Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1.

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.

Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay.

Viruses in the genus Coronavirus are currently placed in three groups based on antigenic cross-reactivity and sequence analysis of structural protein genes. Consensus polymerase chain reaction (PCR) primers were used to obtain cDNA, then cloned and sequenced a highly conserved 922 nucleotide region in open reading frame (ORF) 1b of the polymerase (pol) gene from eight coronaviruses. These sequences were compared with published sequences for three additional coronaviruses. In this comparison, it was found that nucleotide substitution frequencies (per 100 nucleotides) varied from 46.40 to 50.13 when viruses were compared among the traditional coronavirus groups and, with one exception (the human coronavirus (HCV) 229E), varied from 2.54 to 15.89 when compared within these groups. (The substitution frequency for 229E, as compared to other members of the same group, varied from 35.37 to 35.72.) Phylogenetic analysis of these pol gene sequences resulted in groupings which correspond closely with the previously described groupings, including recent data which places the two avian coronaviruses--infectious bronchitis virus (IBV) of chickens and turkey coronavirus (TCV)--in the same group [Guy, J.S., Barnes, H.J., Smith L.G., Breslin, J., 1997. Avian Dis. 41:583-590]. A single pair of degenerate primers was identified which amplify a 251 bp region from coronaviruses of all three groups using the same reaction conditions. This consensus PCR assay for the genus Coronavirus may be useful in identifying as yet unknown coronaviruses.

Vitamin A is required for regulation of polymeric immunoglobulin receptor (pIgR) expression by interleukin-4 and interferon-gamma in a human intestinal epithelial cell line.

The secretory immunoglobulin A (IgA) antibody response to infections of mucosal surfaces requires transport of IgA from the basal to apical surface of mucosal epithelial cells by a specific transport protein, the polymeric immunoglobulin receptor (pIgR). We have tested the hypothesis that the vitamin A metabolite all-trans retinoic acid (RA) is required for the regulation of pIgR expression by the cytokines interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) in HT-29 cells, a well-differentiated human epithelial cell line derived from a colonic carcinoma. pIgR expression is upregulated by IFN-gamma and IL-4 when HT-29 cells are grown in normal media, but this upregulation was significantly lower when cells were grown in vitamin A-depleted media. Treatment with RA at concentrations from 10(-9) to 10(-5) mol/L restored normal levels of pIgR expression. The percentages of cells expressing cell-surface pIgR after 24, 48 and 72 h of treatment with RA, IL-4 and IFN-gamma were 66 +/- 10, 90 +/- 5 and 92 +/- 1, respectively, significantly higher than the percentages seen without RA treatment, which were 32 +/- 2.3, 72 +/- 1.2 and 30 +/- 7, respectively. In addition, the intensity of fluorescence of pIgR-positive cells was significantly higher in the RA-treated cultures than in the cultures without RA treatment. Similarly, pIgR mRNA levels (adjusted for beta-actin mRNA levels) in RA-supplemented cultures were 404, 105 and 949% higher at 24, 48 and 72 h, respectively, than were pIgR mRNA levels in identical cultures grown in the absence of RA. These data indicate that RA strongly interacts with IL-4 and IFN-gamma to regulate pIgR expression in HT-29 cells, suggesting that vitamin A may be required for proper in vivo regulation of IgA transport in response to mucosal infections.

Vitamin A deficiency has different effects on immunoglobulin A production and transport during influenza A infection in BALB/c mice.

