Inhibitory effect of phorbol ester on carbachol-induced signal transduction in cultured canine tracheal smooth muscle cells

Regulation of the increases in inositol 1,4,5-trisphosphate (IP₃) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i) by activation of protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by carbachol led to IP₃ formation and caused an initial transient peak of [Ca²⁺]_i followed by a sustained elevation in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 µM) for 30 min blocked the carbachol-induced IP₃ formation and Ca²⁺ mobilization. Following preincubation, carbachol-induced Ca²⁺ mobilization recovered within 24 h. The concentrations of PMA that gave half-maximal inhibition of carbachol-induced IP₃ formation and increase in [Ca²⁺]_i were 7 and 4 nM, respectively. Prior treatment of TSMCs with staurosporine (1 µM), a PKC inhibitor, inhibited the ability of PMA to attenuate carbachol-induced responses. Inactive phorbol ester, 4-phorbol 12,13-didecanoate at 1 µM, did not inhibit these responses to carbachol. The K_d and B_max of the muscarinic receptor for [³H]N-methylscopolamine binding were not significantly changed by PMA treatment. PMA also decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter, the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit phosphoinositide hydrolysis and consequently attenuate the [Ca²⁺]_i increase or inhibit both responses independently. The inhibition by PMA of carbachol-induced responses was inversely correlated with membranous PKC activity.     

Anticonvulsant effects of magnesium sulfate in hippocampal-kindled rats

The objective of the present study was to determine whether magnesium sulfate has anticonvulsant actions in the hippocampal-kindled rat model of epilepsy. Fully kindled rats received acute intraperitoneal injections of magnesium sulfate (270 mg/kg), phenytoin (20 mg/kg) or saline in random order. Electrical seizure duration, behavioral seizure stage and duration of postictal EEG depression were examined 15, 30 and 60 min after injection. In an additional group of rats, kindled seizures were measured before and after chronic (2 h) intraperitoneal injections of magnesium sulfate versus saline. There was a significant decrease in electrical seizure duration (p<0.01) and behavioral seizure stage (p<0.01) with acute magnesium sulfate injections compared to saline injections. Phenytoin had no statistically significant effects on hippocampal-kindled seizures. Chronic magnesium sulfate treatment significantly reduced behavioral seizure stage at 2, 24, and 48 h postinjection (p<0.05), but did not affect seizure duration. There was a significant time by treatment effect for magnesium sulfate on postictal EEG depression (p<0.01). We conclude that in this model of hippocampal epilepsy-induced (kindled) rats, magnesium sulfate has significant anticonvulsant effects.     

Efficacy of 6-amino-2-n-pentylthiobenzothiazole onTrichophyton in vitro and in vivo

In vitro and in vivo antifungal activities of synthetically parepared 6-animo-2-n-pentylthiobenzothiazole (APB) againstTrichophyton strains were studied. APB inhibited the growth of 3Trichophyton strains at 65 µg/ml. 2-Mercaptobenzothiazole was not effective at 125 µg/ml and ketoconazole inhibited the growth at 20–30 µg/ml. Treatment of experimental dermatophytosis in guinea pigs using 2.5% APB cream was studied in comparison to Canesten cream (1% clotrimazole). Dermatophytosis was considerably reduced after both APB and Canesten therapies.     

Targeting to the HIV-1 RRE of the influenza virus NS1 protein effector domain as a potent, specific anti-HIV agent

The Rev axis of HIV autoregulation is one of two critical viral regulatory pathways required for expression of viral genomic and mRNA and for replication. Consequently it is an attractive therapeutic target. Previous studies have investigated the anti-HIV efficacy of targeting to the RRE (the viral RNA target sequence of the Rev axis) a trans-dominant negative inhibitor mutant Rev, M10. In this study we have fused a portion of the influenza virus NS1 protein (which normally inhibits polyA(+) mRNA transport and splicing) to the Rev M10 gene while deleting the NS1 poly(A) binding region. The resulting chimera demonstrates specific and enhanced inhibition of viral-RRE-containing RNA expression.     

Comparative studies on the diagnosis of hepatitis E virus antibodies with ORF-2 and ORF-3 proteins expressed in insect cells

Hepatitis E is a worldwide health problem, especially in developing countries. The virus genome contains three different open reading frames (ORFs): ORF-1, which is believed to encode nonstructural proteins, and ORF-2 and ORF-3, which are believed to encode structural proteins. Presently, serologic tests for the detection of human antibodies to hepatitis E virus (HEV) infection are primarily based on the ORF-2 structural protein expressed inEscherichia coli, insect cells or synthetic peptides. We report here the comparative studies on the diagnosis of HEV infection with full-length ORF-2 and ORF-3 proteins expressed in insect cells. We found that 31 of 74 (42%) sera were positive for IgM antibody to HEV (anti-HEV) using the ORF-2 protein as an antigen, as compared to 6 of 74 sera (8%) using the ORF-3 protein as an antigen (p<0.001). Similarly, 49 of 74 sera (66%) were positive for IgG anti-HEV utilizing the ORF-2 protein versus 12 of 74 sera (16%) when the ORF-3 protein was used (p<0.001). These results suggest that the recombinant ORF-2 protein is more sensitive as a diagnostic antigen for detecting antibodies to HEV in both acute-phase and convalescent-phase sera than ORF-3 protein.     

Enzymatic activity of metal-binding proteins in epidermal cells

Summary Enzymatic activity was investigated in metal-binding proteins from rat epidermal cells. Tris-HCl buffer soluble and KSCN solubilized proteins were extracted stepwise from granular and cornified cells of 2-day old rat epidermis. Each extract was separately applied to a Cu²⁺ or Zn^2– chelate Sepharose 6B column and the proteins were eluted with buffers of different pHs and finally with EDTA solution. Metal chelate-binding proteins were found in both soluble and solubilized proteins but there was a larger amount in the latter. Affinity of the proteins to bind with Cu²⁺ chelate was greater than that with Zn²⁺ chelate. In Tris-HCl buffer extract, histidase activity was detected in Cu²⁺ chelate-binding proteins, but not in Zn²⁺ chelate-binding proteins. Acid phosphatase, cysteine proteinase, dipeptidase, cathepsin D, -galactosidase, gelatin hydrolase, and superoxide dismutase did not bind to metal chelates although these enzymes, except acid phosphatase, were inhibited by Cu²⁺, but not by Zn²⁺. In contrast, KSCN solubilized metal chelate-binding proteins showed plasminogen activator, acid phosphatase, and gelatin and casein hydrolases while histone hydrolase did not bind to either chelate column. Since metal-binding proteins in rat epidermal cells have been shown previously to be histidine- and cysteine-rich proteins concentrated in keratohyalin granules, interaction of metals and the structural proteins with certain enzymes may be involved in the regulation of epidermal cell functions.