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Biomedical subjects

R M Bell

Publications and source records attributed to R M Bell.

At least 91 records · Page 5Linked to original sources
PubMed1992

Stepping through the drug use sequence: longitudinal scalogram analysis of initiation and regular use.

Using a new technique for tracing the sequence of use over time, this study examined the pattern of drug involvement among 4,145 West Coast adolescents over the 4-year span from Grades 7-10. During the period covered, the mid- to late 1980s, we found no evidence that cocaine has become a gateway drug. However, the data provided some support for treating cocaine initiation as a separate stage that precedes the onset of hard drugs other than pills. The analysis also showed that increased involvement with legal drugs constitutes an important step in the transition to hard drug use for most adolescents. Weekly alcohol use followed marijuana use and preceded use of all other illicit drugs for Hispanic, White, and Black youth. However, it followed use of hard drugs for Asians. Weekly smoking formed a distinct stage between initial use of pills and other hard drugs for non-Hispanic Whites. The results underscore the importance of prevention efforts aimed at curbing the transition to regular use of alcohol and cigarettes, as well as their initial use.

Adolescent↗
PubMed1992

Complications of nonoperative management of blunt hepatic injuries.

Few, if any, complications have been reported with the nonoperative management of selected hepatic injuries diagnosed by computed tomographic (CT) scan in hemodynamically stable patients. This retrospective study was designed to evaluate complications associated with this form of management. Twenty-six patients (21%) of 128 patients with blunt hepatic injuries were treated nonoperatively over a 3-year period. All patients were hemodynamically stable at the time of admission and had hepatic injuries identified by CT scans of the abdomen. Five patients (19%) developed complications associated with nonoperative therapy. Of these, two patients had minor hepatic injuries (grades 1-2) and three had major (grades 3-5) hepatic injuries. Two patients (one with minor and one with major hepatic injury), developed free intraperitoneal biliary leaks and required operative repair. Three patients (one with minor and two with major hepatic injuries) developed large subcapsular bilomas with resultant hepatic dysfunction. These patients were successfully managed with percutaneous CT-guided drainage. There were no deaths in our study population with nonoperative therapy. The complications of hepatic injuries initially managed by expectant observation were treated operatively or by percutaneous CT-guided drainage. Repeated CT evaluation to follow the progress of liver fracture and the occasional use of hepatobiliary scans for the identification of biliary leaks have proven useful in our experience.

Adolescent↗
PubMed1992

Structural characterization of ordered arrays of sn-glycerol-3-phosphate acyltransferase from Escherichia coli.

Overproduction of the sn-glycerol-3-phosphate acyltransferase in Escherichia coli leads to incorporation of this integral membrane protein into ordered tubular arrays within the cell. Freeze-fracture-etch shadowing was performed on suspensions of partially purified tubules and whole bacteria. This procedure revealed the presence of ridges and grooves defining a set of long-pitch left-handed helical ridges. The long-pitch helices represented chains of acyltransferase dimers. Tubules observed within the cell were often closely packed, with an apparent alignment of grooves and ridges in adjacent tubules. Fracture planes passing through the tubules indicated the presence of a bilayer structure, with some portion of the enzyme being associated with the membrane. The major portion of the enzyme extended from the hydrophilic surface, forming a large globular structure that, in favorable views, displayed a central cavity facing the cytoplasm. Computer analysis of shadowed tubules revealed that the left-handed helices were six stranded, with a pitch of 1,050 A (105.0 nm) and a spacing of 75 A (7.5 nm) between acyltransferase dimers along the chains. Analysis of the predicted secondary structure failed to reveal obvious transmembrane segments, suggesting that very little of the protein was inserted into the bilayer.

Cytoplasm↗
PubMed1991

Protein kinase C contains two phorbol ester binding domains.

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.

Amino Acid Sequence↗
PubMed1991

Characterization of active and latent forms of the membrane-associated sn-glycerol-3-phosphate acyltransferase of Escherichia coli.

