The homo-dimeric form of ADP-ribosyl cyclase in solution.

ADP-ribosyl cyclase is a multi-functional enzyme that catalyzes the formation of two Ca2+ signaling molecules, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). X-ray crystallography of three different crystal forms shows that it is a non-covalent dimer. Chemical cross-linking and dynamic light scattering were used in this study to determine if the cyclase is also a non-covalent dimer in solution. Treatment of the cyclase in dilute solution (0.05 mg/ml) with dimethylsuberimidate resulted in complete conversion to a species with molecular weight about twice that of the monomeric cyclase. Prolonged cross-linking of the cyclase at four times higher concentration produced also only the covalently linked dimers and no multimer formation was observed. The cross-linked dimer retained full enzymatic activity and readily catalyzed the formation of cADPR from NAD, NAADP from NADP, cyclic ADP-ribose phosphate from NADP, and cyclic GDP-ribose from nicotinamide guanine dinucleotide. Analysis of the autocorrelation functions obtained from dynamic light scattering measurements indicated the cyclase solution (2 mg/ml) was composed of a single molecular species and its diffusion coefficient was measured to be 7. 4x10-7 cm2/s. Computer modeling using the crystallographic dimensions of the non-covalent cyclase dimer, a donut shaped molecule with a central cavity and overall dimensions of 7x6x3 nm, gave a value for the diffusion coefficient essentially the same as that measured. These results indicate the cyclase is a non-covalent dimer in solution.

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle.

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.

Interaction of vecuronium with piperacillin or cefoxitin evaluated in a prospective, randomized, double-blind clinical trial.

Interactions between beta-lactam antibiotics, particularly acylaminopenicillins, and vecuronium, a widely used muscle relaxant, leading to prolonged neuromuscular blockade have been reported in studies of experimental animals and in a few clinical case reports. In the clinical reports, however, confounding factors always existed. A clinical trial to evaluate interactions between vecuronium and cefoxitin or piperacillin was conducted. Patients having major operations requiring both muscle relaxants as part of general anesthesia and prophylactic antibiotics were entered into the trial and randomly assigned to receive either cefoxitin or piperacillin. The electromyographic twitch response was measured before and after administration of the antibiotic. Five of 27 evaluable patients had minor prolongation of the time to recovery of baseline twitch. No prolonged neuromuscular blockade was observed. There were no differences in responses between the two antibiotic treatment groups. Cefoxitin and piperacillin administered pre- or intra-operatively are not associated with clinically important prolongation of muscle relaxation induced by vecuronium. The potential for prolongation of neuromuscular blockade induced by vecuronium through concomitant administration of piperacillin or cefoxitin as antibiotic prophylaxis was investigated in a clinical trial of 30 patients having major abdominal operations. Quantitative measurement of neuromuscular blockade was done using the electromyographic twitch response to a supramaximal current stimulus.

Glycoalkaloid and nitrate content of potatoes as affected by method of selenium application.

A comparison of two methods of selenium application, banding and foliar spray, of sodium selenite (Na2SeO3) on total glycoalkaloid (TGA) and nitrate nitrogen (NO3-N) was studied during each of two consecutive years. The levels of application used were 0.0, 1.6 (0.75), 3.36 (1.5), and 5.6 (2.5) kg/ha (ppm soil). Both TGA and NO3-N were significantly reduced by application of 1.5 and 2.5 ppm of sodium selenite. Tuber selenium levels were significantly increased at all levels of application, using either banding or foliar spray, but were well below the toxic range for human consumption. Banding resulted in greater uptake of Se, and greater decreases in TGA and NO3-N as compared to foliar spray.

Mass spectrometer failure: an unusual cause.

A potentially serious problem is inherent in the current design of the mass spectrometer multiplex system. In spite of the filter on the sampling tube, foreign material can be aspirated into this tube, causing malfunction of the system. Prevention of this problem is discussed, and precautions are given.

The effect of midazolam on median nerve somatosensory evoked potentials.

OBJECTIVE: To quantify the effect of an induction dose of midazolam on median nerve somatosensory evoked potentials. METHODS: We studied 10 patients undergoing lumbar spine surgery. After an induction dose of intravenous midazolam was given, MNEPs were collected for ten minutes. After ten minutes the patients were intubated and their anesthetic was supplemented with 0.5% isoflurane, narcotic, and N2O. RESULTS: We found a clinically significant decrease in amplitude and an insignificant delay in latency. CONCLUSION: When midazolam is used as an anesthetic induction agent, a decrease in amplitude can be expected.

High-level expression of recombinant Aplysia ADP-ribosyl cyclase in offhia pastoris by fermentation.

