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L Kedes

Publications and source records attributed to L Kedes.

At least 55 records · Page 3Linked to original sources
PubMed1995

Cloning of tropomodulin cDNA and localization of gene transcripts during mouse embryogenesis.

Tropomodulin (Tmod) is a tropomyosin-binding protein involved in the structuring of actin filaments. This report describes Tmod expression in distinct patterns during embryonic development in a wider variety of adult and embryonic vertebrate tissues than previously reported. Identical Tmod cDNAs were cloned from mouse brain, skeletal muscle, heart, and hematopoeitic cells. Genomic blotting demonstrates that Tmod is encoded by a single gene, which has a 1077-bp open reading frame that is highly homologous to that of the human erythrocyte. The spatial and temporal expression of the Tmod gene was examined during mouse embryogenesis using in situ hybridization. Tmod mRNA is present by 9.5 days postcoitum (p.c.) in the developing rostral somites, coincident with expression of contractile protein genes in myotomes, suggesting that Tmod may play an important role in sarcomeric thin filament organization in skeletal muscle. While the expression of Tmod mRNA in cardiac muscle is earlier than that in skeletal muscle, its appearance in the heart also coincides with the expression of genes for thin filament proteins and correlates with initial myocardial contractions at 8.0 days p.c. Tmod mRNA is not detected in developing smooth muscle of the gut, but Tmod mRNA is expressed in hematopoeitic cells in yolk sac and developing liver. The sensory ganglia and epithelia of the inner ear express Tmod mRNA as do other sensory neurons such as those in the olfactory epithelium. Expression levels in the brain are much lower prenatally than postnatally. These findings show that Tmod expression in many cell types is developmentally regulated, suggesting that the interaction of actin filaments with this tropomyosin binding protein is an important process in tissue and cell differentiation.

Amino Acid Sequence↗
PubMed1995

Upregulation of angiotensin II type 1 receptor gene expression in chronic renovascular hypertension.

Although the renin-angiotensin system has been implicated in the pathogenesis of renovascular hypertension (RVH), blood pressure does not parallel serum levels of renin or angiotensin II (AII) in chronic RVH. Upregulation of angiotensin II type 1 receptor (AT1) gene expression may explain this paradox and clarify the pathogenesis of chronic hypertension in RVH. To investigate this hypothesis, we studied changes in AT1 mRNA levels in rat kidney in a two-kidney, one-clip (2K1C) rat model of RVH. Animals were sacrificed at 1 or 10 weeks postoperatively. Blood pressure was measured with a tail cuff photosensor. Relative gene expression was quantitated by dot blotting total RNA, hybridizing with a cDNA probe for AT1, and quantitating signal intensity with scanning densitometry. A significant increase in blood pressure (BP) was observed at 1 week postoperatively (delta BP: 2K1C = +24 mm Hg, n = 3; controls = +7 mm Hg, n = 3; P < 0.05), and at this time relative AT1 mRNA levels actually decreased in the clipped kidney (P < 0.05). Hypertension intensified 10 weeks postoperatively (delta BP: 2K1C = +46 mm Hg, n = 20; controls = -17 mm Hg, n = 7; P < 0.005) and, remarkably, was paralleled by an almost sevenfold upregulation of AT1 mRNA levels in the clipped kidney (P < 0.005) and more than eightfold in the unclipped kidney (P < 0.005) of 2K1C animals. Upregulation of renal AT1 gene expression could lead to increased AT1 receptor production, hypersensitivity to AII, and chronic hypertension in RVH.

Animals↗
PubMed1995

Impaired agonist-induced calcium signaling in hepatocytes from chronic renal failure rats.

