with SLE. Hypomethylated IL10 and IL1R2 genes may provide potential epigenetic markers as clinical predictors for autoimmune diseases.

Changes in the incidence of candidaemia during 2000-2008 in a tertiary medical centre in northern Taiwan.

Candidaemia is associated with high mortality and high healthcare costs. The incidence of candidaemia in Taiwan rose markedly during the period 1980-2000. We conducted this hospital-based surveillance study in order to explore the secular trend in incidence of candidaemia during the period 2000 to 2008. In our study, Candida spp. were the fourth most common cause of bloodstream infections, with a 30-day crude mortality rate of 36.7%. Candida albicans was the most common species identified, although mortality rate did not differ significantly among species. The incidence of candidaemia began to decrease in 2004. Risk factors related to higher mortality included longer hospital stay before onset of candidaemia, liver cirrhosis, malignancy, end-stage renal disease requiring renal dialysis, dependence on mechanical ventilation and urinary catheterisation.

Over-expression of the Endocan gene in endothelial cells from hepatocellular carcinoma is associated with angiogenesis and tumour invasion.

Endocan plays a role in tumour angiogenesis and tumour growth. The aim of this study was to detect the expression of endocan in hepatocellular carcinoma (HCC) tumour-associated endothelial cells and to correlate endocan expression with clinicopathological parameters and tumour angiogenesis. Tumour tissues and surrounding non-cancerous hepatic parenchyma from 42 primary HCC patients were studied. Endothelial cells were isolated using magnetic microbeads conjugated with anti-CD31 and endocan expression was evaluated by real-time reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. Endocan was significantly over-expressed in endothelial cells isolated from HCC tumours compared with corresponding non-cancerous liver tissues. In addition, the endocan mRNA level was significantly correlated with the serum alpha-fetoprotein level, intra-tumoural microvessel density, vascular endothelial growth factor mRNA, and vascular and venous invasion. The over-expression of endocan in tumour endothelial cells was closely related to the process of angiogenesis and pathogenesis in HCC, and suggests that endocan might be a useful marker for HCC progression.

C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro.

With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result raises the possibility that C-INSL7 acts as a signaling molecule independent from INSL7 in the rat CNS.

Levamisole enhances immune response by affecting the activation and maturation of human monocyte-derived dendritic cells.

Levamisole is a synthetic phenylimidazolthiazole that was first introduced in 1966 as an anti-helmintic agent. Current studies have been focused upon its effect on immune response and on cancer treatment. We examined the molecular mechanisms of levamisole in the activation and maturation of human monocyte-derived dendritic cells (DC) and human T cells. Treatment of DC with levamisole increased the presentation of CD80, CD86, CD83 and human leucocyte antigen D-related (HLA-DR) molecules on the cell membrane, as well as the production of interleukin (IL)-12 p40 and IL-10. Levamisole-treated human DC also enhanced T cell activation towards type 1 T helper immune response by inducing interferon-gamma secretion. Neutralization with antibodies against Toll-like receptor (TLR)-2 inhibited levamisole-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-2 in signalling DC upon incubation with levamisole. The inhibition of nuclear factor-kappaB, extracellular signal-regulated kinases 1/2 or c-Jun N-terminal kinases pathways also prevented the effects of levamisole on DC in producing IL-12 p40 or IL-10. Taken together, levamisole could enhance immune response towards T helper 1 development through the activation of dendritic cells or T cell aspects.

The effect of triptolide on apoptosis of glioblastoma multiforme (GBM) cells.

Triptolide, derived from the traditional Chinese herb, Tripterygium wilfordii, sensitizes cancer cells to apoptosis. Glioblastoma multiforme (GBM), which accounts for most cases of central nervous malignancy, has a very poor prognosis and lacks effective therapeutic inventions. We, therefore, investigated the effects of different concentrations of, and different periods of exposure to, triptolide on cell proliferation and apoptosis in the glioma cell lines, U251MG and U87MG, and in normal human fetal astrocytes. Cell proliferation was investigated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay and growth curve analysis, and apoptosis was assessed from genomic DNA fragmentation. Triptolide showed dose-dependent inhibition of cell proliferation and induction of apoptosis in glioma cells. It also increased the ratio of the pro-apoptotic protein, Bax, to the anti-apoptotic protein, Bcl-2. Since U87MG has the wild-type p53 gene whereas U251MG harbours a mutated p53 gene, our results indicate that triptolide induces apoptosis in GBM cells via a p53-independent pathway. The dose-dependent inhibition of cell proliferation and induction of apoptosis by triptolide may involve upregulation of Bax and downregulation of Bcl-2.