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Biomedical subjects

R M Bell

Publications and source records attributed to R M Bell.

At least 127 records · Page 7Linked to original sources
PubMed1989

Does open fasciotomy contribute to morbidity and mortality after acute lower extremity ischemia and revascularization?

A retrospective review was undertaken of 127 lower extremity fasciotomies performed for compartment syndrome after acute ischemia and revascularization in 73 patients with vascular trauma and 49 patients with arterial occlusive disease. One hundred twelve (88%) fasciotomies were performed early (at the time revascularization); 15 (12%) were delayed because of late compartment syndrome diagnosis. Ninety-four (77%) patients had more than one accepted indication for fasciotomy. Double-incision fasciotomy was used in 98 (77%) extremities, single-incision fasciotomy was used in 19 (15%), and fasciotomy-fibulectomy was used in 10 (8%). Fasciotomies were closed in 88 (69%) patients an average of 14 days after surgery. Seven patients needed multiple skin grafting procedures or myocutaneous flaps to close the wound; none compromised limb salvage. Five other patients had minor wound infections that resolved. Functional status returned to preoperative levels by the time of discharge from the hospital in 59 (48%) patients. Thirty-one (24%) patients had residual lower extremity disability related to delayed union of the fracture (five), chronic neuropathy (20), leg swelling (one), or ischemic nonhealing fasciotomy wounds (three); two patients had unrelated disabilities. Fourteen (11%) amputations were required for refractory limb ischemia; two (1.6%) were required for wet gangrene of the foot, which infected the fasciotomy site; the others had open noninfected incisions. Eighteen (15%) patients died of cardiopulmonary failure or multisystem failure or both, without fasciotomy-related problems. Open fasciotomy for compartment syndrome after acute lower extremity ischemia and revascularization was associated with an increased risk of minor wound morbidity. However, limb loss and death resulted from persistent ischemia and underlying systemic disease processes or injuries, but not from open fasciotomy wound complications.

Adolescent↗
PubMed1989

Elevation of 1,2-diacylglycerol in ras-transformed neonatal liver and pancreas of transgenic mice.

Expression of the activated Harvey-ras (H-ras) oncogene in cultured cells is associated with an elevated steady-state concentration of 1,2-diacylglycerol (DG), an intracellular second messenger capable of promoting cell division. To explore the biochemistry of ras expression in vivo, we measured DG in ras-transformed neonatal liver and pancreas of transgenic mice. DG was elevated over 2-fold in these tissues compared to controls, but was not elevated in transgenic neonatal liver expressing normal H-ras, the nuclear oncogene myc, or the Simian Virus 40 T-antigens. DG was also not elevated in ras-induced lung adenomas in transgenic mice. These findings demonstrate an association between activated ras expression and DG concentration in neonatal tissue, but suggest that marked elevation of DG is not necessary for the development of ras-induced tumors in lung.

Animals↗
PubMed1989

A facile enzymatic synthesis of sphingosine-1-phosphate and dihydrosphingosine-1-phosphate.

A procedure is described to prepare sphingosine-1-phosphate by treatment of sphingosylphosphocholine with phospholipase D, isolated from Streptomyces chromofuscus. The phosphorylated long chain bases were purified by selective precipitation and differential extraction. Milligram quantities can be obtained in a yield of about 70%. Application of the procedure to dihydrosphingosylphosphocholine results in the synthesis of dihydrosphingosine-1-phosphate.

Choline↗
PubMed1988

The sn-1,2-diacylglycerol ethanolaminephosphotransferase activity of Saccharomyces cerevisiae. Isolation of mutants and cloning of the EPT1 gene.

