Sequencing and phylogenetic analysis of the coding region of six common rotavirus strains: evidence for intragenogroup reassortment among co-circulating G1P[8] and G2P[4] strains from the United States.

The segmented genome of rotaviruses provides an opportunity for rotavirus strains to generate a large genetic diversity through reassortment; however, this mechanism is considered to play little role in the generation of mosaic gene constellations between Wa-like and DS-1-like strains in genes other than the neutralization antigens. A pilot study was undertaken to analyze these two epidemiologically important strains at the genomic level in order to (i) identify intergenogroup reassortment and (ii) to make available additional reference genome sequences of G1P[8] and G2P[4] for future genomics analyses. The full or nearly complete coding region of all 11 genes for 3 G1P[8] (LB2719, LB2758, and LB2771) and 3 G2P[4] (LB2744, LB2764, and LB2772) strains isolated from children hospitalized with severe diarrhea in Long Beach, California, where these strains were circulating at comparable rates during 2005-2006 are described in this study. Based on the full-genome classification system, all G1P[8] strains had a conserved genomic constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1 and were mostly identical to the few Wa-like strains whose genome sequences have already been determined. Similarly, the genome sequences of the 3 G2P[4] strains were highly conserved: G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-E2-H2 and displayed an overall lesser genetic divergence with reference DS-1-like strains. While intergenogroup reassortment was not seen between the G1P[8] and G2P[4] strains studied here, evidence for intragenogroup reassortment events was identified. Similar studies in the post-rotavirus genomic era will help uncover whether intergenogroup reassortment affecting the backbone genes could play a significant role in any potential vaccine breakthrough events by evading immunity of vaccinated children.

Rate-limiting steps in the beta-adrenergic stimulation of cardiac calcium current.

Fast-flow perfusion and flash photolysis of caged compounds were used to study the activation kinetics of L-type calcium current (ICa) in frog cardiac myocytes. Rapid exposure to isoproterenol (Iso) for 1 s or approximately 1 min produced similar kinetics of increase in ICa with an initial lag period of approximately 3 s, followed by a monophasic rise in current with a half-time of approximately 20 s. Epinephrine, as well as caged Iso, produced increases with similar kinetics. The fact that ICa increased significantly even after short Iso applications suggests that agonist binding to the receptor is rapid and that the increase in ICa is independent of free agonist. To dissect the kinetic contributions of various steps in the cAMP-phosphorylation cascade, the kinetics of the responses to caged cAMP and caged GTP gamma S and fast perfusion of forskolin, acetylcholine, and propranolol were compared. The response to caged cAMP exhibited no lag period, but otherwise increased at a rate similar to that produced by Iso and reached a peak at approximately 40 s after flash photolysis. This suggests that the lag period itself is due to a step before cAMP accumulation, but that activation of protein kinase and phosphorylation of the calcium channel are relatively slow. A lag period was also observed when ICa was stimulated by flash photolysis of caged GTP gamma S and when adenylyl cyclase was activated directly by rapid perfusion with forskolin. The lag period observed with forskolin may be due to slow binding of forskolin. The lag period was not due to the time required for cAMP to reach a threshold concentration, because a similar lag was observed in response to Iso in cells having ICa previously stimulated submaximally by internal perfusion with a low concentration of cAMP. These results suggest that the lag period can be attributed to a step associated with activation of adenylyl cyclase and cAMP accumulation.

The role of a PDGF-activated nonselective cation channel in the proliferative response.

Murine fibroblasts have a 28 pS calcium- and voltage-insensitive NSC that becomes quiescent at G0 arrest and is rapidly and specifically activated by PDGF. Activation is produced by the discrete loss of long channel closures. The NSC can be rapidly and reversibly blocked with the NSAID flufenamic acid, through a prostaglandin-independent mechanism. The cell cycle (not viability) is blocked concomitantly with NSC block. A somatic cell mutant with altered NSC conductance has been isolated and used to clone the genomic locus of the channel. The mutant growth phenotype adds further support to the participation of NSC conductance in cell cycle control.

A comparative analysis of the time course of cardiac Ca2+ current response to rapid applications of beta-adrenergic and dihydropyridine agonists.

