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.     

Endothelial nitric oxide synthase uncoupling as a key mediator of melanocyte malignant transformation associated with sustained stress conditions

Melanoma cell lines and cells corresponding to premalignant melanocytes were established by our group after subjecting a nontumorigenic murine melanocyte lineage, melan-a, to sequential cycles of anchorage blockade. Previous results showed that in melan-a cells the superoxide level increases after such procedure. Superoxide production during melanocyte de-adhesion was inhibited by L-sepiapterin, the precursor of eNOS cofactor BH4, and increased by the inhibitor of BH4 synthesis, DAHP, hence indicating a partial uncoupling state of eNOS. The eNOS uncoupling seems to be maintained in cells derived from melan-a, because they present decreased nitric oxide and increased superoxide levels. The inhibition of superoxide production in Tm5 melanoma cells with L-sepiapterin reinforces their eNOS-uncoupled state. The maintenance of oxidative stress seems to be important in melanoma apoptosis resistance because Mn(III)TBAP, a superoxide scavenger, or L-sepiapterin renders Tm5 cells more sensitive to anoikis and chemotherapy. More importantly, eNOS uncoupling seems to play a pivotal role in melanocyte malignant transformation induced by sustained anchorage impediment, because no malignant transformation was observed when L-NAME-treated melanocytes were subjected to sequential cycles of de-adhesion. Our results show that uncoupled eNOS contributes to superoxide production during melanocyte anchorage impediment, contributing to anoikis resistance and malignant transformation.     

Optimisation of a novel series of selective CNS penetrant CB(2) agonists

A series of benzimidazole CB₂ receptor agonists were prepared and their properties investigated. Optimisation of the three benzimidazole substituents led to the identification of compound 23, a potent CB₂ full agonist (EC₅₀ 2.7 nM) with excellent selectivity over the CB₁ receptor (>3000-fold). Compound 23 demonstrated good CNS penetration in rat. Further optimisation led to the identification of compound 34 with improved selectivity over hERG and excellent CNS penetration in rat.     

Clustering of nuclei in multinucleated hyphae is prevented by dynein-driven bidirectional nuclear movements and microtubule growth control in Ashbya gossypii

During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.     

Effects of menstrual cycle and physical training on heat loss responses during dynamic exercise at moderate intensity in a temperate environment

We evaluated the effects of the menstrual cycle and physical training on heat loss (sweating and cutaneous vasodilation) responses during moderate exercise in a temperate environment. Ten untrained (group U) and seven endurance-trained (group T) women (maximal O2 uptake of 36.7+/-1.1 vs. 49.4+/-1.7 ml.kg-1.min-1, respectively; P<0.05) performed a cycling exercise at 50% maximal O2 uptake for 30 min during both the midfollicular and midluteal menstrual phase in a temperate environment (ambient temperature of 25 degrees C, relative humidity of 45%). In group U, plasma levels of estrone, estradiol, and progesterone at rest and esophageal temperature (Tes) during exercise were significantly higher during the midluteal than during the midfollicular phase (P<0.05). Sweating rate and cutaneous blood flow (measured via laser-Doppler flowmetry) on the chest, back, forearm, and thigh were lower during the midluteal than during the midfollicular phase during exercise. Tes threshold for heat loss responses was significantly higher and sensitivity of the heat loss responses was significantly lower in the midluteal than in the midfollicular phase, regardless of body site. These effects of the menstrual cycle in group U were not observed in group T. The sweating rate and cutaneous blood flow were significantly higher in group T than in group U, regardless of menstrual phase or body site. Tes threshold for heat loss responses was significantly lower and sensitivity of heat loss responses was significantly greater in group T than in group U in the midluteal phase; however, sensitivity of the sweating response was significantly greater in the midfollicular phase. These results suggest that heat loss responses in group U were inhibited in the midluteal phase compared with in the midfollicular phase. Menstrual cycle had no remarkable effects in group T. Physical training improved heat loss responses, which was more marked in the midluteal than in the midfollicular phase.     

