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S D Kahl

Publications and source records attributed to S D Kahl.

At least 19 recordsLinked to original sources
PubMed1999

Differential distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat brain.

Urocortin, a novel 40 amino acid neuropeptide, is a member of the corticotropin-releasing factor family. With 45% homology to corticotropin-releasing factor, urocortin binds with similar affinity to the corticotropin-releasing factor- and corticotropin-releasing factor-2 receptors and may play a role in modulating many of the same systems as corticotropin-releasing factor. To assess whether urocortin and corticotropin-releasing factor are localized in the same regions of the brain, we compared the distribution of urocortin- and corticotropin-releasing factor-like immunoreactivities in the rat central nervous system. Polyclonal antibodies to rat corticotropin-releasing factor and rat urocortin were generated and utilized to map the distribution of corticotropin-releasing factor- and urocortin-like immunoreactivities throughout the rat forebrain and brainstem. Characterization of the antibodies by radioimmunoassay showed no cross-reactivity with related peptides. Male Sprague-Dawley rats were treated with colchicine for 18-24 h. Following colchicine treatment, the rats were perfused with paraformaldehyde-lysine-periodate fixative and their brains removed. Serial coronal sections were taken throughout the rat brain and processed for either corticotropin-releasing factor- or urocortin-like immunoreactivity. Urocortin-like immunoreactivity shows a discrete localization within several regions including the supraoptic nucleus, the median eminence, Edinger-Westphal nucleus and the sphenoid nucleus. This is in contrast to the more abundant corticotropin-releasing factor-like immunoreactivity. Regions containing high levels of corticotropin-releasing factor immunoreactivity include the lateral septum, paraventricular nucleus of the hypothalamus, median eminence and locus coeruleus. There are a few regions that contain both urocortin-immunoreactive and corticotropin-releasing factor-immunoreactive cells, such as the supraoptic nucleus and the hippocampus. Therefore, urocortin and corticotropin-releasing factor appear to have different distribution patterns which may be indicative of their respective physiological functions.

Animals↗
PubMed1999

BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins.

We describe a new, sensitive, rapid, and nonradioactive method involving the use of the commercially available BOCILLIN FL, a fluorescent penicillin, as a labeling reagent for the detection and study of penicillin-binding proteins (PBPs). This method allowed rapid detection of 30 ng of a purified PBP protein under UV light and of 2 to 4 ng of the protein with the aid of a FluorImager. This method also allowed rapid determination of the PBP profiles of Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae. The PBP profiles obtained are virtually identical to those reported previously with 3H-, 14C-, or 125I-labeled penicillin. Using this method enabled us to determine the 50% inhibitory concentrations of the penicillin-sensitive and -resistant PBP2x proteins of S. pneumoniae for penicillin G, thereby allowing a direct evaluation of their relative affinities for penicillin G. Finally, this method also allowed us to compare relative affinities of a PBP2x protein for different beta-lactam antibiotics with the aid of fluorescence polarization technology and to monitor a PBP2x protein during purification.

Bacteria↗
PubMed1998

Characterization of [125I-Tyr0]-corticotropin releasing factor (CRF) binding to the CRF binding protein using a scintillation proximity assay.

We describe the characterization of high affinity [125I-Tyr0]-human CRF binding to purified recombinant human CRF-binding protein (CRF-BP) using a scintillation proximity assay (SPA). For this stable nonseparation technique developed in 96 well microtiter plates, biotinylated CRF-BP is captured by streptavidin-coated SPA beads for the detection of bound [125I-Tyr0]-CRF. Unbound [125I-Tyr0]-CRF represented little or no signal in the assay. Total binding observed was greater than 5000 cpm with a nonspecific signal of < 100 cpm determined in the presence of excess unlabeled human CRF. A comparison of the SPA method with a charcoal precipitation method confirmed that the biotinylation procedure did not adversely affect affinity of the CRF-BP for [125I-Tyr0]-CRF. Saturation binding analysis yielded an apparent equilibrium dissociation constant (Kd) of 208 +/- 5.0 pM (+/- S.D., n = 3). An inhibition constant (Ki) for unlabeled CRF was calculated to be 0.22 +/- 0.03 nM (+/- S.D., n = 8) and a pharmacological profile for eight CRF-related neuropeptides gave a rank potency similar to previously reported results. Finally, the assay variability was assessed with intra- and inter-plate coefficients of variation which were less than 5% each.

Animals↗
PubMed1997

High-throughput screening: advances in assay technologies.