The intrinsically active, sn-glycerol-3-phosphate acyltransferase present in membranes prepared from both wild type Escherichia coli and from strains which overproduce the enzyme can be kinetically distinguished from a latent enzyme species which is unmasked by solubilization and reconstitution. Both membrane-associated and solubilized/reconstituted enzyme preparations exhibited cooperativity with respect to sn-glycerol-3-phosphate and palmitoyl-coenzyme A substrates; positive cooperativity in membranes toward palmitoyl-coenzyme A (napp = 4) and negative cooperativity toward sn-glycerol-3-phosphate (napp = 0.75) were significantly altered upon solubilization and reconstitution. Since the degree of alteration increased with the amount of sn-glycerol-3-P acyltransferase present in the membranes, a detergent-dissociable homooligomerization of the sn-glycerol-3-phosphate acyltransferase was considered as an underlying mechanism. This possibility was investigated by changing the protein-to-Triton X-100 ratio of homogeneous enzyme prior to reconstitution and then analyzing the subsequent migration of samples on a Sephacryl S-300 sizing column. The elution positions were consistent with monomeric and dimeric polypeptide bound to micelles of Triton X-100. Hill coefficients for monomeric, reconstituted enzyme preparations were comparable to those obtained for the active, membrane-associated sn-glycerol-3-phosphate acyltransferase. The reduced cooperativity of dimeric, reconstituted enzyme preparations correlated closely to the Hill coefficient values obtained for latent, solubilized/reconstituted sn-glycerol-3-phosphate acyltransferase from membranes of Escherichia coli which overproduce the enzyme. The physiological significance of these findings is discussed.

Cell Membrane↗
PubMed1991

sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae. Nucleotide sequence of the EPT1 gene and comparison of the CPT1 and EPT1 gene products.

The complete nucleotide sequence of the Saccharomyces cerevisiae EPT1 gene, a structural gene encoding an sn-1,2-diacylglycerol ethanolamine- and cholinephosphotransferase (Hjelmstad, R. H., and Bell, R. M. (1988) J. Biol. Chem. 263, 19748-19757), was determined. The 2123-nucleotide extent of DNA sequenced contained an open reading frame encoding 391 amino acids interrupted by an intron near its 5' end. Northern hybridization analysis detected a single 1.4-kilobase transcript. The inferred 44,525-dalton EPT1 gene product exhibited 54% amino acid sequence homology to the cholinephosphotransferase product of the yeast CPT1 gene. Predictive structural analysis of the EPT1 gene product revealed close structural similarity to the CPT1 gene product with respect to membrane topography, features of secondary structure, and transmembrane asymmetry. Regional protein homologies were identified between the EPT1 gene product and several enzymes as well as the nicotinic acetylcholine receptor. Comparative analysis of this set of protein homologies and the related set of protein homologies to the CPT1 gene product permitted identification of a presumptive active site region which contains highly conserved and divergent subregions and a common mononucleotide binding site.

Amino Acid Sequence↗
PubMed1991

sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae. Mixed micellar analysis of the CPT1 and EPT1 gene products.

The Saccharomyces cerevisiae CPT1 and EPT1 genes are structural genes encoding distinct sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases. A haploid cpt1 ept1 double null mutant lacked detectable choline- and ethanolaminephosphotransferase activity but was viable for growth, establishing that these enzymes are nonessential. The activities of the CPT1 and EPT1 gene products were independently studied in membranes prepared from strains mutant in the cognate locus using mixed micellar assays. Both enzymes absolutely required phospholipid cofactors; half-maximal activation was observed at low mole fractions, suggesting that a small number of phospholipid molecules are required. The activities of the CPT1 and EPT1 gene products were compared with respect to dioleoylglycerol dependence, CDP-aminoalcohol specificity, phospholipid activation, and inhibition by CMP. The EPT1 gene product utilized CDP-ethanolamine, -monomethylethanolamine, -dimethylethanolamine, and -choline to significant extents, while the CPT1 gene product manifested relative specificity for CDP-choline and -dimethylethanolamine. The CPT1 and EPT1 gene products exhibited differing properties with respect to phospholipid activation, but this difference was dependent on the CDP-aminoalcohol substrate. In contrast, the two enzymes could be distinguished on the basis of their dioleoylglycerol dependencies, activation by Mg2+, and CMP inhibition profiles regardless of the CDP-aminoalcohol substrate employed. These studies provide the first definitive kinetic properties of individual choline- and ethanolaminephosphotransferases.

Cytidine Diphosphate↗
PubMed1991

Mechanism of protein kinase C activation by phosphatidylinositol 4,5-bisphosphate.

