Prolonged voluntary wheel running reveals unique adaptations in mdx mice treated with microdystrophin constructs ± the nNOS-binding site.

We tested the effects of prolonged voluntary wheel running on the muscle function of mdx mice treated with one of two different microdystrophin constructs. At 7 weeks of age mdx mice were injected with a single dose of AAV9-CK8-microdystrophin with (gene therapy 1, GT1) or without (gene therapy 2, GT2) the nNOS-binding domain and were assigned to one of four gene therapy treated groups: mdxRGT1 (run, GT1), mdxGT1 (no run, GT1), or mdxRGT2 (run,GT2), mdxGT2 (no run, GT2). There were two mdx untreated groups injected with excipient: mdxR (run, no gene therapy) and mdx (no run, no gene therapy). A third no treatment group, Wildtype (WT) received no injection and did not run. mdxRGT1, mdxRGT2 and mdxR performed voluntary wheel running for 52 weeks; WT and remaining mdx groups were cage active. Robust expression of microdystrophin occurred in diaphragm, quadriceps, and heart muscles of all treated mice. Dystrophic muscle pathology was high in diaphragms of non-treated mdx and mdxR mice and improved in all treated groups. Endurance capacity was rescued by both voluntary wheel running and gene therapy alone, but their combination was most beneficial. All treated groups increased in vivo plantarflexor torque over both mdx and mdxR mice. mdx and mdxR mice displayed ∼3-fold lower diaphragm force and power compared to WT values. Treated groups demonstrated partial improvements in diaphragm force and power, with mdxRGT2 mice experiencing the greatest improvement at ∼60% of WT values. Evaluation of oxidative red quadriceps fibers revealed the greatest improvements in mitochondrial respiration in mdxRGT1 mice, reaching WT levels. Interestingly, mdxGT2 mice displayed diaphragm mitochondrial respiration values similar to WT but mdxRGT2 animals showed relative decreases compared to the no run group. Collectively, these data demonstrate that either microdystrophin construct combined with voluntary wheel running increased in vivo maximal muscle strength, power, and endurance. However, these data also highlighted important differences between the two microdystrophin constructs. GT1, with the nNOS-binding site, improved more markers of exercise-driven adaptations in metabolic enzyme activity of limb muscles, while GT2, without the nNOS-binding site, demonstrated greater protection of diaphragm strength after chronic voluntary endurance exercise but decreased mitochondrial respiration in the context of running.

blaIMP-27 on transferable plasmids in Proteus mirabilis and Providencia rettgeri.

OBJECTIVES: A carbapenem-resistant Providencia rettgeri (PR1) isolate was recovered from a wound infection in Missouri, USA. This isolate possessed an EDTA-inhibitable carbapenemase that was unidentified using the Xpert CARBA-R assay. Our objective was to elucidate the molecular determinant of carbapenem resistance in this isolate. We then sought to test the transmissibility of blaIMP-27 loci in clinical P. rettgeri and Proteus mirabilis isolates. METHODS: In October 2016 the novel ambler Class B carbapenemase blaIMP-27, was reported in two different Proteus mirabilis (PM185 and PM187) isolates. Broth mating assays for transfer of carbapenemase activity were performed for the three clinical isolates with recipient sodium azide-resistant Escherichia coli J53. Antibiotic susceptibility testing and phenotypic carbapenemase activity testing were performed on the clinical isolates, J53 and transconjugants using the Kirby-Bauer disc diffusion method according to CLSI guidelines. Plasmid DNA from PM187, PR1 and their transconjugants were used as input for Nextera Illumina sequencing libraries and sequenced on a NextSeq platform. RESULTS: PR1 was resistant to both imipenem and meropenem. PM187 and PR1 could transfer resistance to E. coli through plasmid conjugation (pPM187 and pPR1). pPM187 had a virB/virD4 type IV secretion system whereas pPR1 had a traB/traD type IV secretion system. CONCLUSION: Two of three blaIMP-27-bearing clinical isolates tested could conjugate resistance into E. coli. The resulting transconjugants became positive for phenotypic carbapenemase production but did not pass clinical resistance breakpoints. blaIMP-27 can be transmitted on different plasmid replicon types that rely on distinct classes of type IV secretion system for horizontal transfer.