Some hormones exert their action by inducing a rise in cytosolic calcium [Ca2+]i (calcium signal), and therefore, a blunting in hormone-induced calcium signal would engender resistance to the action of the hormone. Chronic renal failure (CRF) is associated with resistance to the action of a variety of hormones, a rise in [Ca2+]i and decrease in the amount of mRNA of one hormone receptor, the PTH-PTHrP receptor. We examined the calcium-signal induced by PTH, angiotensin II, vasopressin and glucagon in hepatocytes from CRF animals, evaluated the effect of the basal level [Ca2+]i on the calcium signal and explored the effect of [Ca2+]i on the mRNA of the receptors of these agonists. Hepatocytes from CRF rats have elevated basal levels of [Ca2+]i and display significantly reduced calcium signals induced by all these hormones, while the calcium signals were normal in PTX-CRF animals and those treated with verapamil both of which have normal levels of [Ca2+]i despite CRF. The calcium signals induced by dibutyryl cyclic AMP and G protein activator (GTP gamma S) were normal in hepatocytes from CRF animals despite the high levels of [Ca2+]i. Northern blotting experiments revealed that the levels of the mRNA of the receptors of PTH-PTHrP, angiotensin II and vasopressin were significantly reduced in hepatocytes from CRF animals but PTX-CRF rats and those treated with verapamil had either significantly greater or even normal amounts of the mRNA of these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Doxorubicin-induced Id2A gene transcription is targeted at an activating transcription factor/cyclic AMP response element motif through novel mechanisms involving protein kinases distinct from protein kinase C and protein kinase A.

We have recently shown that doxorubicin (Dox), an antineoplastic drug and an inhibitor of terminal differentiation of myogenic and adipogenic cells, induces expression of Id, a gene encoding a helix-loop-helix transcriptional inhibitor. In this study we have investigated the molecular mechanisms underlying Dox-induced Id2A expression. We have also attempted to determine whether the genetic responses to Dox are related to the UV response, a well-characterized set of reactions to UV and DNA-damaging compounds that is partly mediated by AP-1. Transient transfection of a series of deletions and point mutation derivatives of the human Id2A promoter sequence shows that two closely spaced and inverted short elements similar to an activating transcription factor (ATF) binding site or a cyclic AMP response element (CRE) are necessary and sufficient for a full response to Dox. We refer to this element as the IdATF site. Sequences containing an IdATF site conferred Dox inducibility on a minimal heterologous promoter. An electrophoretic mobility shift assay showed nuclear proteins specifically interacting with the IdATF sequence. While oligonucleotides containing either legitimate ATF/CRE or AP-1 binding sequences competed for binding, antibody supershift experiments suggested that neither CREB/ATF-1 nor AP-1 are major factors binding to IdATF. Several independent criteria suggest that Dox inducibility was independent of Ca2+/phospholipid-dependent protein kinase (protein kinase C), cyclic AMP-dependent protein kinase (protein kinase A), and tyrosine kinase. Moreover, we found that Dox also induces transcription from promoters of immediate-early genes through an AP-1-independent pathway. Taken together, our results suggest that Dox elicits a novel genetic response distinct from the classical UV response.

Antibiotics, Antineoplastic↗
PubMed1995

Myoblast transfer of human erythropoietin gene in a mouse model of renal failure.

Anemia is an invariable consequence of end-stage renal failure (ESRF) and recombinant erythropoietin has dramatically improved the quality of life of patients with ESRF. As an alternative approach, we developed a myoblast gene transfer system for the systemic delivery of human erythropoietin (EPO). We recently reported that transplantation of 4 x 10(7) cells of a C2 myoblast cell clone that stably secretes high level of functional human EPO, increased hematocrit from 44.6 +/- 3.0 to 71.2 +/- 7.9(%) in 2 wk, and the increase was sustained for at least 12 wk in nude mice. A renal failure model was created by a two-step nephrectomy in nude mice, and myoblasts were transplanted 3 wk after the second nephrectomy, when mean blood urea nitrogen level had increased from 26.3 +/- 6.1 to 85.4 +/- 24.0 (mg/dl) and the hematocrit had decreased from 45.2 +/- 2.7 to 33.9 +/- 3.7(%). After transplantation, the hematocrit markedly increased to 68.6 +/- 4.2(%) 2 wk, and to 68.5 +/- 4.0(%) 7 wk after the transplantation. Serum human EPO concentration determined by ELISA indicated a persistent steady EPO production from the transplanted muscle cells 8 wk after the transplantation. The fate of transplanted myoblasts in uremic mice was monitored by transplanting the EPO-secreting clone which had also been transduced with BAG retrovirus bearing the beta-galactosidase gene. 8 wk later, X-gal positive myofibers were detected in the entire transplanted area. The results demonstrate that myoblasts can be transplanted in uremic mice, and that myoblast gene transfer can achieve sufficient and sustained delivery of functionally active EPO to correct anemia associated with renal failure in mice.

