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H F Epstein

Publications and source records attributed to H F Epstein.

At least 37 records · Page 2Linked to original sources
PubMed1995

Evidence that apoB-100 of low-density lipoproteins is a novel Src-related protein kinase.

Protein-tyrosine kinases of signal transduction pathways occur and function intracellularly. In contrast, the low-density lipoprotein (LDL) particle circulates in plasma, where its function is to solubilize and transport lipid. Recently, several reports showed that LDL may have a role in signal transduction. We have identified a region in the apoB-100 primary structure which shows similarity to Src-homology-1 (SH1) domains, the kinase region of protein-tyrosine kinases. Results obtained in protein kinase assays of highly purified LDL showed that only the apoB-100 was phosphorylated, suggesting that apoB-100 has the capacity to undergo autophosphorylation like known protein-tyrosine kinases. Phosphorylation was not observed for any other apolipoprotein in LDL or for any component of high-density lipoprotein and lipoprotein [a]. Our results suggest that apoB-100 may be a novel and functional member of the src protein kinase family.

Amino Acid Sequence↗
PubMed1994

Phosphorylation reactions of recombinant human myotonic dystrophy protein kinase and their inhibition.

The predicted protein kinase activity of the cloned gene product of the human myotonic dystrophy locus has been experimentally verified. Affinity-purified recombinant DM protein kinase became phosphorylated itself and transphosphorylated histone H1. These activities were not present in the bacterial host cells and were exhibited by DMPK and DMPKH, recombinant proteins which contain the protein kinase domain but exhibit distinct sizes, 43 and 66 kDa, respectively. DMPKH was further purified by velocity sedimentation on sucrose gradients; both activities migrated with the recombinant protein at 41 S, consistent with discrete multimeric particles. Phosphoamino acid analysis showed that threonine (predominantly) and serine were phosphorylated in both DMPKH and histone H1. Although PKA and PKC are the known types of protein kinase with closest sequence homology to the DM protein kinase domain, purified DMPKH was inhibited by 4 mM but not 0.04-0.4 mM H7 and H8, which inhibit PKA and PKC with Ki's of 0.4-15 microM. Specific inhibitors of other classes of multifunctional serine/threonine protein kinases such as casein kinases I (CKI-7) and II (heparin) and calcium/calmodulin-dependent protein kinase II (KN-62) did not inhibit DMPKH. DMPKH did not phosphorylate membrane-associated phosphoproteins such as acetylcholine receptor or spectrin which are known to be substrates for PKA, PKC, and CKI and -II, respectively. These experimental results suggest that the active center of the recombinant human myotonic dystrophy protein kinase may have properties distinct from the well-studied classes of serine/threonine protein kinases, in contrast to predictions based upon primary structure alone.

Base Sequence↗
PubMed1993

Myosin and paramyosin of Caenorhabditis elegans embryos assemble into nascent structures distinct from thick filaments and multi-filament assemblages.

The organization of myosin heavy chains (mhc) A and B and paramyosin (pm) which are the major proteins of thick filaments in adult wild-type Caenorhabditis elegans were studied during embryonic development. As a probe of myosin-paramyosin interaction, the unc-15 mutation e73 which produces a glu342lys charge change in pm and leads to the formation of large paracrystalline multi-filament assemblages was compared to wild type. These three proteins colocalized in wild-type embryos from 300 to 550 min of development after first cleavage at 20 degrees C on the basis of immunofluorescence microscopy using specific monoclonal antibodies. Linear structures which were diversely oriented around the muscle cell peripheries appeared at 360 min and became progressively more aligned parallel to the embryonic long axis until distinct myofibrils were formed at 550 min. In the mutant, mhc A and pm were colocalized in the linear structures, but became progressively separated until they showed no spatial overlap at the myofibril stage. These results indicate that the linear structures represent nascent assemblies containing myosin and pm in which the proteins interact differently than in wild-type thick filaments of myofibrils. In e73, these nascent structures were distinct from the multi-filament assemblages. The overlapping of actin and mhc A in the nascent linear structures suggests their possible structural and functional relationship to the "stress fiber-like structures" of cultured vertebrate muscle cells.

