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Biomedical subjects

R J Schwartz

Publications and source records attributed to R J Schwartz.

At least 19 recordsLinked to original sources
PubMed1992

Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal alpha-actin transcription in embryonic myoblasts.

Muscle-restricted transcription of the skeletal alpha-actin gene is controlled in part by a positive regulator, serum response factor (SRF), and a negative regulator, F-ACT1, which bind competitively to the most proximal serum response element (SRE1). We show here that F-ACT1 is identical to a transcription factor recently cloned and described as YY1, NF-E1, delta, or UCRBP. We found that although the DNA-binding activity of SRF accumulates during myogenesis, that of YY1 diminishes simultaneously. Myoblasts rendered incapable of differentiation by BrdUrd treatment exhibited the highest level of YY1 and the lowest level of SRF activities. Transfected SRF could directly transactivate the skeletal alpha-actin promoter by overcoming the inhibitory effect of BrdUrd-induced YY1. The transactivation depends on intact SRE DNA elements and requires the DNA-binding/dimerization domain of SRF as well as its C-terminal half rich in serines and threonines. Since the functions of YY1 and SRF appear to be developmentally regulated, the convergence of their binding sites upon the SRE constitutes an integrated mechanism whereby temporal and spatial muscle gene expression may be accomplished.

Actins↗
PubMed1992

Positive and negative control of the skeletal alpha-actin promoter in cardiac muscle. A proximal serum response element is sufficient for induction by basic fibroblast growth factor (FGF) but not for inhibition by acidic FGF.

Like mechanical load in vivo, basic fibroblast growth factor (bFGF) selectively provokes cardiac expression of "fetal" genes including skeletal alpha-actin (SkA). Antithetically, acidic FGF (aFGF) suppresses SkA transcription. To define sites controlling SkA transcription in cardiac muscle cells, rat cardiac myocytes were transfected with internal-deletion and block-substitution mutations in the SkA promoter, including three motifs resembling the fos serum response element (SRE). The upstream, central, and proximal SREs each contributed to basal expression in cardiac myocytes. To determine whether identical elements mediate induction by bFGF versus inhibition by aFGF, the proximal SRE (SRE1) and fos SRE were positioned upstream from a neutral promoter. In cardiac myocytes, both the SRE1 and fos SRE were expressed at levels up to one-third that of the SkA promoter (nucleotides -202 to -11). Neither was expressed in quiescent cardiac fibroblasts. bFGF augmented SRE1-CAT activity, whereas aFGF produced no change; the fos SRE was induced by both. The transcriptional and mitogenic actions of aFGF were contingent on the presence of a putative nuclear translocation motif. Thus 1) the SkA SRE1 and fos SRE each suffice for tissue specificity in cardiac myocytes; 2) unlike the c-fos SRE, the SkA SRE1 is induced selectively by bFGF yet not aFGF; 3) sequences alternative or in addition to the SRE1 are obligatory for aFGF to suppress the SkA promoter; and 4) possible differences in intracellular localization are one basis for divergent actions of aFGF and bFGF in cardiac muscle cells.

Actins↗
PubMed1992

Elements of the smooth muscle alpha-actin promoter required in cis for transcriptional activation in smooth muscle. Evidence for cell type-specific regulation.

To assess the role of cis-acting elements within the smooth muscle alpha-actin gene in smooth muscle cells (SMC), we transfected chicken smooth muscle alpha-actin promoter-chloramphenicol acetyltransferase gene fusion plasmids into SMC derived from rat and chicken aortas. In marked contrast to effects in chicken skeletal myoblasts and fibroblasts, p122CAT (positions -122 to +19), containing two conserved CArG elements, elicited a modest increase in chloramphenicol acetyltransferase reporter activity in chicken SMC. Addition of upstream sequences between -122 and -151 (p151CAT) increased activity in adult chicken SMC. Addition of sequence between positions -151 and -257 (p257CAT) resulted in a 7-fold increase in chloramphenicol acetyltransferase activity over that of p151CAT in rat SMC, but not in chicken SMC. A genomic clone encoding the rat smooth muscle alpha-actin gene was isolated, and the 5'-flanking region was partially characterized. Comparison of primary sequence between rat and chicken promoters showed a conserved E box motif at position -214 in the chicken gene and at position -213 in the rat gene. Results of these studies demonstrate that regions upstream of the conserved CArG elements exert potent regulatory effects on transcription and that SMC require different cis-acting elements than other cell types to transcriptionally regulate this gene.