A colony autoradiographic assay was used to identify nine Saccharomyces cerevisiae mutants defective in in situ ethanolaminephosphotransferase activity (ept mutants). Genetic analysis revealed five complementation groups. The EPT1 gene was cloned by complementation of ept1 using a yeast genomic library and was localized to a 2.1-kilobase region of DNA. An ept1 deletional mutant was constructed and introduced into the chromosome by integrative transformation. The ethanolaminephosphotransferase activities in membranes prepared from ept1 and ept2 mutants were reduced 30- to 90-fold and 2- to 3-fold compared with wild-type activity, respectively; the other ept mutants had activities similar to wild type. In strains transformed with a multicopy EPT1-bearing plasmid, a 22- to 33-fold overproduction of ethanolaminephosphotransferase activity was observed. The sn-1,2-diacylglycerol cholinephosphotransferase activities in membranes prepared from ept1 mutants were reduced 3.5- to 7-fold. In contrast to the residual CMP-sensitive cholinephosphotransferase activity observed in cpt1 mutants (Hjelmstad, R. H., and Bell, R. M. (1987) J. Biol. Chem. 262, 3909-3917), the residual cholinephosphotransferase activity of ept1 mutants was CMP-insensitive. The cholinephosphotransferase activities in strains bearing the EPT1 gene on multicopy plasmids were elevated 13- to 23-fold and were CMP-sensitive. The data indicate that 1) the cloned EPT1 gene most likely represents the structural gene for the yeast ethanolaminephosphotransferase, 2) the EPT1 gene product possesses both ethanolamine- and cholinephosphotransferase activities, and 3) the EPT1 gene is nonessential for growth.

Cloning, Molecular↗
PubMed1988

sn-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (htpR).

Overexpression of the Escherichia coli sn-glycerol-3-phosphate (glycerol-P) acyltransferase, an integral membrane protein, causes formation of ordered arrays of the enzyme in vitro. The formation of these tubular structures did not occur in an E. coli strain bearing a mutation in the htpR gene, the regulatory gene for the heat shock response. The htpR165 mutation was shown by genetic analysis to be the lesion responsible for blockage of tubule formation. Similar amounts of glycerol-P acyltransferase were produced in isogenic htpR+ and htpR165 strains, ruling out an effect of htpR165 on expression of glycerol-P acyltransferase. Further, phospholipid metabolism was not altered in either strain after induction of glycerol-P acyltransferase synthesis. Increased glycerol-P acyltransferase synthesis caused a partial induction of the heat shock response which was dependent upon a wild type htpR gene. The heat shock proteins induced were identified as the groEL and dnaK gene products on two-dimensional gels. These two proteins have been implicated in the assembly of bacteriophage coats. These heat shock proteins appear essential for tubule formation.

Acyltransferases↗
PubMed1988

Regulation of the epidermal growth factor receptor phosphorylation state by sphingosine in A431 human epidermoid carcinoma cells.

The regulation of protein phosphorylation by sphingosine in A431 human epidermoid carcinoma cells was examined. Sphingosine is a competitive inhibitor of phorbol ester binding to protein kinase C (Ca2+/phospholipid-dependent enzyme) and potently inhibits phosphotransferase activity in vitro. Addition of sphingosine to intact A431 cells caused an inhibition of the phorbol ester-stimulated phosphorylation of two protein kinase C substrates, epidermal growth factor (EGF) receptor threonine 654 and transferrin receptor serine 24. We conclude that sphingosine inhibits the activity of protein kinase C in intact A431 cells. However, further experiments demonstrated that sphingosine-treatment of A431 cells resulted in the regulation of the EGF receptor by a mechanism that was independent of protein kinase C. First, sphingosine caused an increase in the threonine phosphorylation of the EGF receptor on a unique tryptic peptide. Second, sphingosine caused an increase in the affinity of the EGF receptor in A431 and in Chinese hamster ovary cells expressing wild-type (Thr654) and mutated (Ala654) EGF receptors. Sphingosine was also observed to cause an increase in the number of EGF-binding sites expressed at the surface of A431 cells. Examination of the time course of sphingosine action demonstrated that the effects on EGF binding were rapid (maximal at 2 mins) and were observed prior to the stimulation of receptor phosphorylation (maximal at 20 mins). We conclude that sphingosine is a potently bioactive molecule that modulates cellular functions by: 1) inhibiting protein kinase C; 2) stimulating a protein kinase C-independent pathway of protein phosphorylation; and 3) increasing the affinity and number of cell surface EGF receptors.

Carcinoma, Squamous Cell↗
PubMed1988

Aminoacridines, potent inhibitors of protein kinase C.