Actins↗
PubMed1993

Thick filament substructures in Caenorhabditis elegans: evidence for two populations of paramyosin.

The thick filaments of the nematode Caenorhabditis elegans contain two myosin heavy chain isoforms A and B and paramyosin, the products of the myo-3, unc-54, and unc-15 genes, respectively. Dissociation of paramyosin from native thick filaments at pH 6.36 shows a biphasic function with respect to NaCl concentration. Electron microscopy of the remaining structures shows 15-nm core structures that label with monoclonal anti-paramyosin antibody at 72.5-nm intervals. Purified core structures also show 72.5 nm repeats by negative staining. Structural analysis of native thick filaments and dissociated structures suggests that the more dissociable paramyosin is removed radially as well as processively from the filament ends. Minor proteins with masses of 20, 28, and 30 kD cosediment stoichiometrically with paramyosin in purified core structures.

Actin Cytoskeleton↗
PubMed1993

Myotonic dystrophy kinase is a component of neuromuscular junctions.

The clinical manifestation of myotonic dystrophy (DM) is correlated to the extent of expansion of an unstable [CTG]n DNA motif. Recent studies have demonstrated that this trinucleotide motif forms part of the last, 3' untranslated exon of a gene which potentially encodes multiple protein isoforms of a serine/threonine protein kinase (myotonic dystrophy protein kinase, DM-PK). We report here on the development of antisera against synthetic DM-PK peptide antigens and their use in biochemical and histochemical studies. Immunoreactive DM-kinase protein of 53 kD is present at low levels in skeletal and cardiac muscle extracts of DM patients and normal controls. Immunohistochemical staining revealed that DM-PK is localised prominently at sites of neuromuscular and myotendinous junctions (NMJs and MTJs) of human and rodent skeletal muscles. Furthermore, very low levels of immunoreactive DM-PK protein are present in the sarcoplasm of predominantly type I fibres in various muscles. Strikingly, presence of the protein can also be demonstrated for NMJs of muscular tissues of adult and congenital cases of DM, with no gross changes in structural organisation. Our findings provide a basis for further characterisation of the role of the kinase in protein assembly processes or signal mediation at synaptic sites and ultimately for the understanding of the complex pathophysiology of DM.

Amino Acid Sequence↗
PubMed1992

Assembly-dependent phosphorylation of myosin and paramyosin of native thick filaments in Caenorhabditis elegans.

Phosphorylation of the thick filament proteins myosin and paramyosin was studied in Caenorhabditis elegans. We have incubated partially purified, native thick filaments with [gamma 32P] ATP in the presence of 50-750 mM NaCl, pH 6.5-8.0. Myosin heavy chain and paramyosin were phosphorylatable only upon solubilization at 450 mM and higher NaCl concentrations. Under conditions preserving native structures, no phosphorylation of these proteins occurred. The phosphorylation required Mg2+ but was unaffected by cAMP, cGMP or Ca2+. The specific inhibitor of cAMP and cGMP kinase catalytic subunits, H8, inhibits the activity. Sedimentation experiments show that the kinase may associate with but is not an intrinsic component of thick filaments. In C. elegans, phosphorylation by the thick filament associated activity of myosin and paramyosin is dependent upon the state of their assembly.

Adenosine Triphosphate↗
PubMed1992

Diagnostic value of ophthalmologic findings in myotonic dystrophy: comparison with risks calculated by haplotype analysis of closely linked restriction fragment length polymorphisms.