Anemia↗
PubMed1994

Serum-inducible factors binding to an activating transcription factor motif regulate transcription of the Id2A promoter during myogenic differentiation.

Expression of Id, a dominant negative regulator of helix-loop-helix transcription factors, is tightly regulated during cellular differentiation. In this study, we have defined the sequences responsible for the transcriptional regulation of the human Id2A gene. 5' deletion and site-specific mutation analyses of the Id2A promoter showed that both an Sp-1 site and an activating transcription factor (ATF)-like site (referred to as IdATF sites) are required for expression in C2 cells, whereas these sites are not essential for the expression in nonmuscle cells, including HeLa and 10T1/2 cells. Gel shift assays revealed nuclear factors with specific binding to IdATF sites in both C2 and HeLa cells, which are efficiently competed by either legitimate ATF- or AP-1 binding sites. DNA binding activity to IdATF sites was clearly decreased during C2 cell differentiation and rapidly reactivated by treatment with either phorbol 12-myristate 13-acetate or serum, which correlated with the induction of endogenous Id2A mRNA expression. In addition, we show that overexpression of the catalytic domain of protein kinase C leads to the activation of the Id2A promoter through IdATF sites in C2 cells. These results suggest a model in which down-modulation of IdATF site binding activity by mitogen depletion contributes to the down-regulation of Id2A gene expression that is essential for myogenic differentiation.

Base Sequence↗
PubMed1994

Neural tropomodulin: developmental expression and effect of seizure activity.

Tropomodulin is a 40.6 kDa tropomyosin-binding protein associated with actin filaments in muscle and the membrane cytoskeleton in erythrocytes. We have detected tropomodulin mRNA and protein in brains of rats by northern and western blot analyses. In situ hybridization of rat brain and spinal cord sections shows tropomodulin expression in the cerebellum, neocortex, hippocampus, and anterior horn of the spinal cord. Tropomodulin expression is first observed around day 15 after birth and increases through day 24. The temporal and spatial changes in tropomodulin expression during cerebellar development parallel those for brain tropomyosin. Tropomodulin mRNA increases in the dentate gyrus of the hippocampus following prolonged seizure activity induced by kainic acid administration; the increase is clearly evident 8 h after initiation of seizures and is still present 1 week later. However, Western blot analysis of tropomodulin protein level in the dentate gyrus before and after seizure induction show only slight increases in tropomodulin protein concentration, suggesting tight regulation of tropomodulin expression at the translational level. The developmental expression of tropomodulin, together with the induction of tropomodulin mRNA production in the dentate gyrus after kainic acid treatment, suggests a role for tropomodulin in neuronal organization and plasticity.

Animals↗
PubMed1994

Structure and expression of the human slow twitch skeletal muscle troponin I gene.

The contractile protein troponin I is encoded by a multigene family whose members are expressed differentially in various classes of muscle fibers. In vertebrates, the "slow" isoform of troponin I is expressed during early heart and skeletal muscle development but is restricted to slow twitch skeletal muscle in the adult. This diverse expression pattern offers an opportunity to study the regulation of a single gene within different developmental contexts. To initiate such studies, we have cloned the gene encoding the human slow twitch skeletal muscle isoform of troponin I and have identified 5'-flanking sequences required for its expression in skeletal muscle cells. The slow troponin I gene spans 12.5 kilobases and is divided into nine exons. In contrast to many muscle-specific genes, the troponin I promoter does not contain consensus CCAAT or TATA elements. Moreover, the sequence from -9 to +11 resembles an "initiator element" previously shown to direct transcription of some tissue-specific genes lacking TATA boxes (Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113; Brand, N. J., Petkovich, M., and Chambon, P. (1990) Nucleic Acids Res. 18, 6799-6806; Weis, L., and Reinberg, D. (1992) FASEB J. 6, 3300-3309). A transcriptional fusion construct, comprising 4.2 kilobases of troponin I 5'-flanking DNA linked to the bacterial chloramphenicol acetyl-transferase gene, exhibited cell type-specific and developmentally regulated expression. A muscle-specific enhancer regulated slow troponin I promoter activity.