Actins↗
PubMed1992

Guidelines for surgical residents' working hours. Intent vs reality.

To comply with voluntary California medical school guidelines, our general surgery residency program reduced in-house call to 1 in 4 nights, and scheduled a 72-hour work-week. We assessed the effectiveness of these changes by prospectively surveying the actual working hours of surgical house staff through completion of a daily schedule for 1 month. Actual in-hospital hours averaged 98 per week, significantly exceeding the scheduled hours, and were greater for interns (100 hours) and junior residents (97 hours) than for chief residents (95 hours). Twenty hours (22%) of nonconference waking hours were spent on so-called scut work. Significant reduction of intern work hours could be accomplished by expansion of ancillary care, allowing more time for direct patient care. The effect on senior house staff hours would be less dramatic but might be sufficient to bring hours into compliance with proposed limits.

General Surgery↗
PubMed1992

The effect of fibrin glue on intraperitoneal contamination in rats treated with systemic antibiotics.

Intraperitoneal fibrin sealant lowers septic mortality in a rat model of peritoneal contamination (2 x 10(6) organism inoculum) at the cost of increased late intraabdominal abscesses. This study utilized parenteral antimicrobials to determine if the protective effect of intraperitoneal fibrin could be achieved without increasing the late abscess formation rate. One hundred and fifty-five rats were divided into four groups. Gelatin capsules containing various dilutions of feces (10(10) CFU/ml) and barium sulfate were placed into the abdomen in all groups. Group I controls had no antibiotics or fibrin. In group II, the capsule was surrounded by a solution of cryoprecipitate, thrombin, and calcium (fibrin "glue"). Groups III (no fibrin, antibiotics) and IV (fibrin, antibiotics) received a broad-spectrum cephalosporin intramuscularly postoperatively and then daily. Surviving rats were sacrificed on the tenth postoperative day. At a moderate volume of fecal inoculum (0.3 ml), fibrin reduced mortality from 100% in the control group to 0% in treated animals (P less than 0.001) that did not receive antibiotics. Abscesses formed in 10% of the surviving fibrin-treated rats which were implanted with 0.1 ml of inoculum. In the 0.2 and 0.3 ml inoculum groups substantially more abscesses occurred (75 and 70%, respectively). The protective effect of fibrin was not manifested in the antibiotic-treated rats since no deaths occurred in either group. At higher and lower inoculum doses, no significant differences between fibrin and control groups were observed in mortality or abscess formation, whether or not antibiotics were given.(ABSTRACT TRUNCATED AT 250 WORDS)

Abscess↗
PubMed1992

Factors associated with outcome in blunt aortic injury: a population-based study.

A population-based study was conducted for two contiguous states representing a population of 9.1 million to determine whether age, injury severity score, major complications, and preexisting conditions contribute to the outcome of patients diagnosed with blunt traumatic aortic injury. A secondary analysis reviewed patients with blunt aortic injury admitted over a six-year period to a trauma center located in one of the states to examine other more detailed factors related to mortality. Age was the only variable that correlated statistically with mortality in both populations analyzed. (Region P = .004; trauma center P = .0012) The severity of injury showed a tendency for decreased survival with increasing injury severity score. The elderly (age > or = 55) in both data sets sustained higher mortality from blunt aortic injury. In the trauma center population, the elderly had more delay in diagnosis than the younger patient population.