To determine diagnostic value of lens opacities in myotonic dystrophy (DM), we examined 98 at-risk members of 9 DM kindreds. Haplotype analysis of restriction fragment length polymorphisms (RFLPs) using ApoC2, CKMM, and pEFD4.2 supported the diagnosis of DM in 33 and excluded the diagnosis in 51 members. The sensitivities of bilateral iridescent lens opacities, posterior cortical lens opacities, orbicularis oculi weakness, low intraocular pressure, ptosis, and ocular myotonia were 46.7, 50.0, 60.6, 59.3, 51.5, and 3.0%, while their specificities were 100.0, 100.0, 98.0, 94.1, 96.1, and 100.0%, respectively. A peripheral pigmentary degeneration and central macular lesions of retina were not found on indirect fundoscopy. In 86.2% of DM patients, bilateral iridescent lens opacities, posterior cortical lens opacities, or both were present. Unilateral iridescent lens opacities occurred in only 3 of our DM patients, and 2 of non-DM relatives showed a few unilateral iridescent particles. Posterior cortical lens opacities in DM patients always affected both eyes in this series. We conclude that 1) bilateral iridescent lens opacities and posterior cortical lens opacities are highly specific for DM and useful for establishing clinical diagnosis of DM, 2) unilateral iridescent lens opacities are infrequent in DM and are seen in some non-DM members, and 3) ocular myotonia and clinical retinopathies are rare in DM.

Adolescent↗
PubMed1992

Human cardiac and skeletal muscle spectrins: differential expression and localization.

We describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid alpha,beta spectrin-reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively. At least one nonerythroid alpha-spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid alpha-spectrin isoform. The 255 kD erythroid beta-spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid beta- and nonerythroid alpha-spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.

Adult↗
PubMed1992

Genetic approaches to understanding muscle development.

The analysis of both naturally occurring and experimentally induced mutants has greatly advanced our understanding of muscle development. Molecular biological techniques have led to the isolation of genes associated with inherited human diseases that affect muscle tissues. Analysis of the encoded proteins in conjunction with the mutant phenotypes can provide powerful insights into the function of the protein in normal muscle development. Systematic searches for muscle mutations have been made in experimental systems, most notably the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. In addition, known muscle protein genes from other organisms have been used to isolate homologs from genetically manipulatable organisms, allowing mutant analysis and the study of protein function in vivo. Mutations in transcription factor genes that affect mesoderm development have been isolated and genetic lesions affecting myofibril assembly have been identified. Genetic experiments inducing mutations and rescuing them by transgenic methods have uncovered functions of myofibrillar protein isoforms. Some isoforms perform muscle-specific functions, whereas others appear to be replaceable by alternative isoforms. Mutant analysis has also uncovered a relationship between proteins at the cell membrane and the assembly and alignment of the myofibrillar apparatus. We discuss examples of each of these genetic approaches as well as the developmental and evolutionary implications of the results.

Animals↗
PubMed1992

cDNA surveying of specific tissue expression of human chromosome 19 sequences.

cDNA surveying is a straightforward approach for identifying sequences in genomic clones expressed in specific tissues. It has been applied to a subchromosomal region of human chromosome 19 (19q13.2-q13.4), a region that contains several known expressed sequences including the locus for myotonic dystrophy (DM). Genomic clones were selected from this region by probing a human placental cosmid library with a chromosome 19q-specific minisatellite sequence, or human genomic clones were isolated from a cosmid library constructed from a human chromosome 19q13.2-q13.3 hamster hybrid cell line using human repetitive DNA as probe. Pooled cDNAs synthesized from RNA of specific tissues characteristically affected in DM were depleted in repetitive sequences and used as hybridization probes against gridded cosmid arrays. DNA from the cDNA-positive cosmid clones was transferred to nylon filters and reprobed with cDNAs to identify restriction fragments that were expressed in these tissues. Hybridizing restriction fragments were subcloned, sequenced, and demonstrated to be nonrepetitive. Primer pairs complementary to subcloned sequences were constructed and used for PCR amplification of cDNA synthesized from RNA of tissues affected in myotonic dystrophy. PCR products were sequenced to verify the identity of expressed genomic DNA and its corresponding cDNA.

Animals↗
PubMed1992

Accumulation and assembly of myosin in hypertrophic cardiomyopathy with the 403 Arg to Gln beta-myosin heavy chain mutation.

The sarcomeric proteins and organization of cardiac myofibrils appeared intact in multiple unrelated patients with hypertrophic cardiomyopathy. In two subjects demonstrating the missense mutation at position 403 (Arg to Gln) in the beta-myosin heavy chain gene, total myosin and immunoreactive beta-myosin heavy chain levels were similar to those found in other patients with hypertrophic cardiomyopathy and various disease control subjects. No alteration in expression of the cardiac alpha-myosin heavy chain gene was observed. These results are consistent with the examined myosin heavy chain mutation, permitting proper accumulation and assembly of myosin while primarily impairing contractile function. The characteristic myocyte disarray would appear likely to be a secondary consequence of the mutations.

