Biomedical subjects

L Kedes

Publications and source records attributed to L Kedes.

At least 37 records · Page 2Linked to original sources

PubMed1997

Molecular mechanisms of doxorubicin-induced cardiomyopathy. Selective suppression of Reiske iron-sulfur protein, ADP/ATP translocase, and phosphofructokinase genes is associated with ATP depletion in rat cardiomyocytes.

Doxorubicin, a cardiotoxic antineoplastic, disrupts the cardiac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mol. Cell. Biol. 15, 6386-6397). We have now identified neonatal rat cardiomyocyte mRNAs rapidly sensitive to doxorubicin, or its congener daunomycin, including transcripts of nuclear genes encoding enzymes critical in production of energy in cardiomyocytes: ADP/ATP translocase, a heart- and muscle-specific isoform; Reiske iron-sulfur protein (RISP), a ubiquitously expressed electron transport chain component; and a muscle isozyme of phosphofructokinase. Loss of these mRNAs following doxorubicin or daunomycin is evident as early as 2 h and precedes significant reduction of intracellular ATP. ATP levels in control cardiomyocytes (17.9 +/- 2.9 nM/mg of protein) fall only after 14 h and reach residual levels of 10.4 +/- 0.9 nM (doxorubicin; p = <0.006) and 6.7 +/- 1.9 nM (daunomycin; p = <0. 001) by 24 h. Loss of mRNAs generating ATP was highly selective since mRNAs for other energy production enzymes, (cytochrome c, cytochrome b, and malate dehydrogenase), and genes important in glycolysis (pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase) were unaffected even at 24 and 48 h. The drugs had no effect on levels of ubiquitously expressed RISP mRNA in fibroblasts. These findings could link doxorubicin-induced damage to membranes and signaling pathways with 1) suppression of transcripts encoding myofibrillar proteins and proteins of energy production pathways and 2) depletion of intracellular ATP stores, myofibrillar degeneration, and related cardiotoxic effects.

Adenosine Diphosphate↗

PubMed1997

Two-step delivery of retroviruses to postmitotic, terminally differentiated cells.

Recombinant replication-defective retroviral vectors are currently the most commonly used vectors for introducing foreign genes into human cells in gene therapy protocols. Their genomes stably incorporate in the host chromosomes of mitotic cells, thus ensuring stable expression. However, the applications of retroviruses to gene therapy are limited by their inability to infect postmitotic cells such as muscle fibers. In an attempt to overcome such limitations, we have developed a novel two-step transduction protocol that allows integration and expression of retroviral genes in differentiated cells. We induced DNA synthesis in terminally differentiated cultured mouse myotubes derived from both established myogenic cell lines and from primary myoblasts. We infected the postmitotic cells with a recombinant replication-defective adenoviral vector encoding the SV40 large T antigen as a mitogen. Subsequently we transduced the adenovirus-infected cells with a Moloney retroviral vector bearing the LacZ gene. Histochemical analysis revealed the coincident expression of LacZ gene in those myotubes that had been induced to synthesize DNA.

Adenoviridae↗

PubMed1997

Differential expression of troponin C genes during tongue myogenesis.

Determination of muscle fiber type is related to the developmental stage of the tissue. Ordinarily the final distribution of fast and slow fibers in a muscle is determined postnatally. Tongue muscle, however, is composed solely of fast-twitch fibers that express only troponin C fast mRNA and fast (type II) myosin heavy chain (MHC) proteins in both the adult and the one-day-old mouse. The fiber-type determination of this muscle was examined during fetal development. Both troponin C fast and slow mRNAs were expressed at initial stages of tongue development at embryonic day 18. However, by embryonic day 16 the troponin C fast transcripts predominated. AT 17 days of embryonic development, TnC fast mRNA was 10 times more abundant than TnC slow, and at 18 days of development the TnC slow mRNA was barely detectable. The tongue muscle myotubes expressed fast, slow, and embryonic MHC isoforms during early embryonic development. At 18 days of gestation, the MHC isoform expressed by the majority of the myotubes was the fast isoform, whereas the slow isoform was present in very few fibers. RT-PCR analysis of the MHC transcripts present throughout tongue development demonstrated expression of the mdms or type IIx MHC in both late fetal and postnatal stages of development. In contrast, the type I/beta slow MHC mRNA was undetectable in the postnatal and adult tongue. The absence of TnC and MHC slow-isoform mRNAs in the newborn mouse tongue suggests that slow isoform genes become dominantly repressed with the TnC-F and MHC type IIx genes remaining transcriptionally active, giving rise to an unusually homogeneous fast-twitch phenotype. The tongue muscle fibers acquire their specific adult-type fiber characteristics during fetal development rather than postnatally.