Adult↗
PubMed1992

Using proteases to avoid false identification of DNA-protein complexes in gel shift assays.

Gel mobility shift assays using crude nuclear extracts may result in the formation of multiple DNA-protein complexes reflected by their discrete gel mobilities. Identification of the multiple complexes can sometimes be complicated by the presence of protease activities in the extract as demonstrated here. We describe a simple protease-mediated partial digestion method that can be coupled with the gel shift assay to overcome the problem. The combined approach enables us to identify gel complexes that arise from protein degradation and therefore is suitable for analyzing those DNA-binding proteins exhibiting prominent protease sensitivity. The method should prove particularly informative in the search for tissue-specific complexes when crude extracts from different sources are compared by the gel shift assay.

Animals↗
PubMed1992

Evaluating the compliance of universal precautions by aeromedical personnel before and after an educational seminar on infectious diseases.

STUDY OBJECTIVE: To measure the compliance of an aeromedical crew with universal precautions and demonstrate what effect education has on utilization. DESIGN: Blinded time-series design. SETTING: Helicopter emergency medical service. TYPE OF PARTICIPANTS: Aeromedical crew consisting of flight nurses, respiratory therapists, and doctors. INTERVENTIONS: A mandatory educational seminar on universal precautions as required by the Centers for Disease Control. MEASUREMENTS AND MAIN RESULTS: Before the educational seminar the flight crew utilized gloves in 42% of patient contacts and goggles 0% of the time. At that time, masks and gowns were not available. The nurses used gloves in 28/72 (39%) of patients, respiratory therapists in 27/71 (38%) of patients, and doctors in 12/19 (63%) of patients. The overall compliance after the education seminar was 61% for gloves and 0% for goggles. The nurses used gloves in 20/36 (56%) of patients, the respiratory therapist in 23/34 (68%) of patients, and the doctors in 11/16 (69%) of patients. CONCLUSIONS: The use of gloves and goggles as preventive measures to protect the aeromedical crew from the potential hazards of body fluid contact and transmission of disease during their treatment of patients is low. Compliance increased after an educational seminar on universal precautions but still remained low. Other modalities, such as quality assurance measures, continuing education, policies, and peer pressure, in addition to education, are necessary.

Aerospace Medicine↗
PubMed1991

Phased cis-acting promoter elements interact at short distances to direct avian skeletal alpha-actin gene transcription.

Recently, site-directed mutagenesis uncovered four positive cis-acting elements in the 5' promoter region of the chicken skeletal alpha-actin gene that directs myogenic tissue-restricted expression. In this study, interactions between the four promoter sites were examined by means of a series of insertion mutations that increased the linker region between adjacent elements by roughly half or complete DNA helical turns. Unexpectedly, transcriptional activity for all three sets of linker mutants, as assayed with a chloramphenicol acetyltransferase reporter gene, was found to vary in a fashion resembling a damped sinusoid with a period of roughly 10 base pairs, where the sinusoidal maxima appeared when length was increased by half-integral number of helix turns. We present a model which states that in the undistorted wild-type 5' flanking sequence, linker domains position each of the four promoter sites on the helix face opposite that of its immediate neighbors; when any of the three linkers is increased by approximately a half-integral number of helix turns, pairs of neighboring promoter sites are brought into alignment. We propose that this is the required orientation for inducing skeletal muscle-specific promoter activity, achieved in the wild-type promoter as a result of protein-induced torsional deformation.

Actins↗
PubMed1991

Effect of aerosolized fibrin solution on intraperitoneal contamination.