Adult↗
PubMed1994

Doxorubicin represses the function of the myogenic helix-loop-helix transcription factor MyoD. Involvement of Id gene induction.

We have shown recently that Doxorubicin (Dox, Adriamycin), a potent broad spectrum chemotherapeutic agent with a major side effect of cardiomyopathy, completely prevents myoblast fusion and accumulation of muscle-specific transcripts in the mouse C2 skeletal muscle cell line. Here we use mouse embryonic fibroblast 10T1/2 cells to demonstrate that Dox represses muscle-specific gene expression by interfering with MyoD activity. As assayed by transient cotransfection, Dox inhibits the ability of MyoD to trans-activate muscle-specific reporter genes. A stable cell system was developed in which MyoD is constitutively expressed in 10T1/2 cells (M10 cells). Dox-treated M10 cells express MyoD from a long terminal repeat-driven vector but fail to activate endogenous MyoD and myogenin loci. Dox did not effect E2A gene transcript levels, but Id mRNA levels are significantly increased in Dox-treated M10 cells. Interestingly, overexpression of E2-5, which forms inactive heterodimers with Id, can overcome the Dox-induced suppression of the trans-activation function of MyoD in 10T1/2 cells. Furthermore, we demonstrate that the 5'-flanking region of the Id2 gene mediates its Dox-inducible transcriptional expression. These findings support a model in which Dox inhibits muscle-specific gene expression by interfering with the function of MyoD protein through, at least in part, induction of Id gene expression. The implications of our results for the molecular mechanisms underlying the myofibrillar loss observed in Dox-induced cardiomyopathy are discussed.

Animals↗
PubMed1994

Tissue restricted gene expression assayed by direct DNA injection into cardiac and skeletal muscle.

We have used direct injection of plasmid DNA into heart and tongue in vivo and transfection into cells in culture to determine (1) whether the pattern of reporter gene expression parallels the cell-type specific expression of endogenous genes, (2) whether the pattern of reporter gene expression approximates that of the same constructs transfected into myocardiocytes and myogenic cells in culture, (3) whether the expression patterns of promoters and enhancers that had been subtly altered by mutations are similar following transfection and direct DNA injection. We utilized reporter gene constructs derived from the two fiber-type specific human troponin C genes: the fast twitch gene (TnCf) and the slow twitch or cardiac gene (cTnC). The endogenous TnCf gene does not express in the heart and the plasmid DNA expresses neither in myocardium after injection nor in transfected cardiomyocytes. However injected TnCf does express vigorously in tongue muscle. Conversely, the endogenous cTnC gene does not express in fast twitch skeletal muscles like tongue and the plasmid DNA does not express in tongue after injection. However, injected cTnC does express in both injected myocardium and in transfected cardiomyocytes. With few discrepancies, various deletions and alterations of the promoters and enhancers of these genes that have previously been defined by transfection into permissive myogenic cells in culture gave parallel expression patterns after injection in myocardium and tongue. Thus we conclude that direct DNA injection appears to provide a method to verify the identities of important cis-acting regulatory regions that have been mapped in cells in culture.

Animals↗
PubMed1994

Persistent erythropoiesis by myoblast transfer of erythropoietin cDNA.

A myoblast gene transfer approach was developed to deliver human erythropoietin (EPO) systemically. We created stable, high-level EPO-producing muscle cell clones by transfecting C2 myoblasts with a plasmid-bearing human EPO cDNA driven by cytomegalovirus enhancer/promoter and selection by G418. Eleven clones secreted EPO into the media as detected by radioimmunoassay. In vitro bioassay using the EPO-dependent human leukemic cell line UT-7/Epo confirmed the functional activity of the secreted EPO. After transplantation of 4 x 10(7) cells from C2-EPO9, the highest producing clone, the hematocrit increased from 43.4 +/- 2.8 to 56.1 +/- 2.7 (%) in 2 weeks in C3H mice that are syngeneic to C2 cells, and from 44.6 +/- 3.0 to 71.2 +/- 7.9 in nude mice. The increased hematocrit gradually returned to the basal level in 4-5 weeks in C3H mice, while it was sustained for at least 12 weeks in nude mice. Human EPO concentrations in the sera from transplanted nude mice were persistently high (31 +/- 24 mU/ml) at 12 weeks. C2 cells transduced with a retrovirus bearing beta-galactosidase gene were transplanted into nude mice, which showed X-Gal-positive myofibers in the transplanted area 3 months after the transplantation. These results demonstrate that myoblast gene transfer can successfully deliver functional human EPO capable of driving sustained erythropoiesis in mice. Thus, long-term EPO delivery for anemic patients may be feasible by myoblast gene transfer.

