Biomedical subjects

R J McCormick

Publications and source records attributed to R J McCormick.

At least 19 recordsLinked to original sources

PubMed1996

The leucine zipper may induce electrophoretic mobility anomalies without DNA bending.

Numerous proteins bend DNA upon binding, a phenomenon of potential significance for regulation of gene expression and chromatin. DNA bending is commonly predicted from the presence of electrophoretic mobility anomalies in protein-DNA complexes. However, as compared with electrophoretic methods, several DNA binding oncoprotein families do not display comparable evidence of DNA bends in x-ray structural studies. Herein, circularization kinetics and affinity measurements with prebent DNA templates were employed to assess bending and DNA structural preferences for Max and other basic helix-loop-helix/leucine zipper proteins. In this way, proteins in the Myc/Max basic helix-loop-helix/ leucine zipper family were found not to bend DNA in solution but to actually stabilize DNA in an unbent configuration that resists circularization. The mobility anomaly was found to be induced by the leucine zipper protein motif, rather than structural distortions of DNA. Thus rigid protein domain structures may induce anomalous electrophoretic mobility. Moreover, the energetic preference of non-DNA bending proteins for unbent templates suggests mechanisms whereby chromatin structure may regulate transcription.

Binding Sites↗

PubMed1996

The semidominant Mi(b) mutation identifies a role for the HLH domain in DNA binding in addition to its role in protein dimerization.

The mouse microphthalmia (mi) locus encodes a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor called MITF (microphthalmia transcription factor). Mutations at mi affect the development of several different cell types, including melanocytes, mast cells, osteoclasts and pigmented epithelial cells of the eye. Here we describe the phenotypic and molecular characterization of the semidominant Microphthalmia(brwnish) (Mi(b)) mutation. We show that this mutation primarily affects melanocytes and produces retinal degeneration. The mutation is a G to A transition leading to a Gly244Glu substitution in helix 2 of the HLH dimerization domain. This location is surprising since other semidominant mi mutations characterized to date have been shown to affect DNA binding or transcriptional activation domains of MITF and act as dominant negatives, while mutations that affect MITF dimerization are inherited recessively. Gel retardation assays showed that while the mutant MITF(Mi-b) protein retains its dimerization potential, it is defective in its ability to bind DNA. Computer modeling suggested that the Gly244Glu mutation might disrupt DNA binding by interfering with productive docking of the protein dimer onto DNA. The Mi(b) mutation therefore appears to dissociate a DNA recognition function of the HLH domain from its role in protein dimerization.

Animals↗

PubMed1996

Collagen crosslinks in longissimus muscle from lambs expressing the callipyge gene.

The objective of this study was to determine whether increased amounts of collagen and (or) hydroxylysylpyridinoline crosslinks in longissimus muscle are partially responsible for decreased tenderness when callipyge lambs are compared to normal lambs. The longissimus muscle was used because we believe tenderness is a greater problem in loin chops of callipyge lambs than it is in other cuts. Ten normal and 10 half-sibling lambs expressing the callipyge gene were compared. Lambs were slaughtered at approximately 8 mo of age when they reached a target weight of 59 kg. Dressing percentages were higher for the leaner callipyge lambs that exhibited larger longissimus muscle areas and heavier leg weights (P < .01). Warner-Bratzler shear values for longissimus muscle from callipyge lambs were higher (P < .01) than those from normal lambs. The higher shear values were not explained by amount of muscle collagen or by hydroxylysylpyridinoline crosslink concentration because both collagen percentage (P < .09) and crosslink concentration (P < .04) were lower in callipyge lambs. Therefore, attempts to modify shear values and make loin chops from callipyge lambs more acceptable to consumers should focus on the myofibrillar fraction of muscle.

Amino Acids↗

PubMed1996

Partial characterization of ovine skeletal muscle proteoglycans and collagen.