Striated muscle activator of Rho signalling (STARS) is reduced in ageing human skeletal muscle and targeted by miR-628-5p.

AIM: The striated muscle activator of Rho signalling (STARS) is a muscle-specific actin-binding protein. The STARS signalling pathway is activated by resistance exercise and is anticipated to play a role in signal mechanotransduction. Animal studies have reported a negative regulation of STARS signalling with age, but such regulation has not been investigated in humans. METHODS: Ten young (18-30 years) and 10 older (60-75 years) subjects completed an acute bout of resistance exercise. Gene and protein expression of members of the STARS signalling pathway and miRNA expression of a subset of miRNAs, predicted or known to target members of STARS signalling pathway, were measured in muscle biopsies collected pre-exercise and 2 h post-exercise. RESULTS: For the first time, we report a significant downregulation of the STARS protein in older subjects. However, there was no effect of age on the magnitude of STARS activation in response to an acute bout of exercise. Finally, we established that miR-628-5p, a miRNA regulated by age and exercise, binds to the STARS 3'UTR to directly downregulate its transcription. CONCLUSION: This study describes for the first time the resistance exercise-induced regulation of STARS signalling in skeletal muscle from older humans and identifies a new miRNA involved in the transcriptional control of STARS.

Emergence of community-associated methicillin-resistant Staphylococcus aureus strains in the neonatal intensive care unit: an infection prevention and patient safety challenge.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause significant morbidity and mortality in neonatal intensive care units (NICUs). We characterized the clinical and molecular epidemiology of MRSA strains colonizing NICU patients. Nasal MRSA isolates (n = 250, from 96 NICU patients) recovered through active surveillance from 2009 to 2014 were characterized with staphylococcal cassette chromosome mec (SCCmec) typing and detection of mupA (marker of high-level mupirocin resistance) and qacA/B (marker associated with chlorhexidine resistance). Factors associated with community-associated (CA-) or healthcare-associated (HA-) MRSA were evaluated. The overall prevalence of MRSA nasal colonization was 3.9%. Of 96 neonates in our retrospective cohort, 60 (63%) were colonized with CA-MRSA strains and 35 (36%) were colonized with HA-MRSA strains. Patients colonized with HA-MRSA were more likely to develop MRSA infections than patients colonized with CA-MRSA (13/35, 37% versus 8/60, 13%; p 0.007), although the interval from colonization to infection was shorter in CA-MRSA-colonized infants (median 0 days, range -1 to 4 versus HA-MRSA-colonized infants, 7 days, -1 to 43; p 0.005). Maternal peripartum antibiotics were associated with CA-MRSA colonization (adjusted odds ratio (aOR) 8.7; 95% CI 1.7-45.0); intubation and surgical procedures were associated with HA-MRSA colonization (aOR 7.8; 95% CI 1.3-47.6 and aOR 6.0; 95% CI 1.4-24.4, respectively). Mupirocin- and chlorhexidine-resistant MRSA was isolated from four and eight patients, respectively; carriage of a mupirocin-resistant strain precluded decolonization. CA-MRSA strains are prominent in the NICU and associated with distinct risk factors. Given community reservoirs for MRSA acquisition and transmission, novel infection prevention strategies are needed.

Evaluation of Oxacillin and Cefoxitin Disk and MIC Breakpoints for Prediction of Methicillin Resistance in Human and Veterinary Isolates of Staphylococcus intermedius Group.