The potential deleterious effects of aerosolized fibrin on contaminated procedures were investigated in a rat model of peritonitis. One hundred forty rats were divided into two groups. In the control group, gelatin capsules containing feces (10(7) bacteria per milliliter) and barium sulfate at various dilutions were placed into the abdomen; in the second experimental group, a solution of cryoprecipitate, thrombin, and calcium was sprayed diffusely into the peritoneal cavity after similar fecal contamination. Fecal inocula with low bacterial concentrations (0.01, 0.1, and 0.15 mL) caused few deaths from peritonitis or abscess formation in either group. Heavy peritoneal contamination (0.25, 0.3, and 0.5 mL) caused early deaths from peritonitis in both groups, with 80% of the deaths due to sepsis in the first 48 hours. However, in the moderately contaminated rats (0.2 mL of fecal inoculate), fibrin aerosol reduced the 10-day mortality from 80% to 10%. In all survivors in the fibrin-treated group, intraperitoneal abscesses developed. With intraperitoneal bacterial concentrations of 2 x 10(6) organisms, early acute mortality from fibrinopurulent peritonitis is decreased at the expense of late, localized, nonlethal abscess formation. Aerosolized fibrin solution must be used with caution in contaminated surgery.

Abscess↗
PubMed1991

TGF-beta 1 and fibroblast growth factors selectively up-regulate tissue-specific fetal genes in cardiac muscle cells.

TGF-beta 1, like basic and acidic fibroblast growth factor (FGF), inhibits differentiated gene expression in skeletal myoblasts. It potentiates FGF-beta 1 down-regulated expression of the alpha-myosin heavy chain gene and the sarcoplasmic reticulum calcium ATPase gene, yet up-regulated expression of the genes for beta-myosin heavy chain, atrial natriuretic factor, and both skeletal and smooth muscle alpha-actin-four transcripts associated with the embryonic heart. TGF-beta 1 did not affect cardiac alpha-actin gene expression. These responses resemble the generalized 'fetal' phenotype seen during hypertrophy triggered by a haemodynamic load. Chick skeletal and cardiac alpha-actin promoter-driven reported genes were transfected into neonatal rat cardiac myocytes. TGF-beta 1 stimulated skeletal alpha-actin transcription, but not transcription from the cardiac alpha-actin promoter. Basic FGF produced the same results as TGF-beta 1, but acidic FGF suppressed expression of both alpha-actin genes; these results were true for purified and recombinant FGFs. Modulation of alpha-actin transcription by growth factors corresponded accurately to control of the endogenous genes. Three positive cis-acting elements were critical for skeletal alpha-actin transcription in cardiac, as well as skeletal, myocytes, particularly the downstream CCAAT box-associated repeat. Thus, TGF-beta 1 and FGFs selectively induce an ensemble of 'fetal' genes and differentially regulate alpha-actin transcription in cardiac muscle cells.

Actins↗
PubMed1991

Evaluation of liver function tests in screening for intra-abdominal injuries.

STUDY OBJECTIVES: To determine the utility of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) in predicting intra-abdominal injury in blunt trauma patients. DESIGN: Descriptive review of 309 blunt trauma admissions during study period. SETTING: A 1,000-bed Level I trauma center in a major metropolitan area. TYPE OF PARTICIPANTS: Consecutive adult blunt trauma admissions to the trauma service. INTERVENTIONS: Serum levels of study enzymes were measured at initial evaluation and subsequent hospitalization. Results of all intra-abdominal evaluations were recorded. MAIN RESULTS: Significantly greater numbers of patients with SGOT and/or SGPT elevated to more than 130 IU/L had associated intra-abdominal injuries as compared with patients with enzyme elevations of less than 130 IU/L (52% versus 8%). All 18 patients with liver injuries had one or both enzymes elevated to more than 130 IU/L. Higher enzyme levels were more frequently associated with liver injury. CONCLUSIONS: Elevation of serum levels of the study enzymes is a marker for intra-abdominal injury. Levels in excess of 130 IU/L are relative indicators of abdominal computed tomography scan. Levels of less than 130 IU/L are unlikely to be associated with liver injury.