Animals↗

PubMed1997

Differential roles of p300 and PCAF acetyltransferases in muscle differentiation.

PCAF is a histone acetyltransferase that associates with p300/CBP and competes with E1A for access to them. While exogenous expression of PCAF potentiates both MyoD-directed transcription and myogenic differentiation, PCAF inactivation by anti-PCAF antibody microinjection prevents differentiation. MyoD interacts directly with both p300/CBP and PCAF, forming a multimeric protein complex on the promoter elements. Viral transforming factors that interfere with muscle differentiation disrupt this complex without affecting the MyoD-DNA interaction, indicating functional significance of the complex formation. Exogenous expression of PCAF or p300 promotes p21 expression and terminal cell-cycle arrest. Both of these activities are dependent on the histone acetyltransferase activity of PCAF, but not on that of p300. These results indicate that recruitment of histone acetyltransferase activity of PCAF by MyoD, through p300/CBP, is crucial for activation of the myogenic program.

Acetyl-CoA C-Acyltransferase↗

PubMed1997

Cell-cycle-specific transcription termination within the human histone H3.3 gene is correlated with specific protein-DNA interactions.

In vitro studies using highly purified calf thymus RNA polymerase II and a fragment spanning the first intron of H3.3 as template DNA have demonstrated the existence of a strong transcription termination site consisting of thymidine stretches. In this study, nuclear run-on experiments have been performed to assess the extent to which transcription elongation is blocked in vivo using DNA probes corresponding to region 5' and 3' of the in vitro termination sites. These studies suggest that H3.3 expression is stimulated following the inhibition of DNA synthesis through the elimination of the transcription elongation block. Interestingly, both the in vivo and in vitro experiments have revealed that the transcriptional block/termination sites are positioned immediately downstream of a 73 bp region that has been over 90% conserved between the chicken and human H3.3 genes. The extreme conservation of this intronic region suggests a possible role in maintaining cis-acting function. Electrophoretic mobility shift experiments show that HeLa cell nuclear extracts contain protein factors that bind specifically to the region of transcription elongation block. Furthermore, we demonstrate a correlation between the protein binding activity and the transcriptional block in cells that have been either arrested at the initiation of S phase or were replication-interrupted by hydroxyurea. DNA footprinting experiments indicate that the region of protein binding is at the 3' end of the conserved region and overlaps with one of the three in-vitro-mapped termination sites.

Animals↗

PubMed1997

The anti-cancer agent distamycin A displaces essential transcription factors and selectively inhibits myogenic differentiation.

The anticancer drug, distamycin A, alters DNA conformation by binding to A/T-rich domains. We propose that binding of the drug to DNA alters transcription factor interactions and that this may alter genetic regulation. We have analyzed the effects of distamycin A upon expression of the muscle-specific cardiac and skeletal alpha-actin genes which have A/T-rich regulatory elements in their promoters. Distamycin A specifically inhibited endogenous muscle genes in the myogenic C2 cell line and effectively eliminated the myogenic program. Conversely, when 10T1/2C18 derived pleuripotential TA1 cells were induced to differentiate in the presence of distamycin A, adipocyte differentiation was enhanced whereas the numbers of cells committing to the myogenic program decreased dramatically. Using the mobility shift assay distamycin A selectively inhibited binding of two important transcription factors, SRF and MEF2, to their respective A/T-rich elements. The binding of factors Sp1 and MyoD were not affected. The inhibition of factor binding correlated with a repression of muscle-specific promoter activity as assayed by transient transfection assays. Co-expression of the myoD gene, driven by a distamycin A-insensitive promoter, failed to relieve the inhibition of these muscle-specific promoters by distamycin A. Additionally, SRF and MEF2 dependent promoters were selectively down regulated by distamycin A. These results suggest that distamycin A may inhibit muscle-specific gene expression by selectively interfering with transcription factor interactions and demonstrate the importance of these A/T-rich elements in regulating differentiation of this specific cell type.

