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Biomedical subjects

E Wilson

Publications and source records attributed to E Wilson.

At least 127 records · Page 7Linked to original sources
PubMed1990

Mutant analysis approaches to understanding calcium signal transduction through calmodulin and calmodulin regulated enzymes.

An example set of site-specific mutagenesis studies of calmodulin has been discussed in terms of strategy and how the results can provide insight into the functioning of calmodulin. A set of common examples for the study of calcium binding and enzyme activation were discussed. Essentially, site-specific mutagenesis in these initial studies is a perturbation approach. From these perturbation studies, structural features can be correlated in future studies with function and mechanisms of action proposed. More importantly, the approach allows efficient testing of proposed mechanisms and further probing of the molecular aspects of the signal transduction pathways. Clearly, the key functional feature that must be addressed in future studies is how the calcium binding steps in the mechanism are coupled to the enzyme activation step, which is the final step of the calmodulin-enzyme binding mechanism.

Amino Acid Sequence↗
PubMed1990

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity.

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.

Amino Acid Sequence↗
PubMed1990

Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.

The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence. Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically. A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium. Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity. However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule. Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.

Amino Acid Sequence↗
PubMed1990

Sedation during spinal anaesthesia: comparison of propofol and midazolam.

Propofol and midazolam were compared in 40 patients undergoing orthopaedic surgery under spinal anaesthesia. An infusion of either 1% propofol or 0.1% midazolam was given at a rate adjusted to maintain a similar level of sedation. The mean time to reach this required level was similar in both groups. Quality and ease of control of sedation were good in all patients. A mean infusion rate of 3.63 mg kg-1 h-1 was required for propofol and 0.26 mg kg-1 h-1 for midazolam. Immediate recovery, as judged by ability to open eyes and recall date of birth, was significantly more rapid following propofol (P less than 0.001). Similarly, restoration of higher mental function was significantly faster following propofol, measured by choice reaction time and critical flicker fusion threshold. Amnesia for the immediate postoperative period was significantly greater after midazolam (P = 0.0001).

Anesthesia Recovery Period↗
PubMed1990

Disk elution method for MICs and MBCs.

A MIC macrodilution broth method in which routine antimicrobial susceptibility testing disks were used as a source of antibiotic was compared with the M7A reference method of the National Committee for Clinical Laboratory Standards for accuracy and reproducibility over five test runs. Gentamicin, piperacillin, and oxacillin were tested by using American Type Culture Collection quality control strains and laboratory isolates for which the MIC of each agent was known. An optimum time for elution of 1 h was first determined by timed elution in broth, confirming levels determined by the TDX method (Abbott, North Chicago, Ill.) for gentamicin and bioassay for piperacillin and oxacillin. MICs determined by the disk elution method demonstrated a reproducibility rate of 97% as well as 97% agreement with the reference method to within +/- 1 dilution; MBCs showed 93% agreement. The disk elution method is reproducible and provides a reliable test system that can be used by any laboratory in which disk susceptibility testing is performed.

Anti-Infective Agents↗
PubMed1990

Aerosolization of recombinant SLPI to augment antineutrophil elastase protection of pulmonary epithelium.

In a variety of lung diseases the respiratory epithelial surface must contend with an increased burden of neutrophil elastase (NE). One candidate for augmenting epithelial anti-NE protection is the secretory leukoprotease inhibitor (SLPI). In vitro evaluation demonstrated that 96 +/- 1% of the recombinant SLPI (rSLPI) molecules were capable of inhibiting NE, with an association rate constant of 7.1 +/- 0.1 X 10(6) M-1.s-1. Evaluation of rSLPI after in vitro and in vivo aerosolization showed that aerosolization did not alter rSLPI. Aerosolization of a single dose of 50 mg rSLPI to sheep resulted in a fourfold increase of the anti-NE capacity in epithelial lining fluid (ELF) at 3 h, with a half-life in ELF of 12 h. After aerosolization some rSLPI appeared in lung lymph. Simultaneous aerosolization of rSLPI and recombinant alpha 1-antitrypsin (rAAT) demonstrated a molar ratio of the concentration in lymph to the concentration in ELF 3 h after the aerosol eightfold higher for rAAT than for rSLPI. Overall, these observations demonstrate that it is feasible to use aerosolized rSLPI to directly augment the anti-NE capacity of the lung, particularly on the pulmonary epithelial surface.

