Noradrenaline inhibits substantia gelatinosa neurons in mice trigeminal subnucleus caudalis via alpha(2) and beta adrenoceptors.

The actions of noradrenaline (NA) in the substantia gelatinosa (SG) are important for their antinociceptive effects. In order to identify the possible mechanisms underlying NA actions in the SG of trigeminal subnucleus caudalis (Vc), the direct membrane effects were examined by gramicidin-perforated patch clamp recording using brain slice preparation from immature mice brainstem. The majority (60/71, 85%) of neurons tested were hyperpolarized by NA application, and these hyperpolarizing effects were mimicked both by the alpha(2) adrenergic agonist, clonidine (18/28, 64%) and the beta adrenergic agonist, isoproterenol (9/24, 38%). NA-induced hyperpolarizing effect was also blocked by the alpha(2) adrenergic antagonist, yohimbine in five out of six neurons tested. However, a minority (5/71, 7%) of neurons tested were depolarized by NA, and these depolarizing effects were mimicked by the alpha(1) adrenergic agonist, phenylephrine (11/26, 42%). NA-induced hyperpolarizing effects were maintained in the presence of tetrodotoxin (TTX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), d,l-2-amino-5-phosphonopentanoic acid (AP5), picrotoxin and strychnine, a Na(+) channel, ionotropic glutamate receptor, GABA(A) and glycine receptor antagonists, respectively, indicating that the effects of NA are direct on the postsynaptic SG neurons. These results indicate that alpha(2) and beta adrenoceptor mediate inhibition, and alpha(1) adrenoceptor mediates facilitation of orofacial nociceptive processing in mouse trigeminal brainstem SG neurons by postsynaptic actions.

Reversal of the TPA-induced inhibition of gap junctional intercellular communication by Chaga mushroom (Inonotus obliquus) extracts: effects on MAP kinases.

Chaga mushroom (Inonotus obliquus) has continued to receive attention as a folk medicine with indications for the treatment of cancers and digestive diseases. The anticarcinogenic effect of Chaga mushroom extract was investigated using a model system of gap junctional intercellular communication (GJIC) in WB-F344 normal rat liver epithelial cells. The cells were pre-incubated with Chaga mushroom extracts (5, 10, 20 microg/ml) for 24 h and this was followed by co-treatment with Chaga mushroom extracts and TPA (12-O-tetradecanoylphorbol-13-acetate, 10 ng/ml) for 1 h. The inhibition of GJIC by TPA (12-O-tetradecanoylphorbol-13-acetate), promoter of cancer, was prevented with treatment of Chaga mushroom extracts. Similarly, the increased phosphorylated ERK1/2 and p38 protein kinases were markedly reduced in Chaga mushroom extracts-treated cells. There was no change in the JNK kinase protein level, suggesting that Chaga mushroom extracts could only block the activation of ERK1/2 and p38 MAP kinase. The Chaga mushroom extracts further prevented the inhibition of GJIC through the blocking of Cx43 phosphorylation. Indeed cell-to-cell communication through gap junctional channels is a critical factor in the life and death balance of cells because GJIC has an important function in maintaining tissue homeostasis through the regulation of cell growth, differentiation, apoptosis and adaptive functions of differentiated cells. Thus Chaga mushroom may act as a natural anticancer product by preventing the inhibition of GJIC through the inactivation of ERK1/2 and p38 MAP kinase.

Flow cytometric measurements of TB-specific T cells comparing with QuantiFERON-TB gold.

BACKGROUND: Interferon-gamma (IFN-gamma) release assays and the detection of IFN-gamma synthesis in the cytoplasm of activated CD4+ T cells by flow cytometry have recently been used for tuberculosis (TB) diagnosis. The aim of this study was to compare the performance of IFN-gamma assay between ELISA (QuantiFERON-TB Gold, QFT) and intracellular cytokine flow cytometry (ICCFC), and to investigate the significance of an optimal gating strategy in ICCFC. METHODS: The CD4+ T cell response to TB antigens was measured using the intracellular cytokine staining technique and four color FC (CD3, CD4, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha)) on whole blood samples. The results were compared with those of QFT. RESULTS: Regarding sensitivity in the TB group, patients in the QFT positive TB group (N = 22) were all ICCFC positive and 9 patients (64%) in the QFT negative TB group (N = 14) were ICCFC positive. In all test tubes (N = 72), sensitivity of "targeted" gating for TNF-alpha+ IFN-gamma+ CD4+ T cells was significantly higher than customary gating (72%, 54%, respectively, P = 0.001). CONCLUSIONS: The diagnostic sensitivity of ICCFC was further confirmed to be much higher than that of QFT. In the ICCFC analysis, TNF-alpha+ IFN-gamma+ CD4+ T cells should be sequentially gated through appropriately defined regions, minimizing interferents and reflecting characteristics of light scatter and marker expressions of CD4+ T cells activated by TB antigens.

[Measurement of amylase in saliva collected by salivette].

BACKGROUND: Saliva is increasingly being used as a specimen for systemic disease as well as for oral health status. Especially, salivary amylase has been studied as an excellent index for psychological stress. Authors evaluated the measurement of salivary amylase activities collected by Salivettes (Sarstedt, Germany). METHODS: Saliva specimens were collected from 13 healthy adults between 10:00 and 11:00 a.m. Participants were asked to gently chew tampons of Salivettes for 1 min. Immediately after collection, all specimens were stored frozen. On the day of testing, they were centrifuged after thawing and diluted with distilled water. Amylase was measured by Dimension RxL Max (Dade Behring Inc., USA). We evaluated precision, linearity, and recovery rate of Salivette. Amylase activities between collection of saliva by Salivette and passive drool were compared, and also the changes of amylase by the storage temperature were evaluated. RESULTS: Intra-run CVs for three levels of amylase were excellent. Between-day CVs and total CVs were good only for mid and high levels. A good linear relationship was found at all diluted levels. Dosing Salivettes with 2 mL, 1.5 mL, and 1 mL yielded sample recovery 85.5+/-2.4%, 82.4+/-1.5%, and 72.2+/-3.1%, respectively and amylase recovery 78.9+/-10.9%, 74.1+/-13.7%, and 37.3+/-26.9%, respectively. Amylase by Salivette and passive drool were correlated well (r=0.757), although they showed a significant difference. Amylase activity was not affected by the storage temperature. CONCLUSIONS: Measurement of salivary amylase using Salivette could be a useful test having good intra-run CVs and linearity. More than 1.5 mL of saliva would be needed to have more than 70% recovery of Salivette.