A 5-HT4 agonist, mosapride, enhances intrinsic rectorectal and rectoanal reflexes after removal of extrinsic nerves in guinea pigs

Distension-evoked reflex of rectorectal (R-R) contractions and rectointernal anal sphincter (R-IAS) relaxations can be generated in guinea pigs through an extrinsic sacral excitatory neural pathway (pelvic nerves) as well as intrinsic cholinergic excitatory and nitrergic inhibitory pathways. The aim of the present study was to create intrinsic R-R and R-IAS reflex models by pithing (destruction of the lumbar and sacral cords; PITH) and to evaluate whether the prokinetic benzamide mosapride, a 5-HT(4) receptor agonist, enhances these reflexes. The mechanical activities of the R-R and R-IAS were recorded in the anesthetized guinea pig on days 2-9 after PITH. Although the basal rectal pressure at distension after PITH was significantly lower than control, the reflex indexes of R-R contractions and synchronous R-IAS relaxations were unchanged between days 4 and 9 after PITH. The frequency of spontaneous rectal and IAS motility were also unchanged. Immunohistochemical studies revealed that the distribution of myenteric and intramuscular interstitial cells of Cajal (ICC) were not altered after PITH. Mosapride (0.1-1.0 mg/kg iv) dose-dependently increased both intrinsic R-R (maximum: 1.82) and R-IAS reflex indexes (maximum: 2.76) from control (1.0) 6-9 days after PITH. The 5-HT(4) receptor antagonist, GR-113808 (1.0 mg/kg iv) decreased the R-R and R-IAS reflex indexes by approximately 50% and antagonized the effect of mosapride (1.0 mg/kg iv). The present results indicate that mosapride moderately enhanced intrinsic R-R and R-IAS reflexes functionally compensated after deprivation of extrinsic nerves, mediated through endogenously active intrinsic 5-HT(4) receptors.     

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.     

Rosmarinic acid inhibits the formation of reactive oxygen and nitrogen species in RAW264.7 macrophages

Antioxidant action of Rosmarinic acid (Ros A), a natural phenolic ingredient in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, was analyzed in relation to the Ikappa-B activation in RAW264.7 macrophages. Ros A inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein synthesis induced by lipopolysaccharide (LPS), and also effectively suppressed phorbol 12-myristate 13-acetate (PMA)-induced superoxide production in RAW264.7 macrophages in a dose-dependent manner. Peroxynitrite-induced formation of 3-nitrotyrosine in bovine serum albumin and RAW264.7 macrophages were also inhibited by Ros A. Moreover, Western blot analysis demonstrated that LPS-induced phosphorylation of Ikappa-Balpha was abolished by Ros A. Ros A can act as an effective protector against peroxynitrite-mediated damage, and as a potent inhibitor of superoxide and NO synthesis; the inhibition of the formation of reactive oxygen and nitrogen species are partly based on its ability to inhibit the serine phosphorylation of Ikappa-Balpha.     

Evidence that estrogen regulates the sex change of honeycomb grouper (Epinephelus merra), a protogynous hermaphrodite fish

Circulating estradiol-17beta (E2) levels decrease precipitously during female to male (protogynous) sex change in fish. Whether this drop in E2 levels is a cause or consequence of sex change is still largely unknown. The present study treated adult female honeycomb groupers (Epinephelus merra) with aromatase inhibitor (AI, Fadrozole), either alone or in combination with E2, to investigate the role of estrogen in protogynous sex change. Control fish had ovaries undergoing active vitellogenesis; the gonads of AI-treated fish had already developed into testes, which produced sperm capable of fertilization. In contrast, co-treatment of fish with E2 completely blocked AI-induced sex reversal. AI treatment significantly reduced circulating levels of E2, whereas the addition of E2 to AI prevented the loss. The plasma androgen (testosterone and 11-ketotestosterone) levels were increased in the AI-treated fish, while the levels in the E2-supplemented fish were low compared to controls. Present results show that E2 plays an important role in maintaining female sex of hermaphrodite fishes, and that the inhibition of E2 synthesis causes oocyte degeneration leading to testicular differentiation in the ovary.     

Anti-citrullinated collagen type I antibody is a target of autoimmunity in rheumatoid arthritis

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, but its autoimmune mechanisms are not clearly understood. Recently, anti-citrullinated peptide antibodies have been specifically observed in sera of RA patients. Furthermore, we identified RA-susceptible variant in a gene encoding citrullinating enzyme, peptidylarginine deiminase type 4 (PADI4). Therefore, we hypothesized that proteins which are modified in RA synovium by PADI4 act as autoantigens. Subsequently, we obtained human collagen type I (huCI) as one of the autoantigens using a RA synoviocyte cDNA library by immunoscreening. We also investigated that the levels of anti-citrullinated huCI were significantly higher in RA patient sera than in normal control sera with high specificity (99%) and positively correlated with the levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies. We concluded that huCI is a novel substrate protein of PADIs and that citrullinated huCI is a candidate autoantigen of RA.