Both isotopic and nonisotopic assay methodologies are employed in high-throughput screening for drug discovery. Recent advances in cell-based and in vitro biochemical assays will be reviewed, with special emphasis on detection technologies amenable to automated 'mix and read' procedures in high-throughput screening. A major trend is the advent of homogenous assay systems which employ fluorescence resonance energy transfer, fluorescence polarization, and fluorescence correlation spectroscopy. Cell-based assay systems have also become popular in high-throughput screens in which active compounds that directly modulate the disease target are identified. Colorimetric and amperometric methods have also been described recently, but are yet to be adapted widely in high-throughput screens.

Colorimetry↗
PubMed1995

Absence of the skeletal muscle sarcolemma chloride channel ClC-1 in myotonic mice.

The voltage-dependent chloride channel ClC-1 stabilizes resting membrane potential in skeletal muscle. Mutations in the ClC-1 gene are responsible for both human autosomal recessive generalized myotonia and autosomal dominant myotonia congenita. To understand the tissue distribution and subcellular localization of ClC-1 and to evaluate its role in an animal model of myotonia, antibodies were raised against the carboxyl terminus of this protein. Expression of the 130-kDa ClC-1 protein is unique to skeletal muscle, consistent with its mRNA tissue distribution. Immunolocalization shows prominent ClC-1 antigen in the sarcolemma of both type I and II muscle fibers. Sarcolemma localization is confirmed by Western analysis of skeletal muscle subcellular fractions. The ADR myotonic mouse (phenotype ADR, genotype adr/adr), in which defective ClC-1 mRNA has been identified, is shown here to be absent in ClC-1 protein expression, whereas other skeletal muscle sarcolemma protein expression appears normal. Immunohistochemistry of skeletal muscle from ADR and other mouse models of human muscle disease demonstrate that the absence of ClC-1 chloride channel is a defect specific to ADR mice.

Amino Acid Sequence↗
PubMed1994

Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine.

Ryanodine receptors/Ca2+ release channels play an important role in regulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work described here, we characterize the interaction of a tritium-labeled, photoactivable derivative of ryanodine (3H-labeled 10-O-[3-(4-azidobenzamido)propionyl]ryanodine ([3H]ABRy)) with the ryanodine receptor of skeletal, cardiac, and brain membranes. Scatchard analysis demonstrates that this ligand binds to a single class of high affinity sites in skeletal muscle triads. Furthermore, competition binding assays of [3H]ryanodine with skeletal, cardiac, and brain membranes in the presence of increasing concentrations of unlabeled ABRy illustrate that this azido derivative of ryanodine is able to specifically displace [3H]ryanodine from its binding site(s). Analysis of the effects of Ca2+, ATP, and KCl on [3H]ABRy binding in triad membranes shows a similar modulation of binding to that seen in these membranes with [3H]ryanodine. Photoaffinity labeling of triads with [3H]ABRy resulted in specific and covalent incorporation of [3H]ABRy into a 565-kDa protein that was shown to be the skeletal muscle ryanodine receptor. Digestion of the labeled ryanodine receptor revealed a [3H]ABRy-labeled 76-kDa tryptic fragment that was identified with an antibody directed against the COOH-terminal of the receptor. These results demonstrate that the 76-kDa COOH-terminal tryptic fragment contains the high affinity binding site for ryanodine.

Adenosine Triphosphate↗
PubMed1994

Radioimmunoassay for the calcium release channel agonist ryanodine.

A novel photo-activatable derivative of ryanodine, 9-hydroxy-21-(4-azidobenzoyloxy)-9-epiryanodine, has been synthesized and conjugated to keyhole limpet hemocyanin for the production of antibodies with high affinity and specificity to ryanodine. The anti-ryanodine antibodies reacted specifically on immunoblots with the azido-ryanodine compound covalently conjugated to bovine serum albumin. A radioimmunoassay specific for ryanodine was developed using the anti-ryanodine antibodies, and a dissociation constant for ryanodine of 1 nM was determined. Half-maximal inhibition constants (IC50) for various ryanodine derivatives were found to range between 3.2 and 200 nM. These IC50 values correlated very well with the IC50 values obtained for the compounds binding to the skeletal muscle membrane receptor. These antibodies should be useful for the characterization of the ryanodine binding site on the sarcoplasmic reticulum Ca2+ release channel.

Animals↗
PubMed1993

Subunit identification and reconstitution of the N-type Ca2+ channel complex purified from brain.

Calcium channels play an important role in regulating various neuronal processes, including synaptic transmission and cellular plasticity. The N-type calcium channels, which are sensitive to omega-conotoxin, are involved in the control of transmitter release from neurons. A functional N-type calcium channel complex was purified from rabbit brain. The channel consists of a 230-kilodalton subunit (alpha 1B) that is tightly associated with a 160-kilodalton subunit (alpha 2 delta), a 57-kilodalton subunit (beta 3), and a 95-kilodalton glycoprotein subunit. The complex formed a functional calcium channel with the same pharmacological properties and conductance as those of the native omega-conotoxin-sensitive calcium channel in neurons.

