Beta2-adrenoceptor agonists induce the mammalian clock gene, hPer1, mRNA in cultured human bronchial epithelium cells in vitro

The mammalian Per1 gene is one of the most important components of circadian clock function of the suprachiasmatic nucleus and peripheral tissues. We examined whether the β₂-adrenoceptor agonists, procaterol and fenoterol, induce human Per1 mRNA expression in human bronchial epithelium. The in vitro stimulation of β₂-adrenoceptor agonists in BEAS-2B cells led to a remarkable increase in the level of hPer1 mRNA. Moreover, fenoterol or procaterol induced the phosphorylation of CREB in BEAS-2B cells as verified by immunoblot analysis. β₂-adrenoceptor agonists induced human Per1 mRNA expression by the signaling pathways of cAMP-CREB in BEAS-2B cells.     

Treatment with beta2-adrenoceptor agonist in vivo induces human clock gene, Per1, mRNA expression in peripheral blood

This study examined whether in vivo exposure to a β₂-adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real-time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.     

Dexamethasone Influences Human Clock Gene Expression in Bronchial Epithelium and Peripheral Blood Mononuclear Cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS‐2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (～07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time‐PCR analysis. DEX stimulation of human BEAS‐2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.     

Expression of clock genes in human peripheral blood mononuclear cells throughout the sleep/wake and circadian cycles

The rhythmic expression of circadian clock genes in the neurons of the suprachiasmatic nucleus (SCN) underlies the manifestation of endogenous circadian rhythmicity in behavior and physiology. Recent evidence demonstrating rhythmic clock gene expression in non-SCN tissues suggests that functional clocks exist outside the central circadian pacemaker of the brain. In this investigation, the nature of an oscillator in peripheral blood mononuclear cells (PBMCs) is evaluated by assessing clock gene expression throughout both a typical sleep/wake cycle (LD) and during a constant routine (CR). Six healthy men and women aged (mean±SEM) 23.7±1.6 yrs participated in this five-day investigation in temporal isolation. Core body temperature and plasma melatonin concentration were measured as markers of the central circadian pacemaker. The expression of HPER1, HPER2, and HBMAL1 was quantified in PBMCs sampled throughout an uninterrupted 72 h period. The core body temperature minimum and the midpoint of melatonin concentration measured during the CR occurred 2:17±0:20 and 3:24 ±0:09 h before habitual awakening, respectively, and were well aligned to the sleep/wake cycle. HPER1 and HPER2 expression in PBMCs demonstrated significant circadian rhythmicity that peaked early after wake-time and was comparable under LD and CR conditions. HBMAL1 expression was more variable, and peaked in the middle of the wake period under LD conditions and during the habitual sleep period under CR conditions. For the first time, bi-hourly sampling over three consecutive days is used to compare clock gene expression in a human peripheral oscillator under different sleep/wake conditions.     

Glucocorticoids Affect 24 h Clock Genes Expression in Human Adipose Tissue Explant Cultures

Aims

to examine firstly whether CLOCK exhibits a circadian expression in human visceral (V) and subcutaneous (S) adipose tissue (AT) in vitro as compared with BMAL1 and PER2, and secondly to investigate the possible effect of the glucocorticoid analogue dexamethasone (DEX) on positive and negative clock genes expression.

Subjects and Methods

VAT and SAT biopsies were obtained from morbid obese women (body mass index≥40 kg/m²) (n = 6). In order to investigate rhythmic expression pattern of clock genes and the effect of DEX on CLOCK, PER2 and BMAL1 expression, control AT (without DEX) and AT explants treated with DEX (2 hours) were cultured during 24 h and gene expression was analyzed at the following times: 10:00 h, 14:00 h, 18:00 h, 22:00 h, 02:00 h and 06:00 h, using qRT-PCR.

Results

CLOCK, BMAL1 and PER2 expression exhibited circadian patterns in both VAT and SAT explants that were adjusted to a typical 24 h sinusoidal curve. PER2 expression (negative element) was in antiphase with respect to CLOCK and in phase with BMAL1 expression (both positive elements) in the SAT (situation not present in VAT). A marked effect of DEX exposure on both positive and negative clock genes expression patterns was observed. Indeed, DEX treatment modified the rhythmicity pattern towards altered patterns with a period lower than 24 hours in all genes and in both tissues.

Conclusions

24 h patterns in CLOCK and BMAL1 (positive clock elements) and PER2 (negative element) mRNA levels were observed in human adipose explants. These patterns were altered by dexamethasone exposure.     

Interaction of glucocorticoid analogues with the human glucocorticoid receptor

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.     

