Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric alpha gamma beta channel architecture

The extracellular domain of the epithelial Na(+) channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl(-) inhibits ENaC activity. To identify sites involved in Cl(-) inhibition, we mutated residues in the extracellular domain of α-, β-, and γENaC that are homologous to the Cl(-) binding site in acid-sensing ion channel 1a and tested the effect of Cl(-) on the activity of ENaC expressed in Xenopus oocytes. We identified two Cl(-) inhibitory sites in ENaC. One is formed by residues in the thumb domain of αENaC and the palm domain of βENaC. Mutation of residues at this interface decreased Cl(-) inhibition and decreased Na(+) self-inhibition. The second site is formed by residues at the interface of the thumb domain of βENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl(-) inhibition yet had no effect on Na(+) self-inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had little or no effect on Cl(-) inhibition or Na(+) self-inhibition. The data demonstrate that Cl(-) inhibits ENaC activity by two distinct Na(+)-dependent and Na(+)-independent mechanisms that correspond to the two functional Cl(-) inhibitory sites. Furthermore, based on the effects of mutagenesis on Cl(-) inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl(-) inhibition, the data support a model in which ENaC subunits assemble in an αγβ orientation (listed clockwise when viewed from the top).     

Ultrastructural study of Mycoplasma pneumoniae in organ culture.

The ultrastructure of Mycoplasma pneumoniae M129 was studied by using specialized staining methods for thin-section transmission electron microscopy. Nucleic acid was shown in the cytoplasmic granules and fibrillar material in the nuclear region. The central filament of the highly structured tip contained basic protein. With one method of fixation, parallel filaments were seen in the central core. M. pneumoniae was enveloped in an extracellular mucoprotein layer that was especially concentrated around its terminal structure.     

Evidence for a second histidine at the active site of succinyl-CoA synthetase from Escherichia coli.

Ethoxyformic anhydride was used to demonstrate the existence of a second important histidine in succinyl-CoA synthetase from Escherichia coli. Differential labeling of the enzyme by [3H]ethoxyformic anhydride gave a stoichiometry of one important histidine per alpha beta catalytic unit. Data are presented suggesting that this residue and an important thiol group on the beta subunit (Collier, G., and Nishimura, J.S. (1978) J. Biol. Chem. 253, 4938-4943) interact with each other during catalysis. A mechanism of action involving these 2 residues is proposed for one of the partial reactions catalyzed by succinyl-CoA synthetase.     

Directed intermixing in multicomponent self-assembling biomaterials

The noncovalent coassembly of multiple different peptides can be a useful route for producing multifunctional biomaterials. However, to date, such materials have almost exclusively been investigated as homogeneous self-assemblies, having functional components uniformly distributed throughout their supramolecular structures. Here we illustrate control over the intermixing of multiple different self-assembling peptides, in turn providing a simple but powerful means for modulating these materials' mechanical and biological properties. In β-sheet fibrillizing hydrogels, significant increases in stiffening could be achieved using heterobifunctional cross-linkers by sequestering peptides bearing different reactive groups into distinct populations of fibrils, thus favoring interfibril cross-linking. Further, by specifying the intermixing of RGD-bearing peptides in 2-D and 3-D self-assemblies, the growth of HUVECs and NIH 3T3 cells could be significantly modulated. This approach may be immediately applicable toward a wide variety of self-assembling systems that form stable supramolecular structures.     

Characterization of N-[3H]Methylcarbamylcholine Binding Sites and Effect of N-Methylcarbamylcholine on Acetylcholine Release in Rat Brain

The present experiments show that N-[3H]-methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro-beta-erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro-beta-erythroidine and d-tubocurarine, but not by alpha-bungarotoxin or by the muscarinic antagonist atropine. The MCC-induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.     

Morphone abstinence and quasi-abstinence effects after phosphodiesterase inhibitors and naloxone.