Actins↗

PubMed1997

The basic domain of myogenic basic helix-loop-helix (bHLH) proteins is the novel target for direct inhibition by another bHLH protein, Twist.

In vertebrates, the basic helix-loop-helix (bHLH) protein Twist may be involved in the negative regulation of cellular determination and in the differentiation of several lineages, including myogenesis, osteogenesis, and neurogenesis. Although it has been shown that mouse twist (M-Twist) (i) sequesters E proteins, thus preventing formation of myogenic E protein-MyoD complexes and (ii) inhibits the MEF2 transcription factor, a cofactor of myogenic bHLH proteins, overexpression of E proteins and MEF2 failed to rescue the inhibitory effects of M-Twist on MyoD. We report here that M-Twist physically interacts with the myogenic bHLH proteins in vitro and in vivo and that this interaction is required for the inhibition of MyoD by M-Twist. In contrast to the conventional HLH-HLH domain interaction formed in the MyoD/E12 heterodimer, this novel type of interaction uses the basic domains of the two proteins. While the MyoD HLH domain without the basic domain failed to interact with M-Twist, a MyoD peptide containing only the basic and helix 1 regions was sufficient to interact with M-Twist, suggesting that the basic domain contacts M-Twist. The replacement of three arginine residues by alanines in the M-Twist basic domain was sufficient to abolish both the binding and inhibition of MyoD by M-Twist, while the domain retained other M-Twist functions such as heterodimerization with an E protein and inhibition of MEF2 transactivation. These findings demonstrate that M-Twist interacts with MyoD through the basic domains, thereby inhibiting MyoD.

Amino Acid Sequence↗

PubMed1997

Molecular mechanisms of myogenic coactivation by p300: direct interaction with the activation domain of MyoD and with the MADS box of MEF2C.

By searching for molecules that assist MyoD in converting fibroblasts to muscle cells, we have found that p300 and CBP, two related molecules that act as transcriptional adapters, coactivate the myogenic basic-helix-loop-helix (bHLH) proteins. Coactivation by p300 involves novel physical interactions between p300 and the amino-terminal activation domain of MyoD. In particular, disruption of the FYD domain, a group of three amino acids conserved in the activation domains of other myogenic bHLH proteins, drastically diminishes the transactivation potential of MyoD and abolishes both p300-mediated coactivation and the physical interaction between MyoD and p300. Two domains of p300, at its amino and carboxy terminals, independently function to both mediate coactivation and physically interact with MyoD. A truncated segment of p300, unable to bind MyoD, acts as a dominant negative mutation and abrogates both myogenic conversion and transactivation by MyoD, suggesting that endogenous p300 is a required coactivator for MyoD function. The p300 dominant negative peptide forms multimers with intact p300. p300 and CBP serve as coactivators of another class of transcriptional activators critical for myogenesis, myocyte enhancer factor 2 (MEF2). In fact, transactivation mediated by the MEF2C protein is potentiated by the two coactivators, and this phenomenon is associated with the ability of p300 to interact with the MADS domain of MEF2C. Our results suggest that p300 and CBP may positively influence myogenesis by reinforcing the transcriptional autoregulatory loop established between the myogenic bHLH and the MEF2 factors.

3T3 Cells↗

PubMed1997

Involvement of phosphorylation in doxorubicin-mediated myofibril degeneration. An immunofluorescence microscopy analysis.

