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D L Bennett

Publications and source records attributed to D L Bennett.

At least 19 recordsLinked to original sources
PubMed1995

Putative capacitative calcium entry channels: expression of Drosophila trp and evidence for the existence of vertebrate homologues.

Capacitative calcium entry is a major pathway through which intracellular calcium stores are refilled after stimulation. It has been suggested that the protein encoded by the transient receptor potential (trp) gene expressed in Drosophila photoreceptors may be homologous with capacitative calcium entry channels. Expression of the trp gene product in Xenopus oocytes led to significant increases in calcium entry only when the intracellular calcium stores were depleted. Previous investigations have found trp to be uniquely expressed in Drosophila photoreceptors, but PCR cloning shows that homologous proteins exist in Calliphora, mouse brain and Xenopus oocytes. It is thus possible that capacitative calcium entry in Xenopus oocytes is mediated by a homologue of trp.

Amino Acid Sequence↗
PubMed1995

The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule.

Evidence suggests that nerve growth factor (NGF) may function as a mediator of some persistent pain states. We have used a synthetic protein, trkA-IgG, to sequester endogenous NGF and block the survival effects of NGF on cultured sensory neurons. We show that administration of trkA-IgG produces a sustained thermal and chemical hypoalgesia and leads to a downregulation of the sensory neuropeptide CGRP (calcitonin gene-related peptide) in treated sensory neurons. Acute administration of the molecule blocks the hyperalgesia that develops with carrageenan-induced inflammation. These data suggest that peripherally produced NGF normally acts to maintain the sensitivity of nociceptive sensory neurons and that, in some inflammatory states, an upregulation of NGF is responsible for alterations in pain-related behaviour. Antagonists of NGF may therefore be of clinical use in treating some chronic pain states.

Animals↗
PubMed1994

Smoothly graded Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores.

Stimulation of cells with Ca(2+)-mobilizing hormones often leads to the generation of temporally and spatially complex changes in the intracellular Ca2+ ion concentration ([Ca2+]i). To understand the mechanisms regulating Ca2+ release from intracellular stores more clearly, we investigated the ability of histamine to release Ca2+ stores under different experimental conditions, using video imaging of single Fura-2-loaded HeLa cells. In Ca(2+)-free medium, stepwise increases in histamine concentration released an increasing proportion of the intracellular Ca2+ pool. This pattern of Ca2+ release is analogous to the "quantal" release of Ca2+ previously observed using permeabilized cells. Quantal Ca2+ release was observed at both 20 and 37 degrees C and was not due to inactivation or desensitization of the Ca2+ release mechanism, since application of histamine in a pulsatile manner, which avoided desensitization of the Ca(2+)-release mechanism, still produced a quantal response. In Ca(2+)-containing medium at both 20 and 37 degrees C, stepwise increases in histamine concentration evoked [Ca2+]i responses where the amplitude was smoothly graded in direct proportion to the histamine concentration. Similar smoothly graded responses were observed from HeLa cells in Ca(2+)-free medium. These data indicate that hormone-evoked Ca2+ release from intracellular stores is limited by the hormone concentration, and that the mechanisms underlying complex [Ca2+]i signals do not lead to an all-or-none release of Ca2+ from the entire intracellular Ca2+ pool. We suggest that the hormone-sensitive intracellular Ca2+ pool is composed of functionally discrete units that are recruited by agonists in a concentration-dependent manner.

Calcium↗
PubMed1994

Postsynaptic effects of long-range afferents in distant segments caudal to their entry point in rat spinal cord under the influence of picrotoxin or strychnine.