Naive or morphine-dependent rats received a single subcutaneous injection of a phosphodiesterase inhibitor; their behavioral responses were then recorded after a small subcutaneous dose of naloxone. In naive rats, the potent phosphidiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) produced acutely a state in which a small dose of naloxone (0.03 to 1.0 mg/kg subcutaneously) precipitated a quasi-morphine abstinence syndrome that was difficult to distinguish from the true abstinence syndrome, precipitated by the same dose of naloxone in rats made dependent on morphine. IBMX also intensified the true morphine abstinence syndrome. The potency with which IBMX, theophylline, caffeine and RO 20–1724 exerted these effects corresponded with their potency as inhibitors of cyclic-3′, 5′-AMP phosphodiesterase in rat brain homogenate. These and previous findings indicate that: (i) morphine-abstinence effects express increased activity of a central cyclic AMP mechanism; and (ii) naloxone can potently stimulate behavior in animals not treated with any opiate drug.     

Venezuelan equine encephalitis virus replicon particles encoding respiratory syncytial virus surface glycoproteins induce protective mucosal responses in mice and cotton rats

Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-γ)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2K^d-restricted CD8⁺ T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV.     

2-Aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridines as broad-spectrum inhibitors of human herpesvirus polymerases

A novel series of 2-aryl-2-hydroxyethylamine substituted 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potent antivirals against human herpesviruses. These compounds demonstrate broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, EBV, and VZV with high specificity compared to human DNA polymerases.     

A modest glucokinase overexpression in the liver promotes fed expression levels of glycolytic and lipogenic enzyme genes in the fasted state without altering SREBP-1c expression

Hepatic genes crucial for carbohydrate and lipid homeostasis are regulated by insulin and glucose metabolism. However, the relative contributions of insulin and glucose to the regulation of metabolic gene expression are poorly defined in vivo. To address this issue, adenovirus-mediated hepatic overexpression of glucokinase was used to determine the effects of increased hepatic glucose metabolism on gene expression in fasted or ad libitum fed rats. In the fasted state, a 3 fold glucokinase overexpression was sufficient to mimic feeding-induced increases in pyruvate kinase and acetyl CoA carboxylase mRNA levels, demonstrating a primary role for glucose metabolism in the regulation of these genes in vivo. Conversely, glucokinase overexpression was unable to mimic feeding-induced alterations of fatty acid synthase, glucose-6-phosphate dehydrogenase, carnitine palmitoyl transferase I or PEPCK mRNAs, indicating insulin as the primary regulator of these genes. Interestingly, glucose-6-phosphatase mRNA was increased by glucokinase overexpression in both the fasted and fed states, providing evidence, under these conditions, for the dominance of glucose over insulin signaling for this gene in vivo. Importantly, glucokinase overexpression did not alter sterol regulatory element binding protein 1-c mRNA levels in vivo and glucose signaling did not alter the expression of this gene in primary hepatocytes. We conclude that a modest hepatic overexpression of glucokinase is sufficient to alter expression of metabolic genes without changing the expression of SREBP-1c.     

Enzyme Activity and Protein of Multiple Forms of Choline Acetyltransferase: Effects of Calyculin A and Okadaic Acid

Choline acetyltransferase (ChAT) appears to exist in multiple forms, three of which can be isolated biochemically as cytosolic (cChAT), ionically-membrane bound (ibChAT) and non-ionic membranous (mChAT). In this study, we first examined whether the quantitative distribution of enzyme protein and enzyme activity was the same. Enzyme activity and ChAT protein distributed similarly: the majority of ChAT activity and protein were found in cChAT followed by mChAT and least activity and amount were in ibChAT. Our second objective was to investigate the effects of calyculin A or okadaic acid on the subcellular distribution of ChAT activity and amount from rat hippocampal formation. Calyculin A and okadaic acid decreased significantly (p < 0.01) cytosolic and membranous ChAT activity; ionically-bound ChAT was not significantly (p > 0.67) different from control. Removal of calyculin A or okadaic acid restored cytosolic ChAT activity (p > 0.9 as compared to control), but not membranous enzyme activity (p < 0.05 as compared to control). The immunoreactive cytosolic ChAT was reduced significantly (p < 0.01) by calyculin A and okadaic acid. Enzyme amount of membranous ChAT was decreased significantly by calyculin A (p < 0.01) and okadaic acid (p < 0.001). Enzyme amount of ionically-bound ChAT was not changed (p > 0.99) by either of these two phosphatase inhibitors. This investigation demonstrates that alterations in ChAT activity of each subfraction parallel changes in enzyme amounts in the same fractions.