Acridine orange, acridine yellow G, and related compounds potently inhibited protein kinase C (Ca2+/phospholipid-dependent enzyme) activity and phorbol dibutyrate binding. Inhibition was investigated in vitro using Triton X-100 mixed micellar assays (Hannun, Y. A., Loomis, C. R., and Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043 and Hannun, Y. A., and Bell, R. M. (1986) J. Biol. Chem. 261, 9341-9347). Inhibition by the acridine derivatives was subject to surface dilution; therefore, the relevant concentration unit is mol % rather than the bulk molar concentration. Fifty percent inhibition of protein kinase C activity occurred at concentrations of these compounds comparable to concentrations of sn-1,2-diacylglycerol (DAG) and phosphatidylserine (PS) required for enzyme activation (i.e. 1-6 mol %). The mechanism of inhibition appeared to be complex: both the catalytic and regulatory sites of protein kinase C were affected. Acridine orange was a competitive inhibitor with respect to MgATP when the catalytic fragment of protein kinase C was employed. Inhibition at the active site was overcome by the addition of Triton X-100 micelles or phospholipid vesicles. When the activity of intact protein kinase C was measured, inhibition was noncompetitive with respect to MgATP. Further kinetic analysis suggested a competitive type of inhibition with respect to PS and DAG implying an interaction of acridine compounds with the regulatory lipid cofactors or with the regulatory domain of protein kinase C. This was further supported by demonstrating inhibition of phorbol dibutyrate binding to both protein kinase C and the lipid-binding domain generated by trypsin hydrolysis. Acridine orange and acridine yellow G also inhibited thrombin-induced 40-kDa phosphorylation in human platelets and phorbol dibutyrate binding to platelets. These effects were also subject to surface dilution. These results suggest that acridine derivatives have multiple interactions with protein kinase C with the predominant effect being inhibition of activation within the regulatory domain of the enzyme. Some of the biologic effects of acridine derivatives including anti-tumor action may occur as a consequence of protein kinase C inhibition.

Acridine Orange↗
PubMed1988

Effect of harvesting methods, growth conditions and growth phase on diacylglycerol levels in cultured human adherent cells.

The cellular mass of sn-1,2-diacylglycerols, which are intracellular second messengers which activate protein kinase C, were quantitatively determined with an enzymatic assay. The method employed to harvest cultured human skin fibroblasts or human epidermal A431 cells prior to extraction of lipid into chloroform/methanol affected diacylglycerol (DAG) levels. Scraping or trypsinization significantly increased DAG levels. A method was devised to allow reliable and reproducible DAG measurements from adherent cells. The addition of methanol prior to scraping was shown to stop cellular metabolism and to permit accurate quantitation. Importantly, this solvent was compatible with cultures grown on plastic. Using this method, growth conditions which could affect DAG levels were investigated. Changes in the osmolality of the culture medium did not affect the DAG levels of A431 cells; exposure of A431 cells to acidic pH or elevated temperature lowered DAG levels. In contrast to fibroblasts, the total DAG levels of A431 cells continued to increase during serum deprivation. The highest DAG levels, normalized to phospholipids, were observed during the exponential growth phase. This ratio dropped when the cultures reached confluency. These experiments also demonstrated that A431 cells possess higher DAG levels than do normal fibroblasts. The function of DAG in cellular regulation is discussed.

Cell Adhesion↗
PubMed1988

Altered growth regulation and enhanced tumorigenicity of NIH 3T3 fibroblasts transfected with protein kinase C-I cDNA.

Transfection of NIH 3T3 cells with plasmids containing rat brain protein kinase C-I (PKC-I) cDNA controlled by strong viral promoter/enhancer elements led to PKC-I gene expression as assessed by Northern analysis, cellular binding of phorbol ester, immunoblotting of cellular PKC, and membrane-associated PKC activity. While transfection did not induce foci, altered growth regulation was observed in established transfectant lines: transfectants displayed reduced dependence on serum for growth, grew to higher saturation densities, and displayed enhanced tumorigenicity when inoculated into nude mice. Continued high-level expression of PKC-I, however, may not be obligatory for the malignant phenotype in vivo. Tumors that retained transfected sequences had lower PKC-I transcript levels than the parental in vitro lines, suggesting an in vivo modulation. Our data show that PKC-I dysregulation leads to altered cell growth regulation and may be functionally equivalent to the action of tumor promoters.