Staphylococcus pseudintermedius is a coagulase-positive species that colonizes the nares and anal mucosa of healthy dogs and cats. Human infections with S. pseudintermedius range in severity from bite wounds and rhinosinusitis to endocarditis; historically, these infections were thought to be uncommon, but new laboratory methods suggest that their true incidence is underreported. Oxacillin and cefoxitin disk and MIC tests were evaluated for the detection of mecA- or mecC-mediated methicillin resistance in 115 human and animal isolates of the Staphylococcus intermedius group (SIG), including 111 Staphylococcus pseudintermediusand 4 Staphylococcus delphini isolates, 37 of which were mecA positive. The disk and MIC breakpoints evaluated included the Clinical and Laboratory Standards Institute (CLSI) M100-S25 Staphylococcus aureus/Staphylococcus lugdunensis oxacillin MIC breakpoints and cefoxitin disk and MIC breakpoints, the CLSI M100-S25 coagulase-negative Staphylococcus (CoNS) oxacillin MIC breakpoint and cefoxitin disk breakpoint, the CLSI VET01-S2 S. pseudintermedius oxacillin MIC and disk breakpoints, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) S. pseudintermedius cefoxitin disk breakpoint. The oxacillin results interpreted by the VET01-S2 (disk and MIC) and M100-S25 CoNS (MIC) breakpoints agreed with the results of mecA/mecC PCR for all isolates, with the exception of one false-resistant result (1.3% of mecA/mecC PCR-negative isolates). In contrast, cefoxitin tests performed poorly, ranging from 3 to 89% false susceptibility (very major errors) and 0 to 48% false resistance (major errors). BD Phoenix, bioMérieux Vitek 2, and Beckman Coulter MicroScan commercial automated susceptibility test panel oxacillin MIC results were also evaluated and demonstrated >95% categorical agreement with mecA/mecC PCR results if interpreted by using the M100-S25 CoNS breakpoint. The Alere penicillin-binding protein 2a test accurately detected all mecA-positive isolates, although for four isolates, cefoxitin induction was required prior to testing. These data demonstrate that the cefoxitin surrogate test does not reliably detect the presence of mecA in S. pseudintermedius isolates and that laboratories should perform oxacillin disk or MIC tests of these isolates when they are encountered.

Comparison and optimization of two MALDI-TOF MS platforms for the identification of medically relevant yeast species.

The rapid identification of yeast is essential for the optimization of antifungal therapy. The objective of our study was to evaluate two matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platforms, the bioMérieux VITEK MS (IVD Knowledgebase v.2.0) and Bruker Biotyper (software version 3.1), for the rapid identification of medically relevant yeast. One hundred and seventeen isolates, representing six genera and 18 species, were analyzed using multiple direct smear methods to optimize identification. Sequence analysis was the gold standard for comparison. Isolates were analyzed with VITEK MS using the direct smear method +/- a 25 % formic acid on-plate extraction. For Biotyper, isolates were analyzed using direct smear without formic acid, and with 25 % and 100 % formic acid on-plate extractions. When all methods were included, VITEK MS correctly identified 113 (96.6 %) isolates after 24 h with one misidentification, and Biotyper correctly identified 77 (65.8 %) isolates using a threshold of ≥2.0 with no misidentifications. Using a revised threshold of ≥1.7, Biotyper correctly identified 103 (88.0 %) isolates, with 3 (2.6 %) misidentifications. For both platforms, the number of identifications was significantly increased using a formic acid overlay (VITEK MS, p < 0.01; Biotyper, p < 0.001), and reducing the Biotyper threshold from ≥2.0 to ≥1.7 significantly increased the rate of identification (p < 0.001). The data in this study demonstrate that the direct smear method with on-plate formic acid extraction can be used for yeast identification on both MS platforms, and more isolates are identified using the VITEK MS system (p < 0.01).

A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog.

The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as the mammalian homologs ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3). The remaining glycoproteins are a triplet of high molecular weight components that are selectively hydrolyzed by the hatching enzyme. We have isolated one of these proteins and cloned its cDNA. The mRNA for the protein was found to be expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was indicated to be a secreted glycoprotein with a characteristic ZP domain in the C-terminal half of the molecule. The N-terminal half was unrelated to any known glycoprotein. Comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. This envelope component has been designated ZPAX.

Metabolites of caspofungin acetate, a potent antifungal agent, in human plasma and urine.

