Biomedical subjects

R S Decker

Publications and source records attributed to R S Decker.

At least 19 recordsLinked to original sources

PubMed1992

Evaluation of multiple parameters of HIV-1 replication cycle in testing of AIDS drugs in vitro.

Evaluation of the activities of antiretroviral agents and an immunoregulatory compound has been made using two models of HIV-1 infection and three measurements of virus expression. Acute infection of Jurkat cells or chronic/inducible infection in U1.1 cells was monitored at multiple time points after drug treatment. The 50% effective concentrations (EC50) of the HIV-1 inhibitors suramin, 3'-azido-3'-deoxythymidine (AZT), and 2',3'-dideoxycytidine, as measured by HIV-1 RNA hybridization in Jurkat cells two days after infection, were comparable to EC50 values obtained in parallel measurements of extracellular p24 levels and percent HIV-1 IF-positive cells. However, these measurements diverged: at seven days after infection the EC50 of AZT was greater than 10 microM when intracellular HIV-1 RNA was assayed, 0.2 microM by IF, and 0.03 microM by p24 assay. Human thymic humoral factor displayed no direct inductive activity in chronic HIV-1 infection in U1.1 cells, while phorbol ester and lymphocyte supernatants induced all parameters. These observations warrant care when interpreting results of only a single assay and suggest that definitive assay of HIV-1 infection requires measurements of multiple parameters of virus expression.

Antiviral Agents↗

PubMed1991

Cell shape and organization of the contractile apparatus in cultured adult cardiac myocytes.

The isolation and culture of adult cardiac myocytes has proved to be an ideal model system to explore myocardial biology at the cellular level. A major criticism of this model, however, has been that organ-specific characteristics such as cell shape and subcellular structural organization cannot be retained in vitro for prolonged periods of time. Encasing freshly isolated myocytes in a matrix of calcium alginate enables one to maintain the rod-like, three-dimensional (3D) shape of the cultured myocyte. Such preparations more closely resemble their in vivo counterparts with respect to the organization of the contractile apparatus, the transverse tubular system and the sarcoplasmic reticulum than do heart cells cultured on a two-dimensional (2D) plastic surface. Stereologic measurements reveal that myofibrillar volume density (VvMYF) decreases in both non-beating preparations over a 2-week interval, but VvMYF is conserved in cells cultured in an alginate matrix when compared to those myocytes maintained on a laminin-coated substratum. The present observations suggest that in the absence of contractile function myofibrillar atrophy appears responsible for the decline in VvMYF in alginate (3D) preparations, whereas atrophy and subcellular remodelling probably mediate the myofibrillar loss and reorganization that develops when adult heart cells are cultured on a 2D surface.

Alginates↗

PubMed1991

Hypertrophy of isolated adult feline heart cells following beta-adrenergic-induced beating.

Catecholamine-induced beating and myocardial hypertrophy were evaluated in isolated adult feline cardiomyocytes maintained in culture for up to 30 days. Adult feline cardiomyocytes were used in this study because they displayed several unique characteristics that facilitated assessment of factors regulating cardiomyocyte hypertrophy in vitro. These characteristics included the following. 1) A single heart provides a high yield of 20-40 x 10(6) calcium-tolerant rod-shaped myocytes. 2) In culture, isolated adult feline cardiomyocytes maintain a stable population of differentiated myocytes that could be maintained without the dramatic loss of cell number, DNA content, or cell structure seen in adult rat cardiomyocyte cultures. 3) Cultured feline cardiomyocytes remained quiescent in culture unless appropriately stimulated to begin beating. 4) Sustained regular beating activity could be readily initiated up to 3 wk in culture by addition of 1 x 10(-5) M isoproterenol, other beta-adrenergic agonists, or agents known to elevate adenosine 3',5'-cyclic monophosphate. Beating could be maintained indefinitely in the presence of isoproterenol, but ceased upon removal of isoproterenol from the medium. Initiation of beating in 7-day-old cultures resulted in a profound restructuring of cardiomyocyte morphology compared with quiescent cultures. Beating heart cells were 66% larger with increased protein content, and they had significantly greater development of striated myofibrillar structure than quiescent myocytes at the same age in culture. We conclude that maintenance of an organized myofibrillar structure in cultured adult cardiac myocytes requires activation of intrinsic beating. Cardiomyocyte hypertrophy also develops following beta-adrenergic activation of beating, but it is unclear whether beating per se is required for inducing hypertrophy in isolated adult cardiomyocytes in vitro.