Loss of myofilaments has been observed in both adaptive cardiac responses (i.e., hypertrophy) as well as in chemotheraputic use of antineoplastic drugs with cardiotoxic side effects (i.e., doxorubicin). An understanding of the degenerative process is a prerequisite for determining approaches to limit the cardiomyopathic changes associated with chronic heart disease or long-term chemotheraputic treatments. However, little is known about the specific events and molecular changes that initiate the degenerative process. To study this process, neonatal rat cardiomyocytes were treated with doxorubicin, which induced rapid and widespread thin-filament degeneration as observed by fluorescence confocal microscopy. Which demonstrated deterioration of sarcomeric thin-filament structure. Changes in the spontaneous beating of cardiomyocytes corresponding with myofibrillar degeneration were apparent using differential interference contrast video microscopy. After finding induction of kinase activity by doxorubicin in cultured cardiomyocytes, the protective effects of specific inhibitors of kinase activity were assessed for their ability to inhibit doxorubicin-induced myofibrillar break-down. Doxorubicin-induced changes appeared similar to the degeneration observed after treatment with a protein kinase activator (phorbol 12-myristate 13-acetate) or a serine-threonine protein phosphatase inhibitor (okadaic acid). Collectively, these results indicate that activation of protein kinase is an important event in the initiation of myofibrillar degeneration by doxorubicin. Further analyses of myofibrillar proteins with respect to biochemical modifications will be necessary to determine if phosphorylation events transmit signal(s) to initiate degeneration.

Animals↗

PubMed1997

Renal mRNA of PTH-PTHrP receptor, [Ca2+]i and phosphaturic response to PTH in phosphate depletion.

Available data indicate that the mRNAs of the PTH-PTH-related protein (PTH-PTHrP) receptor in the kidney, liver and heart are down-regulated in chronic renal failure (CRF). This is due, in major part, to the elevation of cytosolic calcium ([Ca2+]i) in the cells of these organs. If elevation in [Ca2+]i is indeed the culprit, one should be able to demonstrate down-regulation of the mRNA of the PTH-PTHrP receptor in situations without CRF and with low levels of PTH but with elevated [Ca2+]i. Such a combination of events occurs in phosphate depletion (PD). To test this hypothesis, we examined the [Ca2+]i and the concentration of the mRNA of the PTH-PTHrP receptor in the kidneys from 1, 3 and 6 weeks PD, pair-weighed (PW) rats and PD and PW rats treated with verapamil (PD-V, PW-V). To evaluate the effect of a potential rise in [Ca2+]i on urinary phosphate excretion, we also measured the phosphaturic response to PTH and cAMP in all groups of rats after 6 weeks of the dietary intervention. Renal function was normal in all groups of animals. Blood levels of PTH were significantly (p < 0.01) lower in PD and PD-V after 1 week of PD than in PW and PW-V rats, and they remained low throughout the study. The basal levels of [Ca2+]i in the renal proximal tubular cells were normal after 1 week of PD but rose by the third week of the study and remained elevated by the end of the sixth week. These values were significantly (p < 0.01) higher than those in PD-V, PW and PW-V rats. The concentrations of mRNA of the PTH-PTHrP receptor relative to that of the housekeeping gene G3DPH were significantly (p < 0.01) lower in PD rats after 3 and 6 weeks than in the other three groups of rats. The phosphaturic response to PTH or cAMP was significantly (p < 0.01) greater in PD-V rats than in PD animals. The data show that PD is associated with a rise in [Ca2+]i of renal proximal tubular cells and with down-regulation of PTH-PTHrP receptor in the kidney despite low levels of PTH and normal renal function; normalization of the concentration of [Ca2+]i in PD-V rats was associated with normal expression of mRNA of the receptor. These results provide strong support for the proposal that elevated [Ca2+]i down-regulates the mRNA of the PTH-PTHrP receptor even in the absence of CRF and elevated blood PTH levels. The improvement in the phosphaturic response to PTH and cAMP in PD-V rats is consistent with the notion that the elevated [Ca2+]i of the renal cell in PD rats may interfere with the coupling of PTH receptor-adenylate cyclase system and/or with the postreceptor events responsible for the inhibition of phosphate reabsorption by these agonists.