Caspofungin acetate (MK-0991) is a semisynthetic pneumocandin derivative being developed as a parenteral antifungal agent with broad-spectrum activity against systemic infections such as those caused by Candida and Aspergillus species. Following a 1-h i.v. infusion of 70 mg of [(3)H]MK-0991 to healthy subjects, excretion of drug-related material was very slow, such that 41 and 35% of the dosed radioactivity was recovered in urine and feces, respectively, over 27 days. Plasma and urine samples collected around 24 h postdose contained predominantly unchanged MK-0991, together with trace amounts of a peptide hydrolysis product, M0, a linear peptide. However, at later sampling times, M0 proved to be the major circulating component, whereas corresponding urine specimens contained mainly the hydrolytic metabolites M1 and M2, together with M0 and unchanged MK-0991, whose cumulative urinary excretion over the first 16 days postdose represented 13, 71, 1, and 9%, respectively, of the urinary radioactivity. The major metabolite, M2, was highly polar and extremely unstable under acidic conditions when it was converted to a less polar product identified as N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine gamma-lactone. Derivatization of M2 in aqueous media led to its identification as the corresponding gamma-hydroxy acid, N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-L-threonine. Metabolite M1, which was extremely polar, eluting from HPLC column just after the void volume, was identified by chemical derivatization as des-acetyl-M2. Thus, the major urinary and plasma metabolites of MK-0991 resulted from peptide hydrolysis and/or N-acetylation.

Enhanced gastrointestinal diagnosis: light-scattering spectroscopy and optical coherence tomography.

Detection of microscopic and biochemical changes within the mucosa and submucosa are beyond the realm of routine endoscopy. Distinguishing hyperplastic from neoplastic polyps, differentiating malignant from benign ulcers, and detecting mucosal dysplasia in patients with ulcerative colitis or Barrett's esophagus remain within the purview of the gastrointestinal pathologist. Recent developments in tissue spectroscopy and endoscopic optical coherence tomography have the potential to expand significantly our ability to diagnose gastrointestinal disease beyond the capabilities of visible light endoscopy.

Renal transplantation.

Medical and scientific advances have improved the diagnosis and treatment of kidney disorders. Organ transplantation has evolved from an experimental surgery to a medically accepted form of treatment for organ failure. The kidney was the first organ to be successfully replaced by a donor organ, and it is presently the most commonly transplanted organ. Kidney transplantation restores reasonably normal health to patients whose kidneys no longer function, and it frees them from the limitations imposed by dialysis. Improved graft survival rates have further enhanced the desirability of transplantation.

Anatomy and physiology of the kidney.

The kidneys are complex organs, and they are vital in maintaining normal body functions. A human being's survival depends, to a large degree, on the crucial functions and processes performed by the kidneys. The renal system affects all parts of the body by keeping body fluids in balance and other organ systems functioning normally. Renal and urologic disorders may strike anyone at any age and at any time. An estimated 20 million Americans are affected with renal disorders each year.

Pharmacokinetics and disposition of the oxytocin receptor antagonist L-368,899 in rats and dogs.

L-368,899 is a potent, orally-active oxytocin antagonist that completed phase I clinical trials for the prevention of preterm labor. The pharmacokinetics and disposition of L-368,899 were studied in rats (female and male) and dogs (female), the two species used in the toxicology studies. L-368,899 exhibited similar pharmacokinetics in rats and dogs. After iv dosing at 1, 2.5, and 10 mg/kg, the compound had a t1/2 of approximately 2 hr and plasma clearance between 23 and 36 ml/min/kg at all doses and in both species. The exception was female rats at the 10 mg/kg dose where plasma clearance decreased to 18 ml/min/kg. The Vdss was between 2.0 and 2.6 liters/kg for rats and 3.4 to 4.9 liters/kg for dogs. After oral doing, L-368,899 was rapidly absorbed. Mean Cmax values were achieved at <1 hr at the low doses (25 mg/kg in rats and 5 mg/kg in dogs) and between 1 and 4 hr at the higher doses (100 mg/kg in rats and 33 mg/kg in dogs). In bile duct-cannulated female rats, approximately 70% of a radioactive 28 mg/kg dose was recovered in bile and urine within 72 hr post dose. Plasma drug concentrations were higher in female than in male rats especially at the 25 mg/kg dose, where mean AUC values were 4.5-fold higher in the females. In both rats and dogs, plasma drug levels increased more than proportionally with increasing oral dose. In female rats, the mean AUC increased by approximately 8-fold between 25 and 100 mg/kg, while in female dogs, the mean AUC at the 33 mg/kg dose was 12-fold higher than that at 5 mg/kg. Oral bioavailability was estimated at 14% and 18% for the 5 mg/kg dose in female and male rats, respectively, 41% for the 25 mg/kg dose in male rats and 17% and 41%, respectively, for the 5 and 33 mg/kg doses in dogs. Owing to nonlinear kinetics, bioavailability could not be calculated for the other oral doses. L-368,899 was metabolized extensively in both species after iv and oral dosing, with <10% of the dose excreted unchanged. The main route of elimination was via the feces, which contained >70% of the radioactive dose by 48 hr, primarily as metabolites. The gender and dose dependence of the pharmacokinetics of L-389,899 in rats were attributed to gender differences in metabolizing capacity and saturation of hepatic metabolism, respectively. This conclusion was based primarily on results from experiments comparing the rate of in vitro metabolism of L-368,899 in liver microsomes, which showed that the Vmax and KM values for L-368,899 were 4-fold lower in female than in male rat liver microsomes.