1. Previous work has shown that substantial numbers of arriving myelinated afferent nerve fibers travel for many segments caudal to their entry point and terminate in the gray matter of distant segments. This fact is surprising because no monosynaptic post-synaptic responses attributable to these long-range afferents are observable in the distant segments. Evidence has been produced to explain this paradox by showing that impulse transmission is normally blocked in these long-range afferents by the tonic operation of a primary afferent depolarization (PAD) mechanism. Impulse transmission is restored if the PAD mechanism is disabled with gamma-aminobutyric acid antagonists. In this paper we examine the postsynaptic consequence of restoring conduction in the long-range afferents with picrotoxin. Because picrotoxin blocks PAD and increases the excitability of cells, we here contrast its action with that of the glycine antagonist strychnine, which increases the excitability of dorsal horn cells but does not affect PAD. 2. The preparation used throughout these experiments was decerebrate and spinal at T11. On one side dorsal roots T12, T13, and L1 were intact but all more caudal roots on that side were cut. Recordings of single units were made in the L6 segment with a rigid search pattern. The innervated area of skin on the flank was repeatedly stimulated with pressure in a repeated pattern. In the control state, few cells were detected responding in the L6 segment, which was five to seven segments caudal to the intact dorsal roots. After picrotoxin, the number of cells with excitatory receptive fields rose by a factor of 8, whereas there was no change in the number of inhibited cells. By contrast, after strychnine there was no change in the number of excited cells but the number of inhibited cells rose by a factor of 10. Details of the size of receptive fields, adequate stimuli, and location of the responding cells are provided. 3. To determine the number of synapses involved in producing the excitatory responses of L6 cells, we applied electrical stimuli to the peripheral receptive fields and to the L1 dorsal root. In the presence of picrotoxin, many cells were recorded with a latency consistent with monosynaptic connection. Some of these cells responded twice to two stimuli separated by 2 ms, but there was a latency variation between successive responses. Some cells responded with a short fixed latency and twice in 2 ms. No such cells were detected in the control state or in the presence of strychnine.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

The post-translational processing and intracellular sorting of PC2 in the islets of Langerhans.

Proinsulin conversion in the insulin secretory granule is mediated by two sequence-specific endoproteases related to the Kex2 homologues, PC2 and PC3 (Bennett, D. L., Bailyes, E. M., Nielsen, E., Guest, P. C., Rutherford, N. G., Arden, S. D., and Hutton, J. C. (1992) J. Biol. Chem. 267, 15229-15236; Bailyes, E. M., Bennett, D. L., and Hutton, J. C. (1992) Enzyme, in press). Radiolabeling studies using isolated rat islets showed that PC2 was synthesized initially as a 76-kDa glycoprotein which was converted by limited proteolysis to the mature 64-66-kDa form. Conversion was initiated approximately 1 h after synthesis and proceeded via intermediates of 71, 68, and 66 kDa with a t1/2 of 140 min. Release of only the 66- and 64-66-kDa radiolabeled forms of PC2 was induced by glucose and then only at times more than 2 h following synthesis. Proinsulin conversion, by contrast, was more rapid (delay = 30 min, t1/2 = 60 min), and release commenced as soon as 1 h after synthesis with the secreted material being comprised of the precursor, intermediate, and mature forms of insulin. Ultrastructural analysis of islet beta cells showed that PC2 was concentrated in secretory granules. Subcellular fractionation combined with immunoblot analysis showed that insulinoma secretory granules contained only the mature 64-66-kDa form of PC2, whereas fractions enriched in Golgi and endoplasmic reticulum contained a mixture of the 76- and 66-kDa forms of the enzyme. These results indicate that post-translational proteolysis of PC2 is initiated before sorting into the regulated pathway of secretion and that the relative proportions of proinsulin and PC2 packaged into secretory granules will change with physiological conditions.

Amidohydrolases↗
PubMed1992

Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family.

Enzymological studies have implicated two Ca(2+)-dependent endopeptidases in the conversion of proinsulin to insulin; a type 1 activity which cleaves on the C-terminal side of Arg31-Arg32 and a type 2 activity which cleaves C-terminally to Lys64-Arg65 in the proinsulin sequence. The possibility that these enzymes are related to the recently discovered family of mammalian subtilisin-like gene products (furin, PC2, and PC3) and the yeast propheromone-converting enzyme (KEX-2), was investigated. Degenerate oligonucleotide primers flanking the putative catalytic domain within this gene family were used in a polymerase chain reaction to amplify related sequences from rat insulinoma cDNA. One major product of 700 base pairs was obtained which was greater than 99% identical to the corresponding rat PC2 sequence. This cDNA was subcloned into the bacterial expression vector pGEX-3X to generate a recombinant protein for antibody production. Western blot analysis showed the immunoreactivity was prominent in neuroendocrine tissues as a 65-kDa protein. It was concentrated in secretory granule-enriched fractions of insulinoma tissue, where it was present as a readily solubilized monomeric protein. Deglycosylation studies using endoglycosidase H and N-glycanase showed that the 65-kDa protein was comprised of approximately 9% carbohydrate, consistent with the presence of three consensus sequences for N-linked glycosylation in rat PC2. The immunoreactivity co-eluted with the type 2 proinsulin endopeptidase on gel filtration and ion-exchange chromatography and the antisera specifically immunoprecipitated type 2 activity from insulin granule extracts. N-terminal sequence analysis of the immunoreactive protein gave two sequences which corresponded to residues 109-112 and 112-119 of rat PC2. This indicated that posttranslational processing of PC2 itself occurs C-terminally to basic amino acids to produce the mature enzyme. It is concluded that PC2 is the type 2 endopeptidase involved in proinsulin conversion. Localization of PC2 immunoreactivity to other tissues of the diffuse neuroendocrine system suggests that the type 2 endopeptidase also functions in the processing of precursor forms of other prohormones and polypeptide neurotransmitters.