Ovine longissimus dorsi and biceps femoris muscles were analyzed for proteoglycan content, collagen and lysine aldehyde-derived collagen crosslinking concentrations at 2-4 days, six-month-old, and six-year-old stages of development. Tissue extracted proteoglycan molecular sieve distribution on a Sephacryl S-200HR column revealed two proteoglycan populations with estimated relative molecular weight ranges of 200,000 to 250,000 daltons and 23,000 to 70,000 daltons. The molecular sieve distribution was similar between the two muscles within a developmental age, but changed as a function of developmental age. Primary culture from both the longissimus dorsi and biceps femoris muscle liberated proteoglycans into the culture medium. In contrast to the tissue extracted proteoglycans, at the six-year-old stage of development, culture medium liberated proteoglycan Sephacryl S-200HR molecular sieve distribution differed between the two muscles. In both the tissue extracted and medium liberated proteoglycans at all developmental stages, nitrous acid deamination demonstrated the presence of heparan sulfate. Immunoblot analysis of the tissue extracted proteoglycans indicated the presence of decorin at each developmental stage. Longissimus dorsi and biceps femoris collagen concentrations (5.13 +/- 0.9 vs. 5.53 +/- 1.5%, respectively) and crosslink concentrations (0.07 +/- 0.01 moles HP/mole collagen) were initially similar between the two muscles; however, by six-months the muscles differed in both collagen concentration (1.72 +/- 0.5 and 2.53 +/- 0.7%, respectively) and crosslinking (0.24 +/- 0.02 and 0.27 +/- 0.03 moles HP/mole collagen, respectively). At six years of age, both the longissimus dorsi and biceps femoris exhibited slightly elevated collagen concentrations (2.49 and 3.05%, respectively) while crosslinking values were decreased relative to values at six-months of age (0.11 +/- 0.01 and 0.18 +/- 0.01 moles HP/mole of collagen, respectively). The results from this study indicate that skeletal muscle proteoglycans and collagen show developmental changes, which suggests that they are subject to developmental regulation.

Animals↗

PubMed1996

The avian low score normal muscle weakness alters decorin expression and collagen crosslinking.

Extracellular matrix development of chicken pectoral muscle was examined in the Low Score Normal (LSN) genetic muscle weakness and compared to both normal and avian muscular dystrophy (MD). At 20 days of embryonic development significant elevations were noted in LSN total glycosaminoglycan concentration and decorin, while at 14 days, LSN glycosaminoglycan and decorin levels were indistinguishable from the controls. Levels of a large skeletal muscle chondroitin sulfate proteoglycan (M-CSPG) appear to be unaffected. Morphologically, at 20 days, the extracellular matrix space between muscle fibers increased to a level characteristic to that observed in avian muscular dystrophy. At six weeks posthatch a marked increase in LSN collagen crosslinking relative to MD or control tissues was observed, while collagen concentration was not altered. By one year posthatch LSN collagen crosslink levels did not significantly differ from normal tissue. These data support the concept that the LSN muscle weakness is associated with changes in both proteoglycan and collagen characteristics.

Animals↗

PubMed1995

Pre-bending of a promoter sequence enhances affinity for the TATA-binding factor.

TATA-binding protein (TBP) binds the minor groove of the TATA element with the DNA bent 80 degrees towards the major groove. A constrained minicircle strategy has been used to test the effect of DNA topology on the affinity of TBP for the TATA element. We report here that TBP bound to DNA which was slightly pre-bent towards the major groove with 100-fold higher affinity than unbent (linear) DNA of identical sequence and 300-fold higher affinity than DNA pre-bent towards the minor groove. Similar discrimination was observed with the holo-TFIID transcription complex. DNA topology, particularly bending, is determined by many factors including chromatin in cells and may, through changes in the affinity of the TATA factor, be important in the control of transcription.

DNA↗

PubMed1995

Collagen characteristics of skin, fell, and epimysium from rams, wethers, and zeranol-implanted ram lambs.

Eighteen spring-born Columbia ram, wether, and zeranol-implanted ram lambs were examined to determine the influence of castration or zeranol implantation on collagen characteristics of skin, fell, and epimysium and possible relationships between collagen properties of each tissue and difficulty of pelt removal. Pelt removal force was lower in wethers than in rams (P < .05) and intermediate for zeranol-implanted rams. Collagen concentration in skin of rams was greater (P < .05) than that in wethers or implanted rams, but percentage of heat-soluble collagen in skin was higher in implanted rams. Percentage of type III collagen in skin was highest in rams and lowest in wethers (P < .05); that in the skin of implanted rams was intermediate. The fell of wethers contained a higher (P < .05) collagen concentration, higher insoluble collagen amount, and lower percentage of soluble collagen than that of rams or implanted rams. The fell had a lower percentage of type III collagen than that of rams (P < .05). Epimysium collagen concentration of rams and wethers was higher (P < .05) than that of implanted rams; however, percentage of soluble collagen was higher for the implanted rams than for the other classes. Type III collagen percentage in the epimysium did not differ by animal class (P < .05). Zeranol-implanted ram lambs had a higher percentage of soluble collagen in all tissues examined than did non-implanted rams and force required for pelt removal was reduced in implanted rams.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Fibronectin self-association is mediated by complementary sites within the amino-terminal one-third of the molecule.