Multicentric lymphosarcoma with ovarian involvement in a Nubian goat.

Multiple lymph node enlargement and an intra-abdominal mass were diagnosed in a 6-year-old doe. Necropsy revealed lymphosarcoma involving multiple organs, including the ovaries. Lymphosarcoma is rare in goats; ovarian involvement has not previously been reported.

Lithium toxicosis in a cow.

A case of lithium toxicosis is described in a cow that had consumed grease. Clinical signs included increased salivation, ataxia, reduced consciousness, seizures and diarrhea. No treatment was instituted. The grease did not contain high concentrations of other heavy metals or minerals.

Effects of Alzheimer's disease-related beta amyloid protein fragments on enzymes metabolizing phosphoinositides in brain.

Phosphatidylinositol 4-kinase (PI 4-kinase) and phosphatidylinositol 4-phosphate kinase (PIP kinase) were assayed in membranes prepared from samples of human frontal cortex initially frozen at autopsy. PI 4-kinase activity was significantly lower in Alzheimer's disease patients relative to age-matched controls or patients with Parkinson's disease. PIP kinase was not different in Alzheimer's versus age-matched controls. The beta amyloid protein fragment 1-40 inhibited PI 4-kinase activity in assays of control human or rat cortical membranes. Fragments 1-28 and 25-35 could not mimic the effects of fragment 1-40 while a reverse peptide 40-1 was equipotent. The inhibition of PI 4-kinase by fragment 1-40 was competitive with substrate. The beta amyloid protein fragments had diverse effects on phosphoinositide-specific phospholipase C (PI-PLC) as assayed in rat cortical membranes. Low concentrations of fragment 1-40 stimulated, while high concentrations of 1-40 or 40-1 inhibited PI-PLC activity. Fragment 25-35 stimulated PI-PLC nearly 3-fold, while fragment 1-28 had only minor effects on the enzyme. The results suggest alterations in phosphoinositide metabolism in Alzheimer's disease which could affect signal transduction and/or cytoskeletal organization.

Radiation-induction of micronuclei in human peripheral blood lymphocytes: effect of freezing.

Lymphocytes in peripheral blood samples from three donors were irradiated at doses of 0, 1 and 2 Gy with cobalt-60 gamma rays. Whole blood cultures were established immediately after irradiation. Cultures of Ficoll-Paque separated lymphocytes were set up immediately after irradiation, or 3 weeks after storage in liquid nitrogen at -196 degrees C. The frequency of micronuclei was measured in binucleate cytokinesis-blocked cells and the different treatment groups were compared. No clastogenic effect of the lymphocyte separation technique, or of subsequent freezing in liquid nitrogen, was observed.

Modulation of the phospholipase C activity in rat brain cortical membranes by simultaneous activation of distinct monoaminergic and cholinergic muscarinic receptors.