We examined the effect of advanced vitamin A deficiency (serum retinol < or = 0.35 micromol/L, with weight gain significantly lower than in controls with free access to food) on the secretory immunoglobulin A (IgA) response to a mild, upper respiratory tract infection with influenza A virus in BALB/c mice. Mice fed a vitamin A-deficient or control diet were infected intranasally at 11 to 12 wk of age. The influenza-specific salivary IgA response was lower in the vitamin A-deficient mice (0.11 +/- 0.13% of total IgA 4 wk after infection) than in controls with free access to food (2.73 +/- 1.86%, P < 0.0001). In a separate experiment, the response of vitamin A-deficient mice (0.42 +/- 1.51%) was also lower than that of pair-fed controls (3.43 +/- 4.76%, P < 0.0001). In addition, fewer influenza A-specific IgA-secreting plasma cells were found in the salivary glands of vitamin A-deficient mice (geometric mean 3.0%) than in controls with free access to food or in pair-fed controls (geometric mean 8.7%, P < 0.0001). Although the pathogen-specific IgA response was decreased, vitamin A-deficient mice had a significantly higher concentration of total salivary IgA (31.9 +/- 15.9 mg/L) than did the pair-fed controls (14.3 +/- 8.4 mg/L, P < 0.0001). Northern blot analysis of salivary gland RNA revealed that these vitamin A-deficient mice also had greater levels of mRNA of the polymeric immunoglobulin receptor (pIgR), which transports IgA across mucosal surfaces (plgR: beta-actin mRNA ratio = 7.8 +/- 0.8), than did pair-fed control mice (3.7 +/- 0.4, P = 0.0001). These data demonstrate that vitamin A deficiency has contrasting effects on the secretory IgA response to influenza infection, with a principal effect being a decrease in the pathogen-specific response.

Vitamin A deficiency diminishes the salivary immunoglobulin A response and enhances the serum immunoglobulin G response to influenza A virus infection in BALB/c mice.

We examined the effect of vitamin A deficiency on the secretory immunoglobulin (Ig) A and serum IgG response to influenza A virus infections in BALB/c mice. Mice fed a vitamin A-deficient (VAD mice) or a control diet were inoculated with influenza virus at 7 or 9 wk of age when serum retinol concentration had dropped to < or = 0.35 mumol/L in the VAD mice. The influenza-specific salivary IgA response to a mild infection (intranasal inoculation without anesthesia) was not significantly lower in the VAD group (5.3 +/- 2.1% of total IgA 4 wk after infection) than in the control group (10 +/- 11%, P > 0.05). In a separate experiment, this salivary IgA response was significantly lower in the VAD mice (0.3 +/- 0.4% of total IgA) following a more severe infection (intranasal infection while under anesthesia) than it was in control mice (4.2 +/- 4.6% of total IgA, P < 0.0001). In contrast, the concentration of total salivary IgA was uniformly greater in the VAD mice than in the control mice during both the mild infection (VAD, 17 +/- 6 mg/L vs. control, 8 +/- 11 mg/L at 3 wk, P < 0.0001) and the severe infection (VAD, 38 +/- 30 mg/L vs. control, 9 +/- 7 mg/L, P < 0.0001). Similarly, the influenza-specific serum IgG response was also greater in the VAD mice than in the control mice during both the mild infection (VAD, 194 +/- 91 mg/L vs. control, 79 +/- 95 mg/L at 5 wk, P = 0.0002) and the severe infection [VAD median, 202 mg/L (25th, 75th percentiles, 153, 409 mg/L) vs. control, 123 mg/L (42, 165 mg/L), P = 0.0023]. Thus VAD significantly impairs the secretory IgA response to influenza infection but modestly increases the serum IgG response to the same infection.

A rapid protein A binding radioimmunoassay for the evaluation of antiviral agents.

A simple, rapid and quantitative protein A binding radioimmunoassay (RIA) was developed to screen and evaluate antiviral drugs against human cytomegalovirus (HCMV) replication. It was found that (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and human gamma-interferon (IFN) were more effective than 9-[(1-3-dihydroxy-2-propoxy)methyl]guanine (DHPG) and other IFNs against HCMV replication in human fibroblast cells. The block in virus replication was not due to toxic effect of these compounds on human fibroblast cells. The results were in good agreement with those of other workers. The results indicate that protein A binding RIA can be useful for rapid and quantitative screening of compounds effective against HCMV replication.

Polyclonal B-lymphocyte activation of sheep by Mycobacterium phlei against sheep pox virus.

A trypsinized preparation of Mycobacterium phlei, non specific stimulator of immunity (NSI), and Sheep Pox Virus (SPV) were inoculated in different groups of sheep to activate B-lymphocytes and induce SPV neutralizing substance(s). NSI sensitized sheep B-lymphocytes in the presence of NSI or lymphokine elaborated SPV neutralizing substance(s). The SPV sensitized B-lymphocytes also mediated such neutralizing substance(s). Healthy control sheep B-lymphocytes failed to show any appreciable amount of viral neutralizing substance. However, a significant virus neutralizing substance(s) was detected when healthy sheep B-lymphocytes were cultured in presence of NSI antigen along with lymphokines.