A new set of PCR assays for the identification of multiple human adenovirus species in environmental samples.

AIMS: To assess human adenovirus (HAdV) diversity in environmental samples based on sequence comparisons of hexon gene fragments amplified using newly designed HAdV-specific polymerase chain reaction (PCR) assays. METHODS AND RESULTS: Six PCR primer sets were designed based on 56 aligned hexon sequences from NCBI GenBank to amplify different hexon gene sections (241-349 bp) of the six HAdV species. The amplified hexon genes from wastewater samples were cloned, sequenced, and compared with those in publicly accessible databases (i.e. NCBI GenBank) by using the Blast program. A total of 46 analysed positive clones were affiliated to five HAdV serotypes, i.e. 1, 2, 12, 31 and 41. Similarities between the cloned and database hexon sequences ranged from 95.9 to 100% (with an average of 98.1 +/- 1.0%). CONCLUSION: The designed primers showed higher amplification efficiencies when compared with the existing assays. Using the new assays, HAdV species A, C, and F (serotypes 1, 2, 12, 31 and 41 in particular) were identified in the studied municipal wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: The six PCR primer sets developed in this study can be used to efficiently amplify hexon gene fragments in HAdV. Multiple HAdV serotypes identified in the municipal wastewater provide new information about HAdV diversity in environmental samples.

Mechanisms of resveratrol-induced changes in cytosolic free calcium ion concentrations and cell viability in OC2 human oral cancer cells.

Resveratrol is a natural compound that affects cellular calcium (Ca(2+)) homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca(2+) concentrations ([Ca(2+)]i) and viability in OC2 human oral cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)]i, and water-soluble tetrazolium-1 was used to measure viability. Resveratrol evoked concentration-dependent increase in [Ca(2+)]i. The response was reduced by removing extracellular Ca(2+). Resveratrol also caused manganese-induced fura-2 fluorescence quench. Resveratrol-evoked Ca(2+) entry was inhibited by nifedipine and the protein kinase C (PKC) inhibitor GF109203X but was not altered by econazole, SKF96365, and the PKC activator phorbol 12-myristate 13 acetate. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished resveratrol-evoked [Ca(2+)]i rise. Conversely, treatment with resveratrol inhibited BHQ-evoked [Ca(2+)]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished resveratrol-evoked [Ca(2+)]i rise. At 20-100 µM, resveratrol decreased cell viability, which was not affected by chelating cytosolic Ca(2+)with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. Annexin V-fluorescein isothiocyanate staining data suggest that resveratrol at 20-40 µM induced apoptosis in a concentration-dependent manner. Collectively, in OC2 cells, resveratrol induced [Ca(2+)]i rise by evoking PLC-dependent Ca(2+) release from the endoplasmic reticulum and by causing Ca(2+) entry via nifedipine-sensitive, PKC-regulated mechanisms. Resveratrol also caused Ca(2+)-independent apoptosis.

Naproxen-induced Ca2+ movement and death in MDCK canine renal tubular cells.

Naproxen is an anti-inflammatory drug that affects cellular calcium ion (Ca(2+)) homeostasis and viability in different cells. This study explored the effect of naproxen on [Ca(2+)](i) and viability in Madin-Darby canine kidney cells (MDCK) canine renal tubular cells. At concentrations between 50 µM and 300 µM, naproxen induced [Ca(2+)](i) rises in a concentration-dependent manner. This Ca(2+) signal was reduced partly when extracellular Ca(2+) was removed. The Ca(2+) signal was inhibited by a Ca(2+) channel blocker nifedipine but not by store-operated Ca(2+) channel inhibitors (econazole and SKF96365), a protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, and a PKC inhibitor GF109203X. In Ca(2+)-free medium, pretreatment with 2,5-di-tert-butylhydroquinone or thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) pumps, partly inhibited naproxen-induced Ca(2+) signal. Inhibition of phospholipase C with U73122 did not alter naproxen-evoked [Ca(2+)](i) rises. At concentrations between 15 µM and 30 µM, naproxen killed cells in a concentration-dependent manner, which was not reversed by prechelating cytosolic Ca(2+) with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl. Annexin V/propidium iodide staining data suggest that naproxen induced apoptosis. Together, in MDCK renal tubular cells, naproxen induced [Ca(2+)](i) rises by inducing Ca(2+) release from multiple stores that included the endoplasmic reticulum and Ca(2+) entry via nifedipine-sensitive Ca(2+) channels. Naproxen induced cell death that involved apoptosis.

Investigations of the mechanism of the reduction of plasma glucose by cold-stress in streptozotocin-induced diabetic rats.

Exposure to a cold environment may increase the activity of the sympathetic nervous system inducing an elevation of plasma norepinephrine and may result in hyperglycemia. In the present study, we found that a hypoglycemic effect was produced in streptozotocin-induced diabetic rats after cold-exposure at 4 degrees C for 1 h. In addition to the blockade of this hypoglycemic effect by guanethidine (a ganglion-blocking agent) and prazosin (an alpha1-adrenoceptor antagonist), an increase of plasma norepinephrine was also observed in streptozotocin-induced diabetic rats receiving this cold-stress. Participation of sympathetic hyperactivity can thus be considered. Furthermore, naloxone, in a dose (0.5 mg/kg, i.p.) sufficient to block opioid receptors, reversed this hypoglycemia. Also, an increase of plasma beta-endorphin-like immunoreactivity was observed in streptozotocin-induced diabetic rats receiving this cold-stress. Intravenous injection of beta-endorphin into streptozotocin-induced diabetic rats produced a lowering of plasma glucose. Administration of methoxamine at a dose sufficient to activate the alpha1-adrenoceptors produced hypoglycemia and a similar increase of plasma beta-endorphin-like immunoreactivity in streptozotocin-induced diabetic rats. However, plasma beta-endorphin-like immunoreactivity level was not modified by similar treatment with methoxamine or cold-stress in normoglycemic rats. Therefore, beta-endorphin appears to be responsible for the induction of hypoglycemic effects in streptozotocin-induced diabetic rats after cold exposure which is different to the response in normal rats.