A fast perfusion system was used to analyze the kinetics of the response of L-type calcium current (ICa) to rapid exposures to beta-adrenergic or dihydropyridine agonists in whole-cell patch-clamped frog ventricular myocytes. The perfusion system was based on the lateral motion of an array of plastic capillary tubes from which solutions flowed at a velocity of approximately 5 cm/s. Movement from one capillary to the adjacent one occurred in < 20 ms and complete exchange of extracellular solution was achieved in < 50 ms as demonstrated by the block of ICa by fastflow application of Cd during a depolarizing pulse. Fastflow applications of increasing concentrations of isoprenaline (Iso) led to a dose-dependent stimulation of ICa at [Iso] > 1 nM. The response of ICa to Iso always started after a delay of several seconds. The delay duration decreased as [Iso] increased, and was typically approximately 3 s at 10 microM Iso. The rising phase of ICa increase was monophasic and independent of [Iso] > 100 nM. For short applications of Iso (8.8 s), half maximal and maximal stimulation of ICa occurred approximately 20 s and approximately 40 s after the beginning of Iso application, respectively. When Iso was applied during a depolarizing pulse (with Ba as the charge carrier), IBa never increased during that pulse. The kinetics of the ICa response to Iso were not affected by varying the voltage clamp protocols or the ionic composition of intracellular and extracellular solutions. In comparison with the effects of Iso, the stimulatory effect of the dihydropyridine agonist (-)Bay K 8644 on ICa was approximately 15 times faster: delay, half-time to maximal and time to maximal responses were 15 times shorter with (-)Bay K 8644 than with Iso. It is concluded that frog ventricular myocytes respond slowly to a quick application of beta-adrenergic agonists.

Pathogenesis of and immunity to avian influenza A H5 viruses.

In 1997 in Hong Kong, 18 human cases of respiratory illness were caused by an avian influenza A H5N1 virus. Although avian influenza viruses had not previously been known to cause respiratory illness in humans, the H5N1 viruses caused severe illness and death, primarily in individuals aged > 12 years. The introduction of H5N1 viruses into humans raised concerns about the potential of these viruses to cause a pandemic. We have used the BALB/c mouse to better understand the pathogenesis of and immunity to the H5N1 viruses in a mammalian model. Previously, we demonstrated that H5N1 viruses isolated from humans replicated efficiently in the lungs of mice without prior adaptation to this host. Two general phenotypes of pathogenicity of H5N1 viruses, based on high and low lethality for mice, were observed. We now demonstrate that in addition to a lethal outcome, H5N1 viruses with a high pathogenicity phenotype exhibit additional features that include rapid and uncontrolled replication in the lungs of infected mice, dissemination and replication of the virus in other organs, and depletion of peripheral blood leukocytes. The BALB/c mouse model was also used to better understand the parameters of protective immunity to the H5N1 viruses. Prior infection with H5N1 viruses of low pathogenicity or an antigenically related non-pathogenic H5N3 virus protected mice from death by infection with a highly pathogenic HK/483 virus. Serum hemagglutination-inhibition antibody titers of 40 or greater were associated with protection of mice from death. Immunization of mice with baculovirus-expressed recombinant H5 hemagglutinin protein or a previously defined HS-specific synthetic peptide induced MHC class II restricted CTL activity. Mice that had CTL activity but no serum hemagglutination-inhibition antibody were not protected from a lethal challenge with H5N1 virus. These results suggest that antibody is required for protection of mice against lethal challenge with H5N1 viruses of the high pathogenicity phenotype.

Opposite effects of phosphatase inhibitors on L-type calcium and delayed rectifier currents in frog cardiac myocytes.