Animals↗
PubMed1994

PTH-PTHrP receptor mRNA is downregulated in chronic renal failure.

Resistance to the action of PTH is encountered in chronic renal failure (CRF). This was attributed to downregulation of PTH receptor due to the state of secondary hyperparathyroidism of CRF. The present study examined whether the amount of PTH-PTH-related peptide (PTH-PTHrP) mRNA in a traditional (kidney) and a nontraditional (liver) organs for PTH action was reduced in CRF. PTH-PTHrP mRNA was measured in kidney and liver obtained from normal rats, animals with CRF of 6 weeks' duration, normocalcemic parathyroidectomized CFR rats and from CRF and normal rats treated with verapamil. The mRNA of receptor was quantitated with Northern blot analysis of kidney RNA and liver poly A+ RNA. The relative amounts of mRNA of the PTH-PTHrP receptor to that of beta-actin in both kidney and liver of CRF rats were significantly (p < 0.01) lower than in normal animals. Parathyroidectomy of CRF rats was followed either by significant (p < 0.01) improvement (kidney) or normalization (liver) of the receptor mRNA. Treatment of CRF rats with verapamil also significantly (p < 0.01) improved the concentration of the receptor mRNA in the kidney. The data demonstrate that CRF is associated with downregulation of the PTH-PTHrP receptor mRNA in kidney and liver. This defect is partially or completely reversed by parathyroidectomy of the CRF rats or their treatment with verapamil. The decrease in the receptor mRNA would likely result in a decrease in PTH receptor synthesis and consequently PTH receptor numbers, and hence relative resistance to the action of PTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Tropomodulin in rat cardiac muscle. Localization of protein is independent of messenger RNA distribution during myofibrillar development.

Tropomodulin is a 40.6-kD protein that colocalizes with actin filament pointed ends in skeletal muscle. We report the sequence of two partial-length complementary DNA (cDNA) clones of rat cardiac tropomodulin that cover 90% of the coding region. The cDNA sequence is 90% conserved between human and rat, with the predicted amino acid sequence similarity even higher at 95%. Anti-tropomodulin antibodies label a single polypeptide with an apparent mobility of 43,000 in Western blot analysis of rat cardiac muscle. Immunofluorescence experiments using this anti-tropomodulin antibody result in labeling that is coincident with thin filament ends, as demonstrated by double localization with alpha-actinin antibody. Tropomodulin protein is organized into a sarcomeric staining pattern with the earliest appearance of myofibrils in rat cardiocytes. The localization of tropomodulin protein at or near thin filament ends led us to examine the distribution of tropomodulin messenger RNA (mRNA) during myofibrillar development in vitro. Fluorescent in situ hybridization experiments using tropomodulin cDNA probe in cardiocytes that have been cultured for 3 to 5 days show a distribution of large mRNA patches. The cytoplasmic location of tropomodulin mRNA at this time, which bears no relation to the developed myofibrils, suggests that tropomodulin protein is targeted to thin filament ends rather than using localized translational machinery. However, the distribution of tropomodulin mRNA in cultured cardiocytes changes over the next 2 weeks from large perinuclear patches to small concentrations arranged along myofibrils throughout the cell. The reorganization of tropomodulin mRNA throughout the cardiocyte appears to be distinct from the pattern of glyceraldehyde-3-phosphate dehydrogenase mRNA within the same time period. Increasing intracellular density of myofibrils within developing cardiocytes may lead to redistribution of selected mRNAs for localized translation.

Amino Acid Sequence↗
PubMed1993

Two distinct cDNA sequences encoding the human helix-loop-helix protein Id2.