Abdominal Injuries↗
PubMed1991

Phorbol esters selectively downregulate contractile protein gene expression in terminally differentiated myotubes through transcriptional repression and message destabilization.

Chronic exposure of differentiated avian skeletal muscle cells in culture to the phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (PMA), results in the selective disassembly of sarcomeric structures and loss of muscle-specific contractile proteins, leaving cytoskeletal structures and their associated proteins intact. We demonstrate here that these morphological and biochemical changes are accompanied by dramatic and selective decreases in the level of the mRNAs that encode the contractile proteins. We measured the effects of PMA on the transcriptional activity and mRNA stability of four contractile protein genes (alpha-cardiac and alpha-skeletal actin, cardiac troponin C [cTnC], and myosin light chain lf [MLClf]) and two nonmuscle genes (beta-cytoplasmic actin and the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). The transcriptional activity of the alpha-cardiac actin and cTnC genes dramatically decreased by 8 h after the addition of PMA, while other muscle and nonmuscle genes examined showed no change. Pulse-chase experiments of in vivo labeled RNA showed significant reductions in mRNA half-lifes for all the contractile protein mRNAs examined, while the half-lifes of beta-actin and GAPDH mRNA were unchanged. All of the above effects occurred under conditions in which cellular protein kinase C (PKC) levels had been reduced by greater than 90%. The fact that many of the contractile protein genes remained transcriptionally active despite the fact that the cells were unable to accumulate their mRNAs to any significant extent indicated that the treated cells were still committed to skeletal muscle differentiation. The selective changes in the stability of the contractile protein mRNAs suggest that the control of mRNA stability may be part of the normal regulatory program of skeletal muscle differentiation and that this control may be linked to the integrity of the contractile apparatus and mediated by second messenger pathways involving PKC activation.

Animals↗
PubMed1991

Activation of skeletal alpha-actin gene transcription: the cooperative formation of serum response factor-binding complexes over positive cis-acting promoter serum response elements displaces a negative-acting nuclear factor enriched in replicating myoblasts and nonmyogenic cells.

Three upstream CBAR cis-acting promoter elements, containing the inner core CC(A/T)6GG of the serum response element (SRE), are required for myogenic cell type-restricted expression of the avian skeletal alpha-actin gene (K.L. Chow and R.J. Schwartz, Mol. Cell. Biol. 10:528-538, 1990). These actin SRE elements display differential binding properties with two distinct nuclear proteins, serum response factor (SRF) and another factor described here as F-ACT1. SRF is able to bind to all actin SREs with various affinities. This multisite interaction is marked by cooperative binding events in that the two high-affinity proximal and distal SREs facilitate the weak central-site interaction with SRF, leading to the formation of a higher-order SRF-promoter complex. Functional analyses reveal that undisrupted multiple SRF-DNA interactions are absolutely essential for promoter activity in myogenic cells. F-ACT1, present at higher levels in nonmyogenic cells and replicating myoblasts than in myotubes, binds solely to the proximal SRE, and its binding is mutually exclusive with that of SRF owing to their overlapping base contacts. The cooperative promoter binding by SRF, however, can effectively displace prebound F-ACT1. In addition, an intact F-ACT1 binding site acts as a negative promoter element by restricting developmentally timed expression in myoblasts. F-ACT1 may therefore act as a repressor of skeletal alpha-actin gene transcription. This interplay between F-ACT1 and SRF may constitute a developmental as well as a physiologically regulated mechanism which modulates sarcomeric actin gene expression.

Actins↗
PubMed1991

Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter.