The formation of a fibrillar fibronectin (FN) extracellular matrix requires self-association of FN dimers. In this report, we show that the major sites for self-association are the amino-terminal repeats I1-5 and the first type III repeats. Recombinant FNs and fragments were generated by baculovirus expression of cysteine-rich domains and by bacterial expression of type III repeats as fusion proteins with maltose binding protein. When recombinant polypeptides were immobilized on microtiter wells, FN bound to 70-kDa amino-terminal fragment and to fusion proteins containing repeats III1-2 and III1-6 but not to other type III repeats. Similar results were obtained with a gel overlay assay. Binding was concentration-dependent and saturable. The amino-terminal binding site for III1-2 was further localized to repeats I1-5. Therefore, at least two different sites for FN-FN interaction reside near the amino terminus of the molecule. A model for the regulation of FN matrix assembly is proposed based on intramolecular interactions between these amino-terminal sites.

Amino Acid Sequence↗

PubMed1994

Regional differences in LV collagen accumulation and mature cross-linking after myocardial infarction in rats.

To determine the extent of and any regional differences in remodeling response of the extracellular matrix (ECM) to myocardial infarction (MI), moderate-to-large transmural infarcts were surgically produced in left ventricular (LV) free wall of rats. Animals were killed 13 wk after surgery. In comparison to age-matched controls, infarction was associated with an overall increase in heart weight, which included hypertrophy of both the right ventricle and LV. Although the remaining viable myocardium in LV free wall was significantly reduced, the interventricular septum was hypertrophied some 30% compared with control tissues (247 +/- 9 vs. 189 +/- 8 mg). Collagen concentration more than doubled in remaining viable free wall (8.92 +/- 0.59 vs. 3.95 +/- 0.25 mg/100 mg, P < 0.0001), and a smaller but still highly significant 27% increase occurred (P < 0.01) in the more remote septum. Degree of covalent cross-linking of collagen fibrils as assessed by hydroxylysylpyridinoline (HP) concentration also revealed regional differences in response of the ECM to infarction. Although HP concentration was increased 60% in viable free wall (P < 0.05) post-MI, it was unchanged in the septum. With respect to collagen characteristics of the transmural infarct per se, the scar exhibited still further increases in both collagen and HP concentrations compared with the already elevated values for these two parameters in viable free wall. The results indicate that any evaluation of the remodeling response of viable myocardium post-MI must include not only the myocyte but also the ECM, the principal component of which is collagen.

Amino Acids↗

PubMed1993

Copper deficiency alters collagen types and covalent cross-linking in swine myocardium and cardiac valves.

Dietary copper deficiency induces alterations of connective tissue metabolism that are associated with lesions in cardiovascular and other organ systems. To determine the impact of copper deficiency on characteristics of collagen in porcine myocardium and cardiac valves, weaned pigs were fed diets with adequate or deficient levels of copper. Although dietary copper did not affect the concentration of collagen in either myocardium or bicuspid valves, the degree of collagen cross-linking, as assessed by the level of hydroxylysylpyridinoline, was lower in both tissues of copper-deficient pigs. Proportions of type III collagen were increased in the left ventricle and bicuspid valves of copper-deficient pigs. Copper deficiency induced extensive remodeling, however, of the collagen fraction of cardiac interstitium. Reduction in left ventricular collagen cross-linking may provide the stimulus for the development of cardiac hypertrophy, which characterizes severe copper deficiency, by increasing the compliance of the ventricular wall. The shift in the phenotypic profile of collagen that is associated with this cardiac hypertrophy indicates synthesis of new collagen, which could affect collagen cross-linking irrespective of copper status.

Animals↗

PubMed1993

Age and training alter collagen characteristics in fast- and slow-twitch rat limb muscle.

This study evaluated the single and interactive effects of age and training status on selected collagen parameters in two rodent locomotor skeletal muscles contrasting in fiber type composition. Gastrocnemius (GAST) and soleus (SOL) muscles from both trained (10 wk of daily treadmill running) and sedentary young adult (5-mo-old), middle-aged (15-mo-old), and old (23-mo-old) female Fischer 344 rats were evaluated for concentrations of collagen (measured by hydroxyproline concentration ([OH-Pro])) and of the predominant nonreducible lysine aldehyde-derived collagen cross-link hydroxylysylpyridinoline ([HP]). Maximal aerobic capacity was significantly elevated in all three trained groups compared with sedentary