Animals↗
PubMed1988

Sangivamycin, a nucleoside analogue, is a potent inhibitor of protein kinase C.

Protein kinase C functions prominently in cell regulation via its pleiotropic role in signal transduction processes. Certain oncogene products resemble elements involved in transmembrane signaling, elevate cellular sn-1,2-diacylglycerol second messenger levels, and activate protein kinase C. Sangivamycin was unique among the nucleoside compounds tested in its ability to potently inhibit protein kinase C activity. Inhibition was competitive with respect to ATP for both protein kinase C and the catalytic fragment of protein kinase C prepared by trypsin digestion. Sangivamycin was a noncompetitive inhibitor with respect to histone and lipid cofactors (phosphatidylserine and diacylglycerol). Sangivamycin inhibited native protein kinase C and the catalytic fragment identically, with apparent Ki values of 11 and 15 microM, respectively. Sangivamycin was an effective an inhibitor of protein kinase C as H-7, an isoquinolinsulfonamide. Sangivamycin did not inhibit [3H]phorbol-12,13-dibutyrate binding to protein kinase C. Sangivamycin did not exert its action through the lipid binding/regulatory domain; inhibition was not affected by the presence of lipid or detergent. Unlike H-7, sangivamycin selectively inhibited protein kinase C compared to cAMP-dependent protein kinase. The discovery that protein kinase C is inhibited by sangivamycin and other antitumor agents suggests that protein kinase C may be a target for rational design of antitumor compounds.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Post-traumatic sinusitis.

Septic complications following traumatic injury continue to be a contributing factor to morbidity and mortality. Paranasal sinusitis is being recognized as an often occult etiology of fever and sepsis in multiply injured patients. Our series of 11 patients who developed clinically important maxillary sinusitis is presented. Common features of the patients include: 1) nasal instrumentation; 2) craniofacial trauma; 3) concomitant use of steroids; and 4) severe multisystem injury (mean I.S.S., 45.5; T.S., 10.6). A high index of suspicion in patients with nasal tubes who develop unexplained fever or signs of systemic sepsis should prompt appropriate investigation of the paranasal sinuses. Removal of the tubes, antral puncture for irrigation and aspiration for microbiologic culture, topical nasal decongestants, systemic antibiotics based on sensitivity studies, and occasionally, formal surgical sinus drainage contribute to effective therapy.

Adult↗
PubMed1988

Affirmative action in medical education and its effect on Howard and Meharry: a study of the class of 1975.

During the decade of the 1970s, affirmative action programs were introduced in US medical schools for the purpose of increasing the number of black and other minority medical students and of improving the medical care resources for black and other minority communities. Having for many years served as the main sources of black physicians in the US, Howard University College of Medicine and Meharry Medical College School of Medicine were also affected by affirmative action. No previous studies have compared the black graduates from Howard and Meharry with black and other minority graduates from the other US medical schools.The purpose of this study was to compare these medical school graduates in terms of actual choice of specialty, patient characteristics, practice location, and specialty board certification, using the graduating class of 1975. A greater proportion of black graduates from Howard and Meharry chose primary care specialties than did black graduates from other schools, though this difference was not statistically significant. Black graduates from Howard and Meharry had significantly greater proportions of black patients compared with black graduates from other schools, but the same proportion of Medicaid patients.Though not statistically significant, black graduates from Howard and Meharry were less likely to be found practicing in federally designated underserved areas. Black graduates from Meharry were significantly less likely to have achieved specialty board certification, compared with graduates from Howard or the other medical schools. These results illustrate the experience of Howard and Meharry during the era of affirmative action and generally support their critically important role in producing black physicians and enhancing medical care resources in the black community.

Black or African American↗
PubMed1988

Functions of diacylglycerol in glycerolipid metabolism, signal transduction and cellular transformation.