Adolescent↗
PubMed1992

Anticipation in myotonic dystrophy. I. Statistical verification based on clinical and haplotype findings.

To determine whether anticipation in myotonic dystrophy (DM) is a true biologic phenomenon or an artifact of ascertainment bias, we studied 201 members of nine DM kindreds, including 67 individuals with the clinical diagnosis of DM. Of 49 parent-child pairs in which both the parents and the children were clinically affected, the onset of DM occurred in an earlier decade of life in the child than the parent in 44 pairs and in the same decade in five pairs (p < 0.001). To eliminate direct ascertainment bias, we excluded nine pairs involving the index patients. Indirect ascertainment bias due to incomplete penetrance was unlikely, since 55% of the children of DM parents had DM. However, by haplotype analysis of restriction fragment length polymorphisms, we diagnosed DM in one of the 42 asymptomatic children of affected parents and excluded DM in twenty-eight. We estimated that patients with early-onset DM would have produced an additional 25 DM children if they had normal fertility and nuptiality. Assuming that the expected age-of-onset distribution occurs without anticipation in these 25, only seven would have had the onset of DM earlier than their parents. With the corrected result, the child would have been affected earlier than the parent in 53 pairs, and the parent would have been affected at the same age as or earlier than the child in 13 pairs (p < 0.001). Thus, the observed anticipation is unlikely to be totally attributable to ascertainment bias, suggesting the potential importance of biologic mechanisms.

Adult↗
PubMed1992

Anticipation in myotonic dystrophy. II. Complex relationships between clinical findings and structure of the GCT repeat.

We studied the expansion of the GCT repeats within the myotonic dystrophy protein kinase gene in nine myotonic dystrophy (DM) kindreds. Southern blot and polymerase chain reaction analyses of the repeat region demonstrated the expansion in all 62 patients with the diagnosis of DM. Among 43 DM parent-child pairs, age of onset in the child was earlier than in the parent in 36 pairs, in the same decade as the parent in five, and undetermined in two. The clinical anticipation observed in the 36 pairs accompanied an increase in the fragment size in 32, a decrease in two, and no apparent change in two pairs. In the remaining pairs without documented clinical anticipation, the fragment size increased in four, decreased in two, and was apparently unchanged in one. Overall, the size of expansion showed an inverse correlation with the age of onset (p < 0.001). In all seven pairs in which the fragment did not increase in size, the affected parent was male. Two congenital DM children born to affected mothers had expanded DNA greater than 4.5 kb. The differences between parent and child in age of onset significantly correlated with the differences in the expansion size among father-child pairs (p < 0.001) but not mother-child pairs (p > 0.5). Our data suggest that the expansion of the GCT repeats plays an important role in anticipation although other factors, including the sex of the affected parent, may have significant effects on molecular mechanisms of anticipation.

Base Sequence↗
PubMed1992

Phenotypic expression of the myotonic dystrophy gene in monozygotic twins.

Phenotypic expression of the myotonic dystrophy (DM) gene is highly variable even within the same family. To investigate the importance of genetic and nongenetic factors on phenotypic variability, we studied phenotypic expression in two DM twin sets with monozygosity confirmed by DNA polymorphism analysis. Our observations suggest that genetic factors are primarily responsible for modulating pleiotropic expression of the DM gene.

Adult↗
PubMed1991

Molecular analysis of protein assembly in muscle development.

The challenge presented by myofibril assembly in striated muscle is to understand the molecular mechanisms by which its protein components are arranged at each level of organization. Recent advances in the genetics and cell biology of muscle development have shown that in vivo assembly of the myofilaments requires a complex array of structural and associated proteins and that organization of whole sarcomeres occurs initially at the cell membrane. These studies have been complemented by in vitro analyses of the renaturation, polymerization, and three-dimensional structure of the purified proteins.

Actins↗