Amino Acid Sequence↗
PubMed1991

Proprotein-processing endopeptidases of the insulin secretory granule.

Enzymological studies have implicated two Ca2+ dependent endopeptidases in the conversion of proinsulin to insulin: a type 1 activity and a type 2 activity which cleave on the C-terminal side of R31R32 and K64R65 in proinsulin, respectively. These activities were further characterized and their relationship to the mammalian family of subtilisin-like proteases was investigated. PC2 was expressed in neuroendocrine tissues and in insulinoma secretory granule fractions predominantly as a 65kDa protein. On anion-exchange chromatography of solubilized granules, PC1/3 immunoreactivity comigrated with a peak of type 1 activity whereas PC2 immunoreactivity coeluted with the peak of type 2 endopeptidase activity. PC2 antiserum gave a specific immunoprecipitation of type 2 activity from insulin granule extracts. It was concluded that the PC2 gene-product has type 2 endopeptidase activity.

Amino Acid Sequence↗
PubMed1989

Comparison of integrated electromyographic activity and lumbar curvature during standing and during sitting in three chairs.

The purposes of this study were to monitor the integrated electromyographic activity of the erector spinae (ES) muscles and to measure lumbar curvature (LC) during static and dynamic postures in three chairs--a Balans Multi-Chair (BC), and office chair (OC), and a straight-back chair (SBC)--and during standing. Integrated electromyographic data were recorded in relaxed and erect postures at L2 and L5 in 20 volunteers. Lumbar curvature was measured with a flexible ruler. Analyses of variance for repeated measures (p = .05) and paired t tests were used to compare the IEMG and LC measurements. During relaxed postures, there was more IEMG activity and greater LC in standing than in the OC or the SBC. During erect postures, there was more IEMG activity in standing than in the OC and no difference in LC between chairs. The IEMG activity at L5 was greatest on the left side across chairs. In typing and writing, significant differences in IEMG activity were found between sides, but not between chairs. The LC was greater in the BC than in the SBC in relaxed sitting, typing, and writing. The pattern of IEMG activity is not similar to corresponding LC measurements. Care and prevention of low back injury is a critical focus in physical therapy. The BC could contribute to treatment. Further research is needed to support its use in back care programs.

Adult↗
PubMed1989

Pediatric coin ingestion. A prospective study on the utility of routine roentgenograms.

It has been recommended that all children with a history of coin ingestion immediately undergo roentgenography to locate the coin, regardless of symptoms. We performed a prospective evaluation of these ingestions to determine the risk of asymptomatic esophageal impaction and the need for routine roentgenography. One hundred sixty-two children (mean +/- SD age, 3.6 +/- 2.1 years) were evaluated. All were referred for immediate roentgenography after ingestion and followed up daily by telephone for 5 days. Sixty-six patients (41%) did comply and 96 (59%) did not comply with the roentgenogram recommendation. A coin was visualized in the esophagus of 13 patients (20%); 11 were symptomatic and 2 were asymptomatic at the time of ingestion. Symptomatic patients had a 42% risk of a coin later being located in the esophagus compared with a 5% risk for asymptomatic patients. The asymptomatic patients with lodged coins passed them without difficulty after the administration of oral fluids. Nineteen percent of the patients who did not undergo roentgenography were symptomatic and all became asymptomatic within 24 hours of ingestion. There was no difference in morbidity between the group that underwent roentgenography and the group that did not undergo roentgenography at 5 days after ingestion. Children who are asymptomatic at the time of coin ingestion may not need routine roentgenography if they can tolerate oral fluids and telephone follow-up is available.