1-Methyl-3-isobutylxanthine↗

PubMed1991

Morphometric evaluation of the contractile apparatus in primary cultures of rabbit cardiac myocytes.

Rabbit cardiac myocytes remain quiescent for more than 1 month when cultured at low density. During this period, myofibrillar volume density declines sixfold as myofibrils are disassembled or degraded and are replaced by actin and alpha-actinin-positive, myosin-negative structures that resemble myofibrils but lack thick filaments. Such structures are termed minute myofibrils. The length of the sarcomeres in these altered myofibrils is significantly less than length values obtained from freshly isolated heart cells or from contracting myocytes. A number of high density cultures develop spontaneous, synchronous contraction during the second week of culture. Myofibrillar volume density is stabilized when beating begins, and no further decline is observed in the succeeding weeks of culture. Such contracting myocytes display myofibrils typical of normal heart with no visible evidence of minute myofibrils. The volume density of the transverse tubular system also declines significantly in both beating and nonbeating myocytes, and its reduction appears more closely correlated with cell spreading than with beating per se. No quantitative changes in volume density of mitochondria or sarcoplasmic reticulum could be documented, but the structural organization of the sarcoplasmic reticulum seems to be greatly influenced by the physiological state of the heart cell. The present observations document the importance of mechanical factors in regulating the integrity of the contractile apparatus in cardiac myocytes and emphasize the utility of the cultured heart cell to directly investigate structure-function relations in individual myocytes.

Animals↗

PubMed1990

Morphological analysis of contracting and quiescent adult rabbit cardiac myocytes in long-term culture.

Isolated rabbit ventricular cardiac myocytes adapt readily to primary culture. As the myocytes spread and flatten over the culture substratum, the myofibrillar apparatus retains a "rod-like" orientation. Development of contractile activity is crucial in the maintenance of the integrity of the myofibrillar apparatus during prolonged culture. Myocytes that fail to beat display morphological indications of atrophy; conversely, myocytes that commence beating show no such morphological signs of myofibrillar disorganization. The subcellular organization of other elements of the contractile apparatus, including the transverse tubular system and the sarcoplasmic reticulum, retain their structural relationship with the myofibrils in beating myocytes but not in quiescent cells. Cultured adult myocytes represent an important model to investigate the influence of mechanical factors on the organization and maintenance of the adult cardiac phenotype.

Animals↗

PubMed1990

Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy.

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib-like structure between the cell membrane and the Z-disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three-dimensional arrangement of vinculin in relation to the Z-disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X-Y and X-Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z-disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofibrillogenesis.

Actinin↗

PubMed1990

Use of isolated adult myocytes to evaluate cardiotoxicity. I. Sugar uptake and protein synthesis.

The usefulness of isolated adult cardiac myocytes, both in suspension and in culture, as a model system for measuring cardiotoxicity is evaluated. It has been suggested that isolated adult myocytes should preferably be cultured for such experimentation, as this restores the cells to a physiological preference for fatty acids as a substrate rather than glucose. We show here that the restoration of Ca2+ to cells immediately after isolation results in an artificially enhanced glucose metabolism, as measured by an elevated rate of deoxyglucose uptake. Early restoration of Ca2+ during cell isolation, on the other hand, results in cells with a normal low level of deoxyglucose uptake. We, thus, conclude that cells can be ready for valid toxicity studies immediately after isolation, without the need for culture. The culture of adult feline ventricular cells is also described. These cells, like rabbit but unlike rat, are particularly promising for toxicity studies because they remain quiescent in culture and do not round up. On exposure to norepinephrine, they beat spontaneously and increase their rate of protein synthesis.

Animals↗

PubMed1989

Effects of cysteine protease inhibitors on rabbit cathepsin D maturation.

To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed (24 h) to media containing leupeptin (0-10 mM), E 64 (0-10 mM), or chloroquine (0-50 microM). Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D (Mr 53,000) within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D (Mr 48,000). Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate (not detectable in control fibroblasts). Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.

Animals↗

PubMed1988

Attachment and maintenance of adult rabbit cardiac myocytes in primary cell culture.