Two distinct Id2 cDNA clones, Id2A and Id2B (inhibitor of differentiation), have been isolated from a human heart cDNA library and sequenced. The deduced amino acid sequences show nearly complete identity, except for the nonsense mutation at codon 37 in Id2B. Characterization of the genomic clones revealed that Id2B is encoded by an intronless gene which probably arose after the evolutionary divergence of rats and humans.

Animals↗
PubMed1993

Antineoplastic agent doxorubicin inhibits myogenic differentiation of C2 myoblasts.

Doxorubicin (Dox, adriamycin), an antineoplastic agent that can cause dilated cardiomyopathy, selectively inhibits muscle-specific gene expression in rodent cardiac muscle cells. This study shows that Dox treatment of proliferating C2 myoblasts, an established cell line from mouse skeletal muscle, completely prevents both fusion and accumulation of muscle-specific gene transcripts without significantly altering non-muscle gene transcripts. When added to high density cultures, Dox only blocked myotube formation but did not inhibit induction of muscle-specific genes. Transient transfection into C2 myoblasts showed that the transcriptional expression of chloramphenicol acetyltransferase reporter plasmids regulated by either the cardiac alpha-actin promoter or the muscle creatine kinase enhancer, but not with a viral or beta-actin promoter, was significantly diminished by Dox in a dose-dependent manner. Moreover, exposure of C2 myoblasts to Dox had a profound effect on the expression of regulatory genes critical to the myogenic differentiation program; mRNAs for MyoD and myogenin were dramatically reduced and Id mRNA was concomitantly increased. In addition, there was diminished DNA binding activity of the muscle-specific transcription factor, MEF-2. These results suggest that Dox inhibits myogenesis by preventing muscle-specific gene expression, possibly through affecting the myogenic programs controlled by muscle-specific transcription factors.

Animals↗
PubMed1993

Tropomodulin is highly concentrated at the postsynaptic domain of human and rat neuromuscular junctions.

Tropomodulin is expressed in skeletal muscle and recent studies suggest that tropomodulin is associated with synaptic membranes. Therefore, we have examined neuromuscular junctions by immunofluorescence analysis for tropomodulin localization. Anti-tropomodulin antibodies generated against either the whole protein or a 15-amino acid peptide fragment label human neuromuscular junctions as demonstrated by colocalization with alpha-bungarotoxin label. Tropomodulin labeling also colocalizes with desmin and beta-amyloid proteins which are concentrated in the postsynaptic region. Comparable results are found with immunostaining of neuromuscular junctions in rat diaphragm muscle. These immunofluorescence results suggest that tropomodulin is an integral component of the cytoskeletal lattice associated with the postsynaptic region of neuromuscular junctions.

Animals↗
PubMed1993

Direct DNA injection into mouse tongue muscle for analysis of promoter function in vivo.

The striated muscle of the tongue provides a readily accessible site for the introduction of DNA expression vectors. Parameters were established to use the striated muscle of the tongue as a model system for the examination of gene expression following the direct injection of DNA constructs bearing gene promoter sequences controlling the expression of reporter genes. Plasmid expression vectors were used that contained either constitutive or muscle-specific promoters directing the transcription of reporter genes. Chloramphenicol acetyltransferase (CAT), luciferase, and beta-galactosidase (lacZ) were used as the reporter genes to detect the promoter-specific expression of the injected DNA. The expression of the injected plasmids was directly correlated with the mass of injected DNA and the time of incubation following the injection. Maximal levels of reporter gene expression were observed seven days after the injection, and the expression was maintained for more than two months following injection. Simultaneous injection of two individual expression vectors bearing either CAT or luciferase reporter genes resulted in a dose-dependent level of expression for each of the plasmids. The linearity of the coexpression provided a means to normalize DNA uptake and analyze promoter efficiency. The troponin C-fast enhancer linked to its own promoter directed significantly more CAT expression than an enhancerless SV40 promoter-CAT plasmid, demonstrating that different promoter strengths could be determined in the mouse tongue muscle in vivo. This model system represents a convenient means to approach the functional analysis of muscle gene promoters in vivo.

Animals↗