Child↗
PubMed1988

Clonidine transdermal patch poisoning.

A case of infant clonidine poisoning from a transdermal patch that had been worn for five days by an adult and then discarded is described. The infant became hypotensive with a systolic blood pressure of 38 mm Hg, and a dopamine infusion was required for six hours to maintain adequate blood pressure. The infant was discharged 24 hours after admission. Clonidine toxicity, transdermal delivery system pharmacokinetics, and poison prevention are discussed.

Accidents, Home↗
PubMed1988

Complications and vascular salvage of free-tissue transfers to the extremities.

Complications were examined in 122 free flaps to lower and upper extremities in 104 patients, and vascular salvage was examined in 182 free flaps to lower and upper extremities in 158 patients. All patients were treated by the same surgeon. The overall survival rate of flaps was 96 percent. Complications occurred in 22 percent of the flaps. Complication rates were lower in patients with one free flap than in patients with two. Flaps had more vascular complications than nonvascular. Accompanying skin islands were found to be necessary to monitor vascularized bone transfers in order to avoid flap failure. Flow in the pedicle was reestablished in all flaps, but a higher percentage of flaps with longer ischemic times were lost. Although vascular compromise occurred frequently (15 percent), prompt surgical exploration and reexploration were thought to have greatly increased free-flap survival.

Bone Transplantation↗
PubMed1987

Implementation of mobile decentralized pharmaceutical services in a community teaching hospital.

Administrative strategies that were used to justify mobile decentralized pharmaceutical services in a community teaching hospital are discussed. The report describes the problems prompting the pharmacy's review of its centralized unit dose drug distribution and i.v. admixture services, the evaluation of alternative solutions, the proposal submitted to hospital administrators, the implementation process, and an analysis of fiscal impact. A decentralized system using mobile medication carts was considered the best alternative because it integrated distributive and clinical functions and offered efficiencies in drug distribution; anticipated capital expenditure costs and personnel costs were also lower than the costs of alternatives. The conversion to the new system increased the pharmacy personnel budget by $174,455 annually, representing the addition of 13.8 full-time equivalent (FTE) pharmacists and elimination of 10.4 FTE technicians and interns. The increased personnel budget was offset in fiscal year (FY) 1982-83 by documented savings of $47,000 from pharmacists' clinical activities and by calculated savings of $132,400 in nursing time. Excluding capital expenditures of $19,800 for the medication carts, implementing mobile decentralized services saved the hospital $4945 in FY 1982-83. Mobile decentralized services is a cost-effective approach for providing integrated drug distribution and clinical pharmacy services.

Costs and Cost Analysis↗
PubMed1987

Evaluation of mobile decentralized pharmaceutical services in a community teaching hospital.

Mobile decentralized pharmaceutical services were compared with the previous centralized unit dose drug distribution services in a community teaching hospital. Medication order turnaround time, pharmacist workload activities, number of drug information requests, dose-activity index, and quality of drug distribution and drug administration record keeping were compared under the two systems. The number of drug therapy problems identified and resolved by decentralized pharmacists also was determined. Medication order turnaround time decreased from a mean of 198 minutes to 64 minutes in the centralized and decentralized systems, respectively. The number of drug information requests increased from 0.0055 to 0.05 requests per patient day. The percentage of requests related to adverse drug reactions, drug interactions, therapeutics, pharmacokinetics, and pharmacology increased in the decentralized system. The dose-activity index was 63.5% and 56.9% for centralized and decentralized systems, respectively, with the mean number of doses handled per patient day at 14.4 and 10.7. Decentralized pharmacists spent substantially more time than centralized pharmacists performing educational, therapy-related, and dispensing activities and less time performing clerical and verification activities. Decentralized pharmacists detected a large number of drug therapy problems that probably would have gone undetected in the previous system. The decentralized system met 31 of the 32 quality assurance standards, compared with 20 standards met for the centralized system. The implementation of mobile decentralized services provided greater opportunities for use of the pharmacists' clinical skills and reduced the time allocated to traditional functions.

Drug Information Services↗