Separate induction of the two isozymes of cytochrome P-45011β in rat adrenal zona glomerulosa cells

In the rat adrenal cortex, two isozymes of cytochrome P-450_11β (CYP11B1 and CYP11B2) have been identified. They are encoded by two different genes with a homology much higher in their coding than in their 5′-flanking regions. CYP11B1 is found in all the zones of the gland and catalyzes a single hydroxylation of deoxycorticosterone (DOC) in the 11β- or the 18-position. CYP11B2 is produced exclusively in the zona glomerulosa and catalyzes all three reactions involved in the conversion of DOC to aldosterone. In vivo and in vitro, the expression of the genes encoding CYP11B1 and CYP11B2 is regulated by two separate control systems which appear to operate both independently and interdependently. In vivo, zona glomerulosa expression of CYP11B1 was enhanced by ACTH treatment or potassium depletion and was lowered by potassium repletion. CYP11B2 expression disappeared upon potassium depletion or ACTH treatment, but reappeared during potassium repletion. In vitro, only CYP11B1 activity was detectable and responsive to ACTH treatment in zona glomerulosa cells cultured at a potassium concentration of 6.4 mmol/1. Aldosterone biosynthetic activity and mRNA encoding CYP11B2 could be detected only after at least 1 day of exposure to a high extracellular potassium concentration ( 12 mmol/1).     

Identification of interleukin-13 related biomarkers using peripheral blood mononuclear cells

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma phenotype suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip^® DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-4, an anti-IL-13 monoclonal antibody (mAb) or an isotype control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human wingless-type MMTV integration site family member 5A), that showed increased expression in the IL-13 and IL-4 treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.     

Epigenetic reprogramming,gene expression and in vitro development of porcine SCNT embryos are significantly improved by a histone deacetylase inhibitor—m-carboxycinnamic acid bishydroxamide (CBHA)

Insufficient epigenetic reprogramming of donor nuclei is believed to be one of the most important causes of low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Previous studies have shown that both the in vitro and in vivo development of mouse SCNT embryos could be increased significantly by treatment with various histone deacetylase inhibitors (HDACi), including Trichostatin A, Scriptaid, and m-carboxycinnamic acid bishydroxamide (CBHA), in which only the effect of CBHA has not yet been tested in other species. In this paperweexamine the effect ofCBHAtreatment on the development of porcine SCNT embryos. We have discovered the optimum dosage and time for CBHA treatment: incubating SCNT embryos with 2 μmol/L CBHA for 24 h after activation could increase the blastocyst rate from 12.7% to 26.5%. Immunofluorescence results showed that the level of acetylation at histone 3 lysine 9 (AcH3K9), acetylation at histone 3 lysine 18 (AcH3K18), and acetylation at histone 4 lysine 16 (AcH4K16) was raised after CBHAtreatment. Meanwhile,CBHAtreatment improved the expression of development relating genes such as pou5f1, cdx2, and the imprinted genes like igf2. Despite these promising in vitro results and histone reprogramming, the full term development was not significantly increased after treatment. In conclusion, CBHA improves the in vitro development of pig SCNT embryos, increases the global histone acetylation and corrects the expression of some developmentally important genes at early stages. As in mouse SCNT, we have shown that nuclear epigenetic reprogramming in pig early SCNTembryos can be modified by CBHA treatment.     

Chronopharmacology of morphine in mice

Dosing-time-dependent changes in the effect and toxicity of morphine were examined in mice housed under alternating 12 h light (07:00 to 19:00 h) and dark (19:00 to 07:00 h) cycles. Morphine (0.5 mg/kg) was injected intraperitoneally (i.p.) in animals to assess its beneficial effect (i.e., protection against the kaolin-induced, bradykinin-mediated, writhing reaction) and its toxicity (i.e., alteration of the hepatic enzymes of aspartate aminotransferase [AST] alanine aminotransferase [ALT], and glutathione [GSH] in separate experiments). The magnitude of the analgesic effect of morphine depended on dosing time, with minimum effect at 02:00 h and maximum effect at 14:00 h. The serum hepatic enzyme levels of AST and ALT increased after dosing morphine (100 mg/kg) at 02:00 and 14:00 h. Time courses of these enzymes did not differ between the two trials. However, hepatic GSH, which is involved in the detoxification of chemical compounds, significantly decreased after i.p. morphine injection at 02:00 but not at 14:00 h. Overall, the results suggest that the analgesic effect of morphine is greater after dosing during the resting than during the activity phase of mice that have been induced with bradykinin-mediated pain. Drug-induced hepatic damage as inferred by GSH alteration, however, may be greater after dosing during the active phase.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Efficacy of thymol against Echinococcus granulosus protoscoleces

The aim of the present work was to determine the in vitro protoscolicidal effect of thymol against Echinococcus granulosus. Protoscoleces of E. granulosus were incubated with thymol at concentrations of 10, 5 and 1 μg/ml. The first signs of thymol-induced damage were observed between 1 and 4 days post-incubation. The maximum protoscolicidal effect was found with thymol at 10 μg/ml, viability reduced to 53.5 ± 11.9% after 12 days of incubation. At day 42, viability was 11.5 ± 15.3% and, reached 0% after 80 days. Thymol at concentrations of 5 and 1 μg/ml provoked a later protoscolicidal effect. Results of viability tests were consistent with the tissue damage observed at the ultrastructural level. The primary site of damage was the tegument of the parasite. The morphological changes included contraction of the soma region, formation of blebs on the tegument, rostellar disorganization, loss of hooks and destruction of microtriches. The data reported in this article demonstrate a clear in vitro effect of thymol against E. granulosus protoscoleces.     