The present observations demonstrate that quiescent calcium-tolerant adult rabbit cardiac myocytes can be isolated by collagenase-hyaluronidase perfusion and maintained in primary culture for at least 2 wk. Culturing large numbers of myocytes requires that the freshly isolated cells be attached to a suitable substratum such as laminin, type IV collagen, or fetal bovine serum. The cultured myocytes retain their rod-like morphology for approximately 7 days before gradually spreading into a flattened conformation by 14 days. During the 1st wk of culture, contaminating interstitial cells rapidly proliferate, making cultures unsuitable for long-term study. Pure myocyte populations can be established and maintained if freshly isolated cells are cultured in the presence of cytosine arabinoside (Ara-C, 10 microM). This antimetabolite does not appear to adversely affect high-energy phosphates, since ATP and creatine phosphate (CrP) content of the myocytes is maintained at levels normally found in biopsy samples of rabbit myocardium. These results illustrate that an energetically stable population of adult cardiac myocytes can be maintained in primary culture in sufficient numbers to make them useful for future investigations of myocyte function.

Adenosine Triphosphate↗

PubMed1988

Nonrandom turnover of actin and tubulin in cultured rabbit cardiac fibroblasts.

Total protein fractional rates of growth, synthesis, and degradation were assessed in primary cultures of rabbit cardiac fibroblasts. Differences in fractional growth rates were produced by subculturing cells at low density and growing them to confluence. Total protein fractional degradative rates were then derived by subtracting fractional growth rates from measured fractional synthetic rates (obtained in [3H]leucine pulse-labeling experiments). Actin and tubulin degradation were studied in similar rapidly and slowly growing cultures. [35S]methionine pulse-chase experiments, followed by dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, and densitometry were used to determine the amount of labeled actin and tubulin remaining in cultures at various times during the chase (0-96 h). The indirect study showed a substantially lower total protein fractional degradative rate during rapid vs. slow growth (0.04 +/- 0.13 vs. 0.42 +/- 0.01 d-1 at 2 and 15 days after subculture, respectively; P less than 0.01). At both growth rates, the disappearance of labeled actin and tubulin was delayed, suggesting a more complex model for their degradation than random decay. Serum deprivation of slowly growing fibroblasts increased the rate of disappearance of both proteins by eliminating the delay in their breakdown. Thus the suppression of protein degradation during rapid growth appears to result from the presence of relatively greater amounts of "new" actin and tubulin (and possible other long-lived proteins) that are kinetically distinct from the total intracellular pools of these proteins with respect to their susceptibility to proteolysis.

Actins↗

PubMed1987

Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains.

Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.

DNA Replication↗

PubMed1987

In vitro initiation of DNA replication in simian virus 40 chromosomes.

A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta.

Antigens, Neoplasm↗

PubMed1987

Production of a factor by cultured human heart valves that is immunologically related to interleukin 1.

Analysis of the conditioned medium from cultured human heart valves showed that these tissues secrete a biologically active factor that induces chondrocytes in cultured cartilage to degrade extracellular matrix proteoglycan. This activity was similar to that described for porcine interleukin-1 (catabolin) and a cytokine secreted by cultured porcine heart valves (cardiac catabolic factor). The biological activity of the material in human valve conditioned medium was unaffected by the presence of low doses of cortisol, but its production by cultured valves was impaired by this steroid or benoxaprofen and abolished by cycloheximide. Addition of the conditioned medium to fibroblast monolayers stimulated the secretion of prostaglandin E and the tissue inhibitor of metalloproteinases (TIMP) but not collagenase. Preincubation of the conditioned medium with antiserum raised to the acidic form of porcine interleukin-1 neutralised the proteoglycan degrading stimulus. The material is biologically similar to other cytokines and antigenically related to porcine interleukin-1.

Animals↗

PubMed1987

Protein synthesis and degradation during starvation-induced cardiac atrophy in rabbits.

To determine the relative importance of protein degradation in the development of starvation-induced cardiac atrophy, in vivo fractional synthetic rates of total cardiac protein, myosin heavy chain, actin, light chain 1, and light chain 2 were measured in fed and fasted rabbits by continuous infusion of [3H] leucine. In addition, the rate of left ventricular protein accumulation and loss were assessed in weight-matched control and fasted rabbits. Rates of total cardiac protein degradation were then estimated as the difference between rates of synthesis and growth. Fasting produced left ventricular atrophy by decreasing the rate of left ventricular protein synthesis (34.8 +/- 1.4, 27.3 +/- 3.0, and 19.3 +/- 1.2 mg/day of left ventricular protein synthesized for 0-, 3-, and 7-day fasted rabbits, respectively). Inhibition of contractile protein synthesis was evident by significant reductions in the fractional synthetic rates of all myofibrillar protein subunits. Although fractional rates of protein degradation increased significantly within 7 days of fasting, actual amounts of left ventricular protein degraded per day were unaffected. Thus, prolonged fasting profoundly inhibits the synthesis of new cardiac protein, including the major protein constituents of the myofibril. Both this inhibition in new protein synthesis as well as a smaller but significant reduction in the average half-lives of cardiac proteins are responsible for atrophy of the heart in response to fasting.