1. Application of the phosphatase inhibitors okadaic acid (OA) and microcystin (MC) to frog cardiomyocytes caused large increases in L-type calcium current (ICa) in the absence of beta-adrenergic agonists. The increase occurred without effects on the peak current-voltage relation or voltage-dependent inactivation. OA and MC caused a decrease in amplitude of delayed rectifier current (IK), which is opposite to the increase produced by cAMP-dependent phosphorylation. The decrease occurred without effects on voltage-dependent activation or reversal potential. 2. Analysis of the dose-response relations for OA and MC on ventricular cell ICa were best fitted with a single-site relationship with a K1/2 of 1.58 microM and 0.81 microM, respectively. These data suggest the predominant form of phosphatase active on ICa in this cell type is produced by protein phosphatase 1. Inhibition of phosphatase 2B (calcineurin) was without appreciable effect. 3. Reducing intracellular ATP levels was without effect on basal ICa suggesting that calcium channels may not need to be phosphorylated to open. ATP depletion was able to block completely the ICa increase induced by OA or MC. This demonstrates that the effects of OA and MC on ICa are mediated by a phosphorylation reaction. In contrast, ATP depletion totally abolished IK, suggesting either a requirement for ATP or phosphorylation for basal function of the delayed rectifier channel. 4. Internal perfusion of a peptide inhibitor (PKI(5-22)) of protein kinase A (PK-A) was without effect on basal current levels of ICa or IK, suggesting that this kinase is not phosphorylating these channels under basal conditions. Furthermore, although PKI is capable of completely blocking the response of ICa to isoprenaline or forskolin, PKI does not affect the increase in ICa induced by MC or OA. Inhibition of adenylate cyclase with acetylcholine or inhibition of PK-A with adenosine cyclic 3',5'-(Rp)-phosphothioate (Rp-cAMPS) also had no effect on the response to OA or MC. 5. Application of beta-adrenergic agonist, forskolin or cAMP all produced additional increases in the presence of saturating doses of MC or OA. This supports the hypothesis that PK-A is not mediating the OA response and that phosphatase inhibition does not result in complete phosphorylation of PK-A sites. 6. To attempt to identify the protein kinase activity responsible for OA effects on ICa and IK, several types of protein kinase inhibitors were internally perfused.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical modification of Ca(2+)-activated potassium channels of GH3 anterior pituitary cells.

The effects of amino group specific reagents were examined on single, large-conductance, Ca(2+)-activated, K+ channels in excised membrane patches from GH3 cells. The reagents used include trinitrobenzene sulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and its 4-acetamido derivative, and sulfophenyl-isothiocyanate. These reagents react covalently with peptide terminal amino groups and the epsilon amino groups of lysine residues, thereby removing positive charge. Internal application of 0.1-1.0 mM reagent to inside-out patches irreversibly increases channel open probability. Single-channel conductance and voltage sensitivity are not affected by modification. Analysis of channel openings and closures shows that the increase in open probability is predominantly due to the loss of long-duration closures of the channel; however, the lengths of long-duration openings are increased. After the modification in the presence of Ca2+ was performed, the channel open probability remains large, regardless of the internal Ca2+ concentration. Transitions among several open and closed states of the modified channel are present in the absence of Ca2+, suggesting that many state transitions are not directly dependent on Ca2+ binding or dissociation.

Activation of single-channel currents in mouse fibroblasts by platelet-derived growth factor.

A nonselective cation channel that we characterized in the mouse L-cell membrane becomes quiescent with serum deprivation (arrested cell growth) and rapidly active upon readdition of serum or, specifically, platelet-derived growth factor (PDGF). Using the patch-clamp technique, we find that the predominant channel in the LMTK- cell line is a bursting nonselective cation channel (the NS channel). In cell-attached and inside-out patches, the channel has a conductance of 28 pS; equal selectivity for Na+, K+, and Cs+; and no anion or divalent cation permeability. The channel open probability is voltage insensitive and in inside-out patches does not correlate with intracellular calcium (0.5 nM to 50 microM). When cultures are rendered quiescent by incubation in serum-free medium, channel open probability is virtually 0 as compared to 0.26 (+/- 0.17) in exponentially growing cultures. If mitogenesis is initiated by readdition of serum to quiescent cells while maintaining cell-attached recording, there is a rapid (15-30 s) activation of the channel (n = 12). The open probability of the patch increases (greater than 0.75) for 2-3 min and then decreases. We have attempted applications of several growth factors (fibroblast-derived growth factor, epidermal growth factor, insulin, bombesin, alpha-thrombin, and vasopressin, individually or in combination) but find that only PDGF (5-100 ng/ml; n = 9) produces channel activation. This activation should provide a Na+ entry pathway parallel to that of the Na/H exchanger.

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder.

The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na+ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal 22Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na+ flux was a linear function of mucosal Na+ concentration. Ouabain-sensitive Na+ flux and ouabain-sensitive, Na+-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na+ with the basolateral transport process is assumed to involve the interaction of some number of Na+ ions, n, with a maximal flux, MMAX, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value for MMAX of 287.8 pM cm-2 sec-1 with an intracellular Na concentration of 2.0 mM Na+ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na+-induced current, we calculate a Na+ to K+ coupling ratio of 1.40 +/- 0.07 for the transport process.