Recent studies have shown that two genes regulating myogenesis (MyoD and myogenin) are coexpressed with cardiac alpha-actin during early stages of skeletal muscle development. Myogenin and MyoD are members of a family of regulatory proteins which share a helix-loop-helix (HLH) motif required for dimerization and DNA binding. Myogenin and MyoD form heterodimers with the ubiquitous HLH protein E12 which bind cis-acting DNA elements that have an E box (CANNTG) at their core. E boxes are present in the control regions of numerous muscle-specific genes, although their functional importance in regulating many of these genes has not yet been evaluated. In this report we examine the possibility that myogenin (or MyoD) directly transactivates the cardiac alpha-actin promoter. Heterodimers of myogenin and E12 (or MyoD and E12) specifically bound a restriction fragment extending from -200 to -103 relative to the start of cardiac alpha-actin transcription. Methylation interference footprints pinpointed the site of interaction to an E box immediately adjacent to a previously identified CArG box (CArG3). Site-directed mutations to the DNA-binding site revealed that either an intact E box or an intact CArG3 is required for induction of the cardiac alpha-actin promoter in myoblasts and for transactivation by myogenin in cotransfected fibroblasts. However, deletion and substitution experiments indicate that the complex E box/CArG3 element alone does not confer muscle-specific expression to a minimal promoter. These results suggest that direct and indirect pathways involving multiple cis-acting elements mediate the induction of the cardiac alpha-actin promoter by myogenin and MyoD.

Actins↗
PubMed1991

The vascular smooth muscle alpha-actin gene is reactivated during cardiac hypertrophy provoked by load.

Cardiac hypertrophy triggered by mechanical load possesses features in common with growth factor signal transduction. A hemodynamic load provokes rapid expression of the growth factor-inducible nuclear oncogene, c-fos, and certain peptide growth factors specifically stimulate the "fetal" cardiac genes associated with hypertrophy, even in the absence of load. These include the gene encoding vascular smooth muscle alpha-actin, the earliest alpha-actin expressed during cardiac myogenesis; however, it is not known whether reactivation of the smooth muscle alpha-actin gene occurs in ventricular hypertrophy. We therefore investigated myocardial expression of the smooth muscle alpha-actin gene after hemodynamic overload. Smooth muscle alpha-actin mRNA was discernible 24 h after coarctation and was persistently expressed for up to 30 d. In hypertrophied hearts, the prevalence of smooth muscle alpha-actin gene induction was 0.909, versus 0.545 for skeletal muscle alpha-actin (P less than 0.05). Ventricular mass after 2 d or more of aortic constriction was more highly correlated with smooth muscle alpha-actin gene activation (r = 0.852; P = 0.0001) than with skeletal muscle alpha-actin (r = 0.532; P = 0.009); P less than 0.0005 for the difference in the correlation coefficients. Thus, smooth muscle alpha-actin is a molecular marker of the presence and extent of pressure-overload hypertrophy, whose correlation with cardiac growth at least equals that of skeletal alpha-actin. Induction of smooth muscle alpha-actin was delayed and sustained after aortic constriction, whereas the nuclear oncogenes c-jun and junB were expressed rapidly and transiently, providing potential dimerization partners for transcriptional control by c-fos.

Actins↗
PubMed1991

Targeting of gene expression to skeletal and cardiac muscle of trangenic animals.

The tissue restricted and developmental potentiation of transcription by chicken alpha-skeletal actin promoter regions fused to the reporter gene chloramphenicol acetyl transferase (CAT) were characterized in transgenic mice. Six of eight expressing transgenic mouse lines containing the chicken alpha-skeletal actin promoter fused to CAT resulted in preferential transgene transcription in skeletal muscle tissue, similar to the endogenous mouse alpha-skeletal actin gene. Two of the eight lines departed from the preferred pattern of skeletal muscle expression with primary expression of the transgene in the heart, a tissue containing primarily cardiac actin isoforms. Developmentally, a transition from embryonic heart to fetal and neonatal skeletal muscle expression was produced by the transgene promoter, a pattern of regulation similar to that of the endogenous alpha-skeletal actin gene. Instances of departure of transgene expression from the endogenous gene implied the existance of higher order muscle gene regulatory mechanisms.

Actins↗