Animals↗

PubMed1986

Hominoid triosephosphate isomerase: characterization of the major cell proliferation specific isozyme.

Proliferating cells derived from hominoid species contain electrophoretically separable forms of triosephosphate isomerase (TPI), including a constitutive isozyme and major and minor cell proliferation specific isozymes. Genetic studies have shown that the constitutive and inducible isozymes are products of the same structural gene. A procedure has been developed for the rapid isolation of the constitutive and major proliferation specific TPI isozymes from human lymphoblastoid B cells. [35S]methionine labeled isozymes were purified through several steps of polyacrylamide gel electrophoresis in sufficient quantities for turnover studies and preliminary structural analysis. The intact isozymes were subjected to 23 steps of automated Edman degradation; both preparations yield a [35S] PTH-methionine only at cycle 14, as expected if the protein is TPI. Neither isozyme contains a blocked NH2-terminus and length heterogeneity at the amino terminal does not exist. A comparison of the two purified isozymes on 2-D PAGE confirms that the constitutive isozyme consists of only type 1 subunits while the major proliferation specific isozyme is composed of a type 1 subunit and a unique type 2 subunit. The type 1 and type 2 subunits differ by at least four charge units under native, nondenaturing conditions of electrophoresis but do not differ in molecular mass. The difference between the type 1 and type 2 subunits is covalent, as the difference in isoelectric point between the two subunits is stable to both 2% SDS and 8 M urea. The expression of TPI-2 does not correlate with the existence of the labile asparagine residues. Turnover studies indicate that the level of each subunit is regulated by differences in rates of synthesis rather than degradation but a precursor-product relationship between the subunits was not observed. Thus the mechanism for synthesis of TPI-2 must operate either during mRNA processing or nascent peptide synthesis and then only in cells from hominoid species.

B-Lymphocytes↗

PubMed1986

Lysosomal responses of fetal mouse hearts recovering from anoxia and substrate depletion.

Recovery from a 1 h period of anoxia and substrate deprivation is accompanied by a marked lysosomal response in myocytes of fetal mouse hearts maintained in organ culture. Two classes of subcellular vacuoles form within 5 to 15 min of recovery. One appears to provide lysosomal enzymes for degradation of subcellular particles, while the other segregates organelles within the cytoplasm of the injured myocyte. When the two populations fuse with each other, the degradation of sequestered organelles appears to commence. After 6 h of recovery, intravacuolar degradation appears complete, and the injured myocytes are morphologically indistinguishable from control cells, demonstrating that the breakdown of the partitioned cell organelles is quite efficient. The process can proceed, albeit at a reduced rate, while protein synthesis is inhibited, since cycloheximide only modestly interferes with recovery after reoxygenation. The present results demonstrate that the fetal mouse heart subjected to conditions that simulate some important aspects of ischemia is an excellent model to examine the role of lysosomes during recovery from sublethal injury.

Animals↗

PubMed1986

Influence of agents that alter lysosomal function on fetal mouse hearts recovering from anoxia and substrate depletion.

Recovery of fetal mouse heart myocytes from oxygen and substrate deprivation for 1 h is accompanied by complicated lysosomal and non-lysosomal vacuolar responses which can be subdivided temporally into four distinct phases that include production of lysosomal dense bodies; segregation of damaged subcellular organelles into vacuoles that initially lack lysosomal enzymes; delivery of lysosomal enzymes to these vacuoles through fusion with dense bodies, transforming them into lysosomal autophagic vacuoles and degradation of the sequestered organelles. These events are normally completed within 6 h of the resupply of oxygen and substrate. The progression of these events is influenced significantly by pharmacological interventions that alter lysosomal properties. Chloroquine inhibits all aspects of the lysosomally-related processes as well as the sequestration phase during recovery. Leupeptin delays the lysosomal degradation, presumably by slowing proteolysis. Hydrocortisone permits the engulfment phase and the appearance of lysosomal dense bodies but appears to prevent or postpone the delivery of lysosomal enzymes to many of the large vacuoles and to delay the degradation of sequestered organelles. These observations reveal that segregation of damaged organelles and lysosomally-mediated degradation of these subcellular structures are important events during recovery from ischemic-like injury, and that agents that interfere with normal lysosomal function can prevent or delay some or all of the lysosomal responses that are involved in the recovery process.

Animals↗

PubMed1986

Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.

Animals↗