Animals↗
PubMed1993

Disruption of the dystrophin-glycoprotein complex in the cardiomyopathic hamster.

Cardiomyopathies are a diverse group of primary cardiac diseases, most of which have a poorly understood etiology. One type of hereditary cardiomyopathy is caused by defects in the dystrophin gene in Duchenne and Becker muscular dystrophy patients. Our laboratory has identified a complex of dystrophin-associated proteins in skeletal and cardiac muscle which span the sarcolemma, linking the subsarcolemmal cytoskeleton to the extracellular matrix. The absence of dystrophin in Duchenne muscular dystrophy patients leads to the loss of dystrophin-associated proteins in both skeletal and cardiac muscle, suggesting that a primary loss of one or more dystrophin-associated proteins might lead to other forms of cardiomyopathy. Here we report the specific deficiency of the 50-kDa dystrophin-associated glycoprotein in cardiac and skeletal muscles of the BIO 14.6 strain of cardiomyopathic hamsters, which experience both autosomal recessive cardiomyopathy and myopathy. Other dystrophin-associated proteins are well preserved in myopathic hamster skeletal muscle, but the link between dystrophin and dystroglycan is disrupted. All dystrophin-associated proteins are decreased in abundance in the cardiomyopathic hamster heart, perhaps explaining why the cardiomyopathy is more severe than the myopathy. Thus, the disruption of the dystrophin-glycoprotein complex may play a role in skeletal and cardiac myocyte necrosis of the cardiomyopathic hamster.

Animals↗
PubMed1992

Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle.

Dystrophin is associated with a complex of muscle membrane (sarcolemmal) glycoproteins that provide a linkage to the extracellular matrix protein, laminin. The absence of dystrophin leads to a dramatic reduction of the dystrophin-associated proteins (156DAG, 59DAP, 50DAG, 43DAG and 35DAG) in the sarcolemma of patients with Duchenne muscular dystrophy and mdx mice. Here we demonstrate that dystrophin-related protein (DRP, utrophin), an autosomal homologue of dystrophin, is associated with an identical or antigenically similar complex of sarcolemmal proteins and that DRP and the dystrophin/DRP-associated proteins colocalize to the neuromuscular junction in Duchenne muscular dystrophy and mdx muscle. The DRP and dystrophin/DRP-associated proteins are found throughout the sarcolemma in small-calibre skeletal muscles and cardiac muscle of adult mdx mice. Because these muscles show minimal pathological changes, our results could provide a basis for the upregulation of DRP as a potential therapeutic approach.

Animals↗
PubMed1991

Purification of dystrophin from skeletal muscle.

Dystrophin was purified from rabbit skeletal muscle by alkaline dissociation of dystrophin-glycoprotein complex which was first prepared by derivatized lectin chromatography. Dystrophin-glycoprotein complex was isolated from digitonin-solubilized rabbit skeletal muscle membranes by a novel two-step method involving succinylated wheat germ agglutinin (sWGA) chromatography and DEAE-cellulose ion exchange chromatography. Proteins co-purifying with dystrophin were a protein triplet of Mr 59,000 and four glycoproteins of Mr 156,000, 50,000, 43,000, and 35,000, all previously identified as components of the dystrophin-glycoprotein complex. Alkaline treatment of sWGA/DEAE-purified dystrophin-glycoprotein complex resulted in complete dissociation of the dystrophin-glycoprotein complex. In order to separate dystrophin from its associated proteins, alkaline-dissociated dystrophin-glycoprotein complex was sedimented by sucrose gradient centrifugation. The residual glycoproteins which contaminated peak dystrophin-containing gradient fractions were then removed by WGA-Sepharose adsorption. The resulting protein appeared as a single band with an apparent Mr of 400,000 on overloaded Coomassie Blue-stained gels. The absence of WGA-peroxidase staining on nitrocellulose transfers of the pure protein indicated that the pure protein was devoid of contaminating glycoproteins. Antisera raised against the carboxyl terminus of human skeletal muscle dystrophin (which does not cross-react with the carboxyl terminus of the chromosome 6-encoded dystrophin-related protein) recognized the pure protein as did antisera specific for the amino terminus of human dystrophin. These data indicate that the protein isolated is indeed the intact, predominant skeletal muscle isoform product of the Duchenne muscular dystrophy gene.

Animals↗
PubMed1991

Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and the associated delta peptides.