Cyclic ADP-ribose (cADPR), a Ca2+ mobilizing cyclic nucleotide derived from NAD+, is rapidly emerging as an endogenous modulator of Ca2(+)-induced Ca2+ release mechanisms in various cellular systems. ADP ribosyl cyclase, first isolated from the marine invertebrate Aplysia californica, cyclizes NAD+ to cADPR. In this study we have utilized the methylotrophic yeast Pichia pastoris to express high levels of this enzyme. The cyclase construct consisted of the soluble domain, with isoleucine (25 residues following the initial methionine) as the N-terminus, cloned in frame with the yeast alpha-factor mating signal sequence. Cyclase yeast transformants were screened using the Zeocin (phleomycin from Streptomyces verticillus) selectable marker which resulted in 100% active transformation. All active clones comprised the methanol utilization slow (Muts) phenotype. The protein was expressed using the tightly regulated methanol-inducible alcohol oxidase (AOX1) promoter and the Saccharomyces cerevisiae alpha-factor mating secretion signal. Using high biomass fermentations, up to 300 mg/liter of cyclase was achieved. SDS-PAGE analysis revealed that the heterologous protein comprised nearly 90-95% of the total protein secreted extracellularly. The enzyme characteristics of the recombinant cyclase compared favorably with those of the native enzyme. The yeast expression system can thus produce gram quantities of this novel protein.

Recognition of mixed anesthetic agents by mass spectrometer during anesthesia.

Anesthetic agents are sometimes added to the wrong vaporizer on an anesthesia machine. As a result, the vaporizer may deliver a mixture of anesthetic agents at concentrations inappropriate for use on a patient. However, untoward clinical complications related to vaporizers can be prevented with a time-shared mass spectrometer. This device accurately and rapidly indicates the gases and gas concentrations present in a vaporizer.

Postoperative unilateral facial oedema: a complication of acute flexion of the neck.

A case report describing a complication following the use of the sitting position and an extreme flexed position of the neck is presented. The patient developed unilateral oedema of the face, tongue and soft tissues of the mouth. The trachea was reintubated and the oedema subsided without treatment. The cause of the oedema is believed to be obstruction of the venous drainage of the head and neck.

Structures and activities of cyclic ADP-ribose, NAADP and their metabolic enzymes.

ADP-ribosyl cyclase and CD38 are multi-functional enzymes involved in calcium signaling. Both can cyclize NAD and its guanine analog, NGD, at two different sites of the purine ring, N1 and N7, respectively, to produce cyclic ADP-ribose (cADPR) and cyclic GDP-ribose, a fluorescent but inactive analog. Both enzymes can also catalyze the exchange of the nicotinamide group of NADP with nicotinic acid, producing yet another potent activator of Ca2+ mobilization, nicotinic acid adenine dinucleotide phosphate (NAADP). The Ca2+ release mechanism activated by NAADP is totally independent of cADPR and inositol trisphosphate indicating it is a novel and hitherto unknown Ca2+ signaling pathway. This article summarizes the current results on the structures and activities of cADPR, NAADP and the enzymes that catalyze their syntheses. A comprehensive model accounting for the novel multi-functionality of ADP-ribosyl cyclase and CD38 is presented.

Antagonism of mivacurium neuromuscular block: neostigmine versus edrophonium.

This study was designed to compare the effectiveness of antagonism of mivacurium blockade with either neostigmine, edrophonium, or spontaneous recovery. Thirty ASA physical status I or II patients provided informed consent and were randomized to one of the following groups: Group 1, placebo saline; Group 2, edrophonium (1 mg/kg); and Group 3, neostigmine (70 micrograms/kg) (n = 10/group). All studied patients had anesthesia induced with propofol and maintained with propofol/N2O/fentanyl. Mivacurium bolus of 0.2 mg/kg was used for endotracheal intubation and an infusion titrated to maintain deep levels of block (T1% = 1%-5%) (T1% = first response/control response x 100). The antagonist was injected at a deep level of the block (T1% = 1%-8%) and neuromuscular (NM) recovery was evaluated by train-of-four twitches (TOF). T1% was used during maintenance, whereas both T1% and TOF% (fourth response/first response x 100) were used during recovery. Investigators were blinded to the antagonist used. Plasma cholinesterase activity was measured prior to antagonist administration (0 min), as well as 15, 30, and 60 min after. Plasma cholinesterase activity was decreased to 29% of control at 15 min and remained at approximately 60% of the control after neostigmine administration. Edrophonium did not affect plasma cholinesterase activity. Clinically adequate spontaneous recovery (TOF% > or = 70%) of the mivacurium block with placebo required 15-18 min. On average, clinically adequate antagonism of mivacurium by edrophonium was 50% faster than placebo and 30%-40% faster than with neostigmine. In summary, the speed of antagonism with edrophonium is faster than with neostigmine when antagonizing deep mivacurium NM block.(ABSTRACT TRUNCATED AT 250 WORDS)

A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities.

CD38 is a multifunctional enzyme involved in metabolizing two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purified, and characterized by immunoblot. Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.

Capnography for detection of endobronchial migration of an endotracheal tube.

A patient is described in whom migration of an endotracheal tube into the right main bronchus was suspected when end-tidal carbon dioxide suddenly decreased from 28 to 22 mm Hg. Acute changes with migration of the endotracheal tube into the main bronchus were also studied in an animal experimental model. End-tidal carbon dioxide decreased and tracheal (inflation) pressure increased, with no change in tidal volume. Arterial blood gases showed time-dependent decreases in pH and oxygen tension and an increase in carbon dioxide tension.

Identification of the enzymatic active site of CD38 by site-directed mutagenesis.

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.