External K+ increases Na+ conductance of the hyperpolarization-activated current in rabbit cardiac pacemaker cells.

The hyperpolarization-activated current (I(f)) was recorded from single myocytes dissociated from rabbit sinoatrial node. Although I(f) is usually carried by both Na+ and K+, removal of the minor K+ component from physiological saline suppresses inward component. This inward Na+ current through I(f) channel increases on raising the extracellular K+ concentration. The Na+ conductance relative to K+ conductance (PNa/PK), as measured from the reversal potential, increases and saturates near 5 mM K+. This effect is different from the current increase caused by raising the concentration of carrier ion K+, which saturates at 70 mM with a half-maximal value (K1/2) of 10 mM. It is suggested that the I(f) channel has multiple, interactive binding sites for cation permeation.

Control of the hyperpolarization-activated cation current by external anions in rabbit sino-atrial node cells.

1. Effects of varying concentrations of anions on the hyperpolarization-activated current (I(f)) were studied in myocytes isolated from the rabbit sino-atrial node. Substituting Cs+ for the intracellular K+ clearly separated I(f) from the delayed rectifier K+ current. Control properties, including gating kinetics and ion selectivity, similar to previous studies were obtained. 2. Substitution of extracellular Cl- with larger anions including isethionate, glutamate, acetate, and aspartate, reduced the amplitude of I(f) without changing the reversal potential. Substitution with small anions such as iodide or nitrate supported an intact I(f). These effects were reproduced in the excised outside-out patch conformation. 3. The conductance for I(f) was a saturating function of the extracellular Cl- concentration ([Cl-]o) with an equilibrium binding constant (K1/2) of 11 mM and a slope factor of about 1 when substituted with large anions. Total removal of small anions completely abolished I(f). 4. The voltage-dependent gating of I(f) was not affected by changing ([Cl-]o), suggesting that Cl- modulates conductance properties of I(f). 5. The results indicate that I(f) conductance is unique in that it is dependent on an extracellular anion (Cl-), yet it is carried exclusively by cations, K+ and Na+. These effects are independent of any measurable voltage-dependent gating parameters.

Effects of saxitoxin analogues and ligand competition on sodium currents of squid axons.

Saxitoxin (STX) and several STX analogues from dinoflagellates (genus Protogonyaulax) block sodium conductance in squid giant axons with variable potencies. Toxins, analyzed under voltage clamp, are 21-sulfosaxitoxin, 21-sulfosaxitoxin 11 alpha-hydroxysulfate, 21-sulfosaxitoxin 11 beta-hydroxysulfate, (B1, C1, C2, respectively) and gonyautoxins 2 and 3. The potency sequence for the toxins examined is STX greater than gonyautoxin 3 greater than B1 greater than C2 greater than gonyautoxin 2 much greater than C1. Guanidine, when substituted for sodium in external seawater, reduced the potency of STX to block inward current but did not affect tetrodotoxin activity. Methylguanidine also reduced the ability of STX to block outward sodium current. Inhibitory constants for guanidine and methylguanidine were 116 and 187 mM, respectively. Competition can be explained by binding at or near the toxin binding site but not by surface potential alteration.

Modified M2 proteins produce heterotypic immunity against influenza A virus.

Vaccination with the influenza A transmembrane protein M2 provides enhanced viral clearance and recovery from influenza A virus infection in mice. However, the high degree of hydrophobicity of the protein limits its purification for vaccine purposes. We have attempted to alter the structure of the M2 protein to allow high level recombinant expression in Escherichia coli, to reduce its hydrophobicity and improve protein solubility, thus improving its properties as a vaccine subunit candidate. Constructs investigated include deletion of the transmembrane domain of M2 (residues 26-43) and an extended deletion (residues 26-55). A full-length M2 protein was not pursued because of poor expression, even in the presence of amantadine. Expressed as glutathione S-transferase fusion proteins and used to vaccinate mice, either deletion construct was found to raise M2-specific serum antibodies and enhance viral clearance in mice challenged with homologous and heterologous influenza A viruses. Enzymatic cleavage from the GST fusion domain produces soluble protein giving similar results. The results demonstrate that large alterations of M2 protein structure can improve its isolation and purification characteristics without detracting from its immunogenic properties.