The activation of phospholipase C (PLC) was examined in membranes of rat cerebral cortex simultaneously exposed to monoaminergic receptor and muscarinic receptor agonists after the treatment of membranes with two alkylating agents, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (100 microM EEDQ) and propylbenzilylcholine (10 nM PrBCM). Treatment of membranes with PrBCM results in a selective inactivation of M3 muscarinic receptors, while treatment with EEDQ results in a relative sparing of M1 muscarinic receptors. Stimulation of PLC by GTP gamma S alone in rat cortical membranes had an apparent EC50 of about 0.4 microM, but in the presence of carbachol (1 mM) was 0.09 microM. Treatment of rat cortical membranes with EEDQ or PrBCM did not modify the concentration-response curves for GTP gamma S alone, but the ability of carbachol (1 mM) to shift the EC50 of GTP gamma S was lost in PrBCM-treated membranes. We have previously shown that dopamine, working through D1-like dopamine receptors, alters the PLC response to carbachol by preventing this shift in the apparent EC50 for GTP gamma S16. When we reproduced these experiments in EEDQ- and PrBCM-treated membranes, only in EEDQ-treated membranes was dopamine able to inhibit the PLC response to carbachol. The results indicate that the post-receptor mechanisms of PLC activation are distinct for the putative M1 as opposed to M3 muscarinic receptors in rat cortical membranes. Further, there appears to be a specific interaction between D1 and M3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Transmembrane signaling through phospholipase C in human cortical membranes.

Stimulation of phosphoinositide-specific phospholipase C (PLC) by carbachol, dopamine and serotonin was measured by supplying exogenous [3H]phosphatidylinositol 4,5-bisphosphate to membranes prepared from human cortex dissected and frozen at autopsy. Subjects with Alzheimer's disease, Parkinson's disease or schizophrenia were compared to age-matched controls with no known neurological disorders. Stimulation of PLC by the neurotransmitters was dependent on the presence of GTP gamma S. Carbachol elicited the greatest stimulations of PLC followed by serotonin and then dopamine. The maximal stimulations of PLC evoked by a neurotransmitter were similar for the various categories of subjects except in Parkinson's patients, where dopamine failed to stimulate PLC beyond the activity attained with carbachol. In the presence of carbachol, the sensitivity of PLC to GTP gamma S was significantly increased in Alzheimer's membranes, but not in age-matched controls or Parkinson's. Overall, the experiments demonstrate the feasibility for using the exogenous substrate assay to study the functionality of the phosphoinositide transmembrane signaling system in human brain.

Differential effects of alkylating agents on the multiple muscarinic receptor subtypes linked to activation of phospholipase C by carbachol in rat brain cortical membranes.

Muscarinic cholinergic receptor function in rat brain cortex was characterized by performing binding assays with [3H](-)quinuclidinyl benzilate ([3H]QNB) in parallel with assays of phospholipase C (PLC) activation by carbachol using membrane preparations and exogenous [3H]-phosphatidylinositol 4,5-bisphosphate ([3H]PIP2). Competitive binding studies revealed high- and low-affinity binding sites for the receptor antagonists, pirenzepine, methoctramine and the p-fluoro analog of hexahydro-sila-difenidol (p-F-HHSiD). Carbachol-stimulated [3H]-phosphatidylinositol 4,5-biphosphate breakdown was specifically inhibited by pirenzepine and p-F-HHiSD. The inhibition curves for these antagonists were best described by interactions at two sites. There was quantitative agreement between the antagonist affinity constants and the proportion of high- and low-affinity sites derived in functional and binding studies. The characteristics of the putative subtypes of muscarinic receptors and their stimulation of phospholipase C was examined after treatment with two alkylating agents, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline and propylbenzilylcholine mustard. Loss of receptors was closely correlated with loss of PLC activation by carbachol, without alteration of the EC50 value (21 microM) of this agonist, clearly demonstrating a lack of receptor reserve. When both alkylating treatments were adjusted to induce a decrease of 60% in the maximal number of [3H]QNB binding sites, a similar (60%) reduction in the maximal effect of carbachol on PLC activation was found. However, the characteristics of the remaining receptors after the treatment with the two alkylating agents differ markedly as determined by competition of pirenzepine, p-F-HHSiD and methoctramine for [3H]QNB binding, and for inhibition of carbachol-stimulated phospholipase C by pirenzepine and p-F-HHSiD.(ABSTRACT TRUNCATED AT 250 WORDS)