Modification of superoxide dismutase (SOD) mRNA and activity by a transient hypoxic stress in cultured glial cells.

In order to understand the role of superoxide dismutase (SOD) in response to transient hypoxia or hypoxia-reperfusion in astrocytes, the present study performed an in vitro investigation using rat glial cells in culture. Hypoxia was induced by an incubation with nitrogen gas for 10 min and that followed a further reperfusion with air for 10 min was indicating as hypoxia-normoxia. Activity of SOD was determined by the reduction of nitroblue tetrazolium (NTB). Changes of mRNA for Cu,Zn-SOD or Mn-SOD were also characterized using Northern blotting analysis. Transient hypoxia increased the activity of Mn-SOD but not that of Cu,Zn-SOD in glial cells. Expression of mRNA for SOD was also elevated in cells received hypoxia and the mRNA level for Mn-SOD raised higher than that for Cu,Zn-SOD. In cells received hypoxia-reperfusion, these changes of SOD both the activity and the mRNA level were not observed. Otherwise, the SOD protein amount, both Cu,Zn-SOD and Mn-SOD, identified by Western blotting was not changed in glial cells receiving hypoxic stress or not. The obtained results suggest that gene expression and activity of Mn-SOD in glial cells can be activated in response to the transient hypoxic stress.

Synthesis and vasorelaxing evaluation of alpha-methylidene-gamma-butyrolactone bearing quinolin-2(1H)-one and 3,4-dihydroquinolin-2(1H)-one derivatives.

The main objective of this investigation was to explore the vasorelaxing structure-activity relationships of alpha-methylidene-gamma-butyrolactone bearing quinolin-2(1H)-ones and their 3,4-dihydro derivatives. These target compounds were synthesised in two steps starting from aryl-OH which was treated with a bromomethyl ketone followed by a Reformatsky-type condensation. Quinolin-2(1H)-one alpha-methylidene-gamma-butyrolactones exhibited less vasorelaxing activity than their 3,4-dihydro counterparts. Compounds with a methyl or a phenyl group at the C(gamma) of the lactone were more vasorelaxant than the C(gamma)-fluorophenyl derivatives in the 3,4-dihydroquinolin-2(1H)-one series. When comparing the positional isomers, alpha-methylidene-gamma-butyrolactone substituted at the 7-position of the 3,4-dihydroquinolin-2(1H)-ones were more active than their 6-substituted counterparts, which in turn were more active than the 8-substituted derivatives. The vasorelaxing effect of these 3,4-dihydroquinolin-2(1H)-ones was proved to be dose dependent. Among them, 7-[(2,3,4,5-tetrahydro-4-methylidene-5-oxo-2-phenyfuran-2-yl)methoxy]-quinolin-2(1H)-one (10b) was the most potent with an IC(50) of 9.2 microM on the KCl-induced vasoconstriction of pig coronary arteries.

Stimulatory effect of phenylephrine on the secretion of beta-endorphin from rat adrenal medulla in vitro.

In an attempt to investigate the role of alpha1-adrenoceptors in the regulation of opioid secretion from adrenal gland, phenylephrine was employed to investigate the effect on secretion of beta-endorphin-like immunoreactivity (BER) from adrenal medulla of rat in vitro. Phenylephrine enhanced the BER from isolated adrenal medulla in a concentration-dependent manner and this action was abolished by the antagonists of alpha1-adrenoceptors, prazosin and tamsulosin. Investigations of signal pathway further support that an activation of alpha1-adrenoceptors is responsible for the stimulatory effect of phenylephrine on BER secretion from adrenal medulla. In the presence of U73312, the specific inhibitor of phospholipase C (PLC), phenylephrine-induced change of BER was reduced in a concentration-dependent manner but it was not affected by U73343, the negative control of U73312. Moreover, chelerythrine and GF 109203X diminished the action of phenylephrine at concentration sufficient to inhibit protein kinase C (PKC). In conclusion, our results suggest that an activation of alpha1-adrenoceptors in adrenal medulla by phenylephrine may enhance the secretion of opioids from adrenal gland of rat via signals of PLC-PKC pathway.

Structure-activity relationships of anthraquinones in the decrease of intestinal motility.

The effects of substituted anthraquinones on intestinal motility were evaluated in-vitro using rabbit small intestinal strips. This structure-activity relationship study revealed the critical requirement of a hydroxy group at R2 position. The intestinal motility was inhibited 50% (IC50) by emodine (8 microM), 2-hydroxy anthraquinone (20 microM), 2,6-dihydroxy anthraquinone (25 microM), 2,7-dihydroxy anthraquinone (10 microM), 1,2,4-trihydroxy anthraquinone (80 microM) and 1,2,5,8-tetra-hydroxyanthraquinone (9 microM). The presence of other polar groups at R2 position such as an amino, aldehyde and carboxylic acid group significantly reduced the activity (IC50 360-400 microM). The presence of a methyl group and esterification of the carboxylic acid at R2 position was found to abolish the activity. These data are useful for the future development of anthraquinones as laxative agents.