Upon disulfide bond reduction, the alpha 2-subunit of the dihydropyridine-sensitive Ca2+ channel undergoes a characteristic mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with the concurrent appearance of the three delta peptides delta 1 (25,000 Da), delta 2 (22,000 Da), and delta 3 (17,000 Da). Densitometric scanning of Coomassie Blue-stained gels shows a stoichiometric ration of 1.0:0.31.47:0.08 for the alpha 2-subunit and the delta peptides 1, 2, and 3, respectively. Characterization of the delta peptides using antibodies, photoincorporation of a hydrophobic probe, and lectin staining shows tham to be antigenically similar hydrophobic glycoproteins. Amino-terminal sequence analysis of the delta peptides reveals three identical sequences that match the predicted amino acid sequence of the alpha 2-subunit starting at Ala935. Enzymatic deglycosylation of the reduced alpha 2.delta complex produces individual core peptides of 105,000 and 17,000 Da, respectively. Treatment of skeletal muscle membranes with high pH in the presence of reducing agents is able to extract the larger amino-terminal peptide but not the smaller carboxyl (delta) peptide, consistent with a single transmembrane domain in the carboxyl (delta) region. The data support a model of the alpha 2-subunit in which the propeptide is processed into two chains that remain attached through disulfide linkages.

Animals↗
PubMed1991

Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle.

Dystrophin-related protein (DRP) is an autosomal gene product with high homology to dystrophin. We have used highly specific antibodies to the unique C-terminal peptide sequences of DRP and dystrophin to examine the subcellular localization and biochemical properties of DRP in adult skeletal muscle. DRP is enriched in isolated sarcolemma from control and mdx mouse muscle, but is much less abundant than dystrophin. Immunofluorescence microscopy localized DRP almost exclusively to the neuromuscular junction region in rabbit and mouse skeletal muscle, as well as mdx mouse muscle and denervated mouse muscle. DRP is also present in normal size and abundance and localizes to the neuromuscular junction region in muscle from the dystrophic mouse model dy/dy. Thus, DRP is a junction-specific membrane cytoskeletal protein that may play an important role in the organization of the postsynaptic membrane of the neuromuscular junction.

Animals↗
PubMed1990

Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle.

Dystrophin, the protein encoded by the Duchenne muscular dystrophy (DMD) gene, exists in a large oligomeric complex. We show here that four glycoproteins are integral components of the dystrophin complex and that the concentration of one of these is greatly reduced in DMD patients. Thus, the absence of dystrophin may lead to the loss of a dystrophin-associated glycoprotein, and the reduction in this glycoprotein may be one of the first stages of the molecular pathogenesis of muscular dystrophy.

Animals↗
PubMed1989

Association of dystrophin and an integral membrane glycoprotein.

Duchenne muscular dystrophy (DMD) is caused by a defective gene found on the X-chromosome. Dystrophin is encoded by the DMD gene and represents about 0.002% of total muscle protein. Immunochemical studies have shown that dystrophin is localized to the sarcolemma in normal muscle but is absent in muscle from DMD patients. Many features of the predicted primary structure of dystrophin are shared with membrane cytoskeletal proteins, but the precise function of dystrophin in muscle is unknown. Here we report the first isolation of dystrophin from digitonin-solubilized skeletal muscle membranes using wheat germ agglutinin (WGA)-Sepharose. We find that dystrophin is not a glycoprotein but binds to WGA-Sepharose because of its tight association with a WGA-binding glycoprotein. The association of dystrophin with this glycoprotein is disrupted by agents that dissociate cytoskeletal proteins from membranes. We conclude that dystrophin is linked to an integral membrane glycoprotein in the sarcolemma. Our results indicate that the function of dystrophin could be to link this glycoprotein to the underlying cytoskeleton and thus help either to preserve membrane stability or to keep the glycoprotein non-uniformly distributed in the sarcolemma.

Acetylglucosaminidase↗
PubMed1988

Evidence for the association of dystrophin with the transverse tubular system in skeletal muscle.

Polyclonal antibodies to dystrophin (the protein product of the human Duchenne muscular dystrophy gene) were used to identify and characterize dystrophin in isolated triads from rabbit skeletal muscle. Anti-dystrophin antibodies recognize an approximately 400,000-Da protein in isolated triads or heavy microsomes from skeletal muscle. Treatment of heavy microsomes with buffers containing high salt or EDTA to remove peripheral or extrinsic membrane proteins does not remove dystrophin; however, treatment of intact triads with trypsin shows that dystrophin is extremely sensitive to mild proteolytic digestion. Isolation of junctional complexes from skeletal muscle triads indicates that dystrophin is tightly associated with the triadic junction. Fractionation of the triadic junction into junctional transverse tubular membranes and junctional sarcoplasmic reticulum membranes has shown that dystrophin is enriched in junctional transverse tubular membranes. Thus, our results suggest that dystrophin is a component of the triad junction which is exposed to the cytoplasm and embedded in or attached to the transverse tubular membrane.

Animals↗