Aerosols↗
PubMed1989

Computational and site-specific mutagenesis analyses of the asymmetric charge distribution on calmodulin.

Calmodulin's calculated electrostatic potential surface is asymmetrically distributed about the molecule. Concentrations of uncompensated negative charge are localized near certain alpha-helices and calcium-binding loops. Further calculations suggest that these charge features of calmodulin can be selectively perturbed by changing clusters of phylogenetically conserved acidic amino acids in helices to lysines. When these cluster charge reversals are actually produced by using cassette-based site-specific mutagenesis of residues 82-84 or 118-120, the resulting proteins differ in their interaction with two distinct calmodulin-dependent protein kinases, myosin light chain kinase and calmodulin-dependent protein kinase II. Each calmodulin mutant can be purified to apparent chemical homogeneity by an identical purification protocol that is based on conservation of its overall properties, including calcium binding. Although cluster charge reversals result in localized perturbations of the computed negative surface, single amino acid changes would not be expected to alter significantly the distribution of the negative surface because of the relatively high density of uncompensated negative charge in the region around residues 82-84 and 118-120. However, this does not preclude the possibility of single amino acid charge perturbations having a functional effect on the more intimate, catalytically active complex. The electrostatic surface of calmodulin described in this report may be a feature that would be altered only by cluster charge reversal mutations. Overall, the results suggest that the charge properties of calmodulin are one of several properties that are important for the efficient assembly of calmodulin-protein kinase signal transduction complexes in eukaryotic cells.

Amino Acid Sequence↗
PubMed1989

Incidence and importance of bacteriuria in postoperative, short-term urinary catheterization.

The frequency and importance of bacteriuria in patients requiring short-term postoperative catheterization (24 to 48 hours) is unknown. From a prospective, controlled study of 100 patients requiring catheterization postoperatively, the authors determined that there was a definite risk (6%) of bacteriuria; however, a symptomatic urinary tract infection developed in only 1% of the patients. There was no advantage to using either a latex or silicone catheter. It appears that the risk of symptomatic bacteriuria in this group of patients is very low when a closed urinary drainage system is used under strict, sterile conditions of catheter insertion and with meticulous ongoing catheter care.

Bacteriuria↗
PubMed1988

Modulation of the free sphingosine levels in human neutrophils by phorbol esters and other factors.

Because free long-chain bases have been recently found to have potent pharmacological effects when added to neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623) and other cell types, the levels in human neutrophils were measured by high-performance liquid chromatography. Sphingosine was the major free long-chain base in freshly isolated cells and ranged from 13 to 101 pmol/10(7) cells for different donors (mean +/- S.E. of 50 +/- 5, n = 17). Upon incubation at 37 degrees C, there was a time-dependent increase in free sphingosine (57 +/- 8% in 1 h, n = 17), but no change was seen at 4 or 25 degrees C. The sphingosine was apparently derived from more complex sphingolipids because little (less than 1%) could be accounted for by new synthesis from [14C]serine. Greater increases in free sphingosine were obtained when neutrophils were incubated with serum, plasma, or serum lipoproteins (about 2-fold higher than for cells incubated alone). In contrast, agonists such as phorbol 12-myristate 13-acetate, A23187, arachidonic acid, low concentrations (10 nM) of N-formyl-methionyl-leucyl-phenylalanine, and opsonized zymosan either decreased the amount of free sphingosine or blunted the time-dependent increase. This may be due to enhanced removal of free sphingosine because phorbol 12-myristate 13-acetate-treated cells exhibited an increased conversion of exogenously added [3H]sphinganine to ceramides. Endogenous sphingosine was approximately one-tenth the level found in neutrophils when exogenous long-chain bases were added to inhibit protein kinase C. Hence, depending on the subcellular localization of the endogenous versus exogenous long-chain bases, the amounts of free sphingosine in neutrophils might be sufficient to affect the function of these cells.