Animals↗

PubMed1996

Avoidance of immune response prolongs expression of genes delivered to the adult rat myocardium by replication-defective adenovirus.

BACKGROUND: Gene delivery is a rapidly expanding field with potential applications to every human organ system. Recently, adenoviruses have been used as efficient vectors for in vivo gene transfer into the myocardium. These methods, however, have shown a sharp decline of gene expression after 1 week. To test the hypothesis that an immune-effector mechanism is involved in this decline, we compared the results after injection of adenovirus-5 carrying the beta-galactosidase gene (Ad beta-gal) into the left ventricular myocardium of athymic nude rats (NDRs) versus immunocompetent Sprague-Dawley rats (SDRs). METHODS AND RESULTS: Ad beta-gal (5.0 x 10(9) PFU/mL) was injected into the left ventricle of NDRs (n = 16) and SDRs (n = 22). Hearts were harvested, embedded in paraffin, and sectioned and stained for beta-gal activity, hematoxylin and eosin and picrosirius red at 4, 21, 35, 85, and 120 days. Representative samples were immunostained with antibodies directed at inflammatory markers. beta-gal activity was quantified by digital planimetry and expressed as area of staining (% +/- SEM). Peak beta-gal activity was highest at 4 days, with NDRs displaying significantly greater staining (83 +/- 3.0% versus 54 +/- 8.0%; P = .03). SDRs sustained a rapid drop in activity, such that at 35 (1 +/- 0.19%) and 85 (1 +/- 0.4%) days, only occasional cells stained positive and by 120 days (0.3 +/- 0.0%), activity had been extinguished. NDRs continued to show transgene expression at all time periods (35 and 85 days, 25 +/- 7.1% and 7.4 +/- 2.7%, respectively) and was still readily detected at 120 days. An inflammatory response was limited in NDRs compared with SDRs, in which there was intense mononuclear cell infiltration, with collagen deposition and scar formation. Immunostaining identified the majority of these inflammatory cells as not being of lymphocyte lineage, although small numbers of lymphocytes and phagocytic and activated plasma cells were identified. CONCLUSIONS: Our data suggest that immune-effector mechanisms can severely affect the expression of genes delivered by adenovirus. The present model provides efficient gene expression for at least 120 days without significant inflammatory reaction.

Adenoviridae↗

PubMed1996

Doxorubicin inhibits Tat-dependent transactivation of HIV type 1 LTR.

Tat, the human immunodeficiency virus (HIV)-encoded transcription factor, is vital for HIV replication and transcription. Any drug that inhibits Tat's activity is a valuable candidate for chemotherapeutic applications. We show here that doxorubicin (Dox), a well-known anticancer drug and its derivative, daunomycin, inhibit the ability of Tat to activate the HIV-1 LTR. We contransfected HeLa cells with pSV40TAT and a chloramphenicol acetyltransferase gene driven by an HIV LTR promoter. CAT transcription was vigorously stimulated many fold by Tat production but the effect of Tat was inhibited by Dox in a dose-dependent manner. The transcriptional activation domain of Tat, located in its 67 amino terminal residues, remains Dox sensitive. A TAR-deleted reporter gene with a Gal binding domain is transactivated by a Gal-Tat fusion protein. This transcription complex retains a high level of activity in the presence of Dox, suggesting that Dox primarily affects RNA-Tat, rather than DNA-Tat, mediated transactivation. RNA gel mobility analysis reveals that Dox does not affect the binding of Tat to TAR-RNA in vitro but does increase the binding activity of cellular nuclear proteins with TAR-RNA. Induction or activation of such TAR-binding proteins in cells that might interfere with the activity of Tat could explain the observed inhibitory effects of Dox on Tat-activated transcription. These results suggest that Dox may have chemotherapeutic effects on HIV expression mediated through TAR RNA.

Antibiotics, Antineoplastic↗

PubMed1996

Lens tropomodulin: developmental expression during differentiation.