The mechanism of protein kinase C (PKC) activation by phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI) was investigated by using Triton X-100 mixed micellar methods. The activation of PKC by PIP2, for which maximal activity was 60% of that elicited by sn-1,2-diacyglycerol (DAG), was similar to activation by DAG in several respects: (1) activation by PIP2 and DAG required phosphatidylserine (PS) as a phospholipid cofactor, (2) PIP2 and DAG reduced the concentration of Ca2+ and PS required for activation, (3) the concentration dependences of activation by PIP2 and DAG depended on the concentration of PS, and (4) PIP2 and DAG complemented one another to achieve maximal activation. On the other hand, PIP2 activation of PKC differed from activation by DAG in several respects. With increasing concentrations of PIP2, (1) the optimal concentration of PS required was constant at 12 mol%, (2) the maximal activity at 12 mol% PS increased, and (3) the cooperativity for PS decreased. PIP2 did not inhibit [3H]phorbol 12,13-dibutyrate (PDBu) binding of PKC at saturating levels of PS; however, at subsaturating levels of PS, PIP2 enhanced [3H]PDBu binding by acting as a phospholipid cofactor. PIP did not function as an activator but served as a phospholipid cofactor in the presence of PS. While PIP2, PIP, and PI did not support DAG-dependent PKC activation as phospholipid cofactors, their presence reduced the amount of PS required for maximal activation to as low as 2 mol% from 8 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Noninvasive diagnosis of vascular trauma by duplex ultrasonography.

Duplex ultrasonography was used prospectively in the initial evaluation of 198 patients with 319 potential vascular injuries of the neck and extremities. Patients who were unstable or who had obvious arterial trauma were excluded. Injury was caused by gunshot in 104 (53%), blunt trauma in 42 (21%), stab wound in 34 (17%), and shotgun in 18 (9%). Duplex ultrasonography correctly characterized and localized vascular injuries in 23 patients: arterial disruptions (13), intimal flaps (4), acute pseudoaneurysms (3), arteriovenous fistulas (2), and shotgun pellet arteriopuncture (1). Nineteen other patients had vasospasm (13) or external compression (6) without evidence of intrinsic vessel injury, these 42 studies had true-positive results. Twenty patients underwent arterial repair (13 on the basis of duplex ultrasonography alone), one had primary amputation, three required fasciotomy, and 18 were observed. Two patients with false-negative results had minor shotgun pellet arteriopunctures that were missed by duplex ultrasonography, but neither needed repair. One hundred fifty-three patients had true-negative results on duplex ultrasonography: all clinically had only proximity injuries and easily palpable distal pulses. The result of one duplex ultrasonography study was found to be false-positive on arteriography. The sensitivity of duplex ultrasonography was 95%, the specificity was 99%, and the overall accuracy was 98%. These results closely approximate those reported with the use of exclusion arteriography in the evaluation of similar vascular trauma patients. Furthermore, duplex ultrasonography has no interventional risks and is more cost-effective for screening such injuries than arteriography or exploration. Duplex ultrasonography is a reliable method of diagnosis in patients with potential peripheral vascular injuries.

Adolescent↗
PubMed1990

Expression of the phospholipid-dependent Escherichia coli sn-1,2-diacylglycerol kinase in COS cells perturbs cellular lipid composition.

The Escherichia coli sn-1,2-diacylglycerol (DAG) kinase has been successfully expressed in COS cells. The E. coli dgkA locus which contains the coding sequences for DAG kinase was subcloned into an eukaryotic expression vector, pMT2. COS cells transfected with the vector pMT2dgk expressed the DAG kinase as shown by Western analysis. Immunofluorescence studies revealed that the E. coli DAG kinase was prominently but not exclusively located in the endoplasmic reticulum. In addition, mixed micellar assays in beta-octyl glucoside revealed that membranes prepared from pMT2dgk-transfected COS cells contained over a 1500-fold increase in DAG kinase activity: 107 nmol/min/mg compared with only 0.067 nmol/min/mg for controls. DAG kinase activity from the E. coli enzyme was distinguished from endogenous COS cell activity based on differences in thermolability and the ability of the E. coli enzyme to use ceramide as a substrate. No ceramide kinase activity was detected in control COS cells, so the activity detected in pMT2dgk transfectants must have resulted from the expressed E. coli DAG kinase. The Km values for DAG kinase derived from E. coli and COS cells were nearly identical. Finally, transfected COS cells were labeled with [32P]Pi to investigate possible perturbations in lipid composition induced by the action of the E. coli DAG kinase. Ceramide (generated by the action of sphingomyelinase) was also used to clearly implicate the E. coli enzyme. Levels of ceramide phosphate increased more than 150-fold in pMT2dgk-transfected cells relative to controls. The results of these studies show that the E. coli enzyme expressed in COS cells is active and perturbs lipid composition in the intact cell system; the absolute lipid cofactor requirement of E. coli DAG kinase can be satisfied in COS cells.

Animals↗