Calcimycin↗
PubMed1988

Successful pregnancies from men with retrograde ejaculation with the use of washed sperm and gamete intrafallopian tube transfer (GIFT).

Viable sperm were collected from men suffering from retrograde ejaculation by the technique of rapid washing of semen/urine in a buffered collection solution. The utilization of washed semen/urine from two men with retrograde ejaculation in the GIFT procedure lead to successful conceptions and the birth of two healthy female babies. Thus, the technique of rapid washing of sperm from retrograde ejaculating men coupled with GIFT represents a viable alternative for the attainment of pregnancy.

Ejaculation↗
PubMed1988

Multidisciplinary teams in health care: lessons from oncology and renal teams.

An indepth study conducted on units treating renal disease and cancer clinics determined that multidisciplinary teams are relatively commonplace in these areas. Developing four team organizational structures--sequential, primary, nucleus and dynamic--the authors hypothesize that each varies on a continuum in terms of how highly structured they are. The framework suggests that the ideology of equal participation on the team is the most difficult to sustain, and that difficulties typically arise as organization requirements become more complex. More research seems to be indicated to develop a comprehensive organizational framework, and the probable effect of organizational form on team performance. One of the key issues addressed is team leadership.

Canada↗
PubMed1988

Syringe HIV seroprevalence and behavioural and demographic characteristics of intravenous drug users in Sydney, Australia, 1987.

The contents of needles and syringes returned by intravenous drug users to two Sydney needle and syringe exchange centres were analysed for HIV antibodies by the enzyme-linked immunosorbent assay (ELISA). Reactive and borderline samples were further tested by the Western blot method. Basic demographic and needle sharing data were also collected from the clients of the exchanges. Of a sample of 1544 returned syringes, 48 (3%) were confirmed as containing HIV-infected blood. The proportion of infected syringes at exchange 2 was 6% (33 out of 545), which was significantly greater (P less than 0.05) than the proportion of exchange 1 at 1.5% (15 out of 999). The difference in seroprevalence between the two centres may be related to the behavioural characteristics of the client populations that attended each of the exchanges. The maintenance of a low syringe HIV seroprevalence (1-1.5%) over a 7-month period at one exchange may indicate that the availability of sterile needles and syringes prevented transmission of HIV among the clients of that exchange. HIV antibody testing of the contents of used syringes is a potentially valuable method of monitoring HIV infection among intravenous drug users. In this study, syringe exchange schemes have proved to be suitable venues for investigating the demographic characteristics and risk-taking behaviours of this population.

Adult↗
PubMed1988

A comparison of propofol and midazolam by infusion to provide sedation in patients who receive spinal anaesthesia.

Twenty patients scheduled for orthopaedic surgery under spinal anaesthesia received by intravenous infusion either 1% propofol or 0.1% midazolam at a rate adjusted to maintain adequate sedation as judged on a five-point scale. No other anaesthetic or analgesic drugs were given. The mean time to reach the required level of sedation was similar in both groups and the quality and ease of control of sedation were good in all patients. Mean infusion rates were 3.73 mg/kg/hour for propofol and 0.27 mg/hg/hour for midazolam. Airway maintenance was excellent and there were no side effects other than restlessness of the arms in one patient in each group. Recovery, judged by ability to open the eyes and recall date of birth, was significantly more rapid after propofol than after midazolam (2 and 10 minutes respectively after the end of infusion) and two patients in the latter group were unduly drowsy in the initial postoperative period. Pre- and postoperative amnesia were greater in the midazolam group but no patient had recall of peri-operative events. Psychometric tests showed significantly better recovery of higher mental function after propofol for up to 2 hours after surgery.

Anesthesia Recovery Period↗
PubMed1987

Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils.

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.

Calcimycin↗