Lens epithelial cells undergo a dramatic transformation during the process of differentiation into elongated fiber cells. The membrane-associated actin cytoskeleton is likely to play a critical role in the stabilization and maintenance of the highly elongated fiber cell shape. Tropomodulin is a tropomyosin-binding protein associated with actin filaments in a variety of terminally differentiated cell types where stable actin filament organization is required for cell function. We now present results of studies to determine the temporal expression of tropomodulin in the developing lens. In situ hybridization experiments detected expression of tropomodulin mRNA in the developing mouse lens in elongating cells with a pattern similar to that of the fiber specific beta- and gamma-crystallins. Tropomodulin mRNA expression first appeared around 11.5 days post-coitum in elongating cells in the posterior part of the lens vesicle. At later stages the signal for tropomodulin was present in the elongating cells at the lens equator and in cortical fiber cells; signal was absent from the epithelium. To investigate the possible link between tropomodulin expression and fiber differentiation we used a well-established lens epithelial explant culture system in which fiber differentiation is induced by fibroblast growth factor (FGF). Tropomodulin expression was only observed in FGF-treated explants in conjunction with morphologic changes characteristic of lens fiber cell differentiation. The appearance of tropomodulin during the process of fiber cell differentiation suggests that tropomodulin may be important for stabilization and/or determination of actin filament length.

Animals↗

PubMed1996

Ischemic/reperfused myocardium can express recombinant protein following direct DNA or retroviral injection.

A non-contracting scar following myocardial infarction can adversely affect ventricular topography and hemodynamic function. Gene transfer has the potential to prevent or alter such pathophysiological processes. Normal myocardium is a proven target for delivery of DNA or viral vectors but the potential for gene therapy in ischemic myocardium has not been evaluated. In an initial series of experiments, we determined whether the direct injection of reporter genes into hearts subjected to coronary artery occlusion followed by reperfusion could result in gene expression comparable to the levels observed in non-occluded normal hearts. Anesthetized rats were subjected to 15 min or 60 min of proximal coronary occlusion or sham operation. Luciferase gene under the control of the Rous sarcoma virus promoter was injected directly into the anterior left wall. At 1 week, high expression of luciferase was observed in both the ischemic/reperfused and non-ischemic tissue. Thus DNA transfer by direct injection is possible after ischemic injury and uptake and expression are not impaired. In a second series of experiments, myocardial infarcts in dogs were injected with a beta-galactosidase expressing retroviral vector. LNPOZ. Six to 11 days later frozen sections revealed macroscopically visible expression of beta-galactosidase activity. Not only can foreign genes be taken up by direct injection of DNA or retroviruses into ischemic/reperfused myocardium but they can be transcribed and the protein synthetic machinery of the injured cells can produce recombinant polypeptides that retain enzymatic activity. These results open the way for the investigation of gene therapy in models of ischemia.

Animals↗

PubMed1996

Adenovirus-mediated gene transfer into infarcted myocardium: feasibility, timing, and location of expression.

Gene transfer as a therapeutic modality for the treatment of myocardial ischemia and/or infarction has been proposed as a revolutionary approach to improve collateral circulation, enhance myocardial viability and amplify healing. Our study was undertaken to assess the feasibility, efficiency, anatomic distribution, timing and localization of adenovirus-mediated gene transfer into the vicinity of infarcted myocardium in the adult mammalian heart. We induced myocardial infarction by subjecting rats to 60 min of coronary artery occlusion followed by sustained reperfusion. Gene transfer into the infarction area was performed using direct injection of a replication-defective adenovirus vector encoding the bacterial reporter gene, beta-galactosidase. A total of 5.0 x 10(9) plaque-forming units of virus was delivered into the left ventricular myocardium either immediately (n = 7) or at 7 (n = 6), 22 (n = 5) or 30 days (n = 5) after reperfusion of rat hearts. Control rats received either 50 microliters of saline 13 days after myocardial infarction (n = 2) or were not subjected to infarction and received Adenovirus carrying the beta-galactosidase gene as described above (n = 4). All rats were killed at 7 days after cardiac injection. Hearts were harvested, frozen and sectioned and stained for beta-galactosidase activity and with hematoxylin and eosin. Sections were evaluated by light microscopy. Relative beta-galactosidase activity was measured by digital planimetry and expressed as the ratio of the maximal area of beta-galactosidase staining relative to the total area of the section examined (% +/- S.E.M.). beta-galactosidase gene expression was limited mainly to viable myocytes at the border of the myocardial infarction. The area of transgene expression in the non-infarcted hearts (28 +/- 7%) was significantly higher (P = 0.02) than at any time point studied in infarcted tissues (3.4 +/- 1.2%, 1.4 +/- 1.0%, 2.8 +/- 0.8% and 3.4 +/- 0.9% at reperfusion and at 7, 22 and 30 days after myocardial infarction, respectively). Hearts injected 7 days after infarction had significantly less transgene activity (P = 0.03) with three of five samples displaying no macroscopically visible beta-gal activity. Following viral injection, an inflammatory response consisting of mononuclear cell infiltration was much less intense seven days following injection in non-infarcted control rat hearts than at any of the time points examined for infarcted hearts. Gene transfer into infarcted myocardium, while feasible, was limited by low transfection efficiency when compared to non-infarcted normal myocardium. Transgene expression in the infarcted myocardium appears restricted to residual cardiomyocytes in the periphery. Nevertheless, the ability to introduce genes into these viable peripheral cells might be a useful therapeutic strategy for enhancing neovascularization, collateral flow and healing.