Clearly, cellular DAG levels are regulated at the levels of synthesis, degradation and compartmentalization. This complex regulation enables DAG to perform its two distinct roles: supporting the biosynthesis (and degradation) of glycerolipids, and regulating PKC activity. Further definition is needed as to how DAG fulfills both functions, with particular emphasis on how distinct DAG pools are maintained, the interrelationships between the numerous pathways of DAG metabolism, and the role which elevated DAG plays in cellular transformation. Cellular function and growth control may be profoundly altered by perturbation of DAG metabolism. Defects in the regulation or the activity of enzymes responsible for attenuation of DAG second messengers (eg. DAG kinase and lipase) would be expected to elevate plasma membrane DAG levels. This could lead to persistent PKC activation and cellular transformation. Defects in the enzymes which utilize DAG in the biosynthetic pathway (eg. diacylglycerol acyltransferase, and choline and ethanolamine phosphotransferases) could elevate DAG levels in the ER. One consequence of this could be activation of PKC, perhaps at intracellular sites where activation does not normally occur. DAG undergoes rapid transbilayer movement and can be rapidly transferred between cellular membranes by a facilitated process. Therefore, elevated pools of DAG in the ER may lead to elevated DAG in other membranes (eg. plasma membrane) and PKC activation. These DAG utilizing enzymes may, therefore, represent products of unidentified recessive oncogenes.

Animals↗
PubMed1987

Assembly of the endoplasmic reticulum phospholipid bilayer. Transporters for phosphatidylcholine and metabolites.

Phosphatidylcholine is synthesized on the cytoplasmic surface of the endoplasmic reticulum and transported to the lumenal monolayer by a protein transporter, a phosphatidylcholine "flippase" (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). Since the endoplasmic reticulum contains enzymes involved in phosphatidylcholine turnover that have different locations within the organelle, transport systems may exist for phosphatidylcholine metabolites. To test the hypothesis that rat liver microsomes contain a lysophosphatidylcholine transporter, sn-1-monobutyroylphosphatidylcholine was employed. Since this homolog is highly water-soluble, transport of lysophosphatidylcholine could be measured using standard transport methods. sn-1-Monobutyroylphosphatidylcholine entered the lumenal compartment of microsomal vesicles. Transport was saturable and dependent on time and on amount of microsomes and required an intact permeability barrier. sn-1-Monobutyroylphosphatidylcholine transport was inhibited by treatment of microsomes with trypsin, N-ethylmaleimide, and trinitrobenzene-sulfonic acid. These findings suggest that sn-1-monobutyroylphosphatidylcholine transport is protein-mediated. sn-1-Monobutyroylphosphatidylcholine transported into microsomes was degraded to glycerophosphorylcholine. Glycerophosphorylcholine was also transported across the microsomal membrane. Glycerophosphorylcholine transport was also saturable and dependent on time, amount of microsomes, and an intact permeability barrier but was not inhibited by treatment with trypsin or the two protein modification agents. Thus, separate and distinct transport systems exist for phosphatidylcholine metabolites. Molecular events of phosphatidylcholine turnover in the endoplasmic reticulum are discussed.

Animals↗
PubMed1987

Sphingosine inhibition of agonist-dependent secretion and activation of human platelets implies that protein kinase C is a necessary and common event of the signal transduction pathways.

Sphingosine is a potent inhibitor of [3H]phorbol dibutyrate binding and protein kinase C activity in vitro and in human platelets (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. (1986) J. Biol. Chem. 261, 12604-12609). Preincubation of platelets with sphingosine resulted in the inhibition of platelet secretion and second phase aggregation in response to ADP, gamma-thrombin, collagen, arachidonic acid, and platelet activating factor. Sphingosine did not affect the initial shape change of platelets or the first phase of aggregation in response to these agonists. Ristocetin-induced platelet agglutination was not affected by sphingosine. Sphingosine inhibition of secondary aggregation (secretion and second phase aggregation) was overcome by phorbol dibutyrate and by the cell-permeable protein kinase C activator, dioctanoylglycerol. Furthermore, platelet secretion and irreversible aggregation were induced by protein kinase C activators in platelets that had been "primed" to undergo initial shape change and first phase aggregation by low concentrations of agonists. These results suggest that protein kinase C activation is a necessary component in the signal transducing pathways that lead to platelet activation. Higher concentrations of agonists, however, induced irreversible aggregation and partial secretion in the presence of sphingosine, suggesting the existence of protein kinase C-independent pathways for platelet activation. These results demonstrate the utility of sphingosine as a pharmacologic tool in probing the role of protein kinase C in signal transduction.

Adenosine Diphosphate↗