可利霉素通过调控巨噬细胞极化影响黑色素瘤的增殖*

To investigate whether carrimycin (CAM) affects the occurrence and development of melanoma by regulating the polarization of macrophages, and the following related cell biology assays were used to examine its function. Methods: The effect of CAM on macrophage polarization was detected by real-time quantitative polymerase chain reaction (Q-RT-PCR) and Western blot. Flow cytometry and Cell Counting Kit-8 were used to detect the effect of CAM on mouse macrophages in vitro and in vivo phagocytosis and proliferation. Cell line-derived xenograft model was constructed via B16-F10 to evaluate the anti-tumor effect of CAM on melanoma. Results: In the mRNA level, CAM could up-regulate the levels of TNF-α and iNOS in M1 and down-regulate the level of Arg-1 in M2. In the protein level, CAM can increase the expression of p-STAT1 and decrease the expression of p-STAT3. In the cell line-derived xenograft model, these data shown that the occurrence and CAM development of melanoma was inhibited after CAM treatment, the tumor inhibition rate was 41.6%, and promoted the increase of the number of M1 macrophages (P<0.05). Conclusion: CAM promotes the increase in the number of M1 macrophages in vivo and inhibits the progression of melanoma, suggesting that CAM may achieve anti-tumor effects by inducing the polarization of macrophages to M1.     

Induction of oxidative cell damage by photo-treatment with zinc meta N-methylpyridylporphyrin

We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP^4 + ) can act as photosensitizers with efficacy comparable to that of hematoporphyrin derivative (HpD) in preventing cell proliferation and causing cell death in vitro. To better understand the biochemical basis of this activity, the effects of photo-activated ZnTM-3-PyP^4 +  on GSH/GSSG ratio, lipid peroxidation, membrane permeability, oxidative DNA damage, and the activities of SOD, catalase, glutathione reductase, and glutathione peroxidase were evaluated. Light exposure of ZnTM-3-PyP^4 + -treated colon adenocarcinoma cells caused a wide spectrum of oxidative damage including depletion of GSH, inactivation of glutathione reductase and glutathione peroxidase, oxidative DNA damage and peroxidation of membrane lipids. Cell staining with Hoechst-33342 showed morphological changes consistent with both necrotic and apoptotic death sequences, depending upon the presence of oxygen.     

Enhancement of myeloid cell growth by benzene metabolites via the production of active oxygen species

In low concentrations, benzene and its metabolite hydroquinone are known to have diverse biological effects on cells, including the synergistic stimulation with GM-CSF of hematopoietic colony formation in vitro, stimulation of granulocytic differentiation in vitro and in vivo, and general suppression of hematopoiesis in vivo. These chemicals are also known to be active in the induction of active oxygen species. We used several assays to determine the effects of benzene metabolites (hydroquinone, benzenetriol, benzoquinone) and active oxygen species (xanthine/xanthine oxidase) on cell growth and cell cycle kinetics of the human myeloid cell line HL-60. HL-60 cells treated with these chemicals for 2 h in PBS showed increased growth over untreated controls in a subsequent 18h growth period in complete media. Incorporation of ³H-thymidine was also increased proportionately by these treatments. Catalase treatment abrogated the increased cell growth of all chemicals, suggesting an oxidative mechanism for the effect of all treatments alike. Cell cycle kinetics assays showed that the growth increase was caused by an increased recruitment of cells from G₀/G₁ to S-phase for both hydroquinone and active oxygen, rather than a decrease in the length of the cell cycle. Benzene metabolite's enhancement of growth of myeloid cells through an active oxygen mechanism may be involved in a number of aspects of benzene toxicity, including enhanced granulocytic growth and differentiation, stimulation of GM-CSF-induced colony formation, apoptosis inhibition, and stimulation of progenitor cell mitogenesis in the bone marrow. These effects in sum may be involved in the benzene-induced “promotion” of a clonal cell population to the fully leukemic state.     

Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus

Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus. In A. fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified. One gene (AflMDR1) was isolated from A. flavus and is the apparent homologue to AfuMDR1. AfuMDR1and AflMDR1 encode proteins of molecular weights 148 000 and 143 000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites. These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site. The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1. The second gene identified in A. fumigatus, AfuMDR2, encodes a protein of molecular weight 85 000, containing four putative transmembrane domains and an ATP binding domain. The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae. Expression of AFUMDR1 in S. cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.