Adenoviruses, Human↗

PubMed1996

Chicken skeletal muscle tropomodulin: novel localization and characterization.

Tropomodulin is a 40.6-kDa isoform-specific tropomyosin-binding protein which inhibits actin filament elongation from the slow-growing (pointed) end and localizes at or near the pointed ends of thin filaments in rat skeletal muscle. Immunofluorescent localization using affinity-purified anti-tropomodulin antibodies in avian myofibril preparations demonstrates novel immunoreactivity at the Z-disc in addition to the previously reported localization at the periphery of I-Z-I brushes where actin filaments terminate. Identical results were obtained using antibody preparations generated against either bacterially expressed tropomodulin or human erythrocyte tropomodulin. Chicken muscle preparations contain Mr 43000 polypeptides which bind antibodies generated against tropomodulin in Western blot analysis, as well as 125I-labeled tropomyosin in blot overlays. Tropomodulin mRNA expression in adult muscle was confirmed by RNase protection assays, and the sequence of our tropomodulin cDNA amplified from chicken muscle mRNA preparations by polymerase chain reaction closely matches clones selected by chicken muscle cDNA library screening. The novel immunolocalization we report raises new possibilities for the role of tropomodulin in the organization of avian skeletal muscle at the Z-disc. We conclude that tropomodulin is likely to be important in striated muscle biology as a structural component in the Z-disc region which participates in the process of thin filament organization and assembly.

Actins↗

PubMed1996

Differences between MyoD DNA binding and activation site requirements revealed by functional random sequence selection.

A method has been developed for selecting functional enhancer/promoter sites from random DNA sequences in higher eukaryotic cells. Of sequences that were thus selected for transcriptional activation by the muscle-specific basic helix-loop-helix protein MyoD, only a subset are similar to the preferred in vitro binding consensus, and in the same promoter context an optimal in vitro binding site was inactive. Other sequences with full transcriptional activity instead exhibit sequence preferences that, remarkably, are generally either identical or very similar to those found in naturally occurring muscle-specific promoters. This first systematic examination of the relation between DNA binding and transcriptional activation by basic helix-loop-helix proteins indicates that binding per se is necessary but not sufficient for transcriptional activation by MyoD and implies a requirement for other DNA sequence-dependent interactions or conformations at its binding site.

3T3 Cells↗

PubMed1995

Sequences of the 5'-flanking region of the human helix-loop-helix protein-encoding Id2A gene, and promoter activity regulated by serum and c-Jun/AP-1.

The 5'-flanking region of the human Id2A gene between nt -1213 and +36 from the putative transcription start point was sequenced. Transient transfection studies showed that the region between nt -834 and +30 directs the expression of a reporter gene and contains regulatory elements responsive to serum and c-Jun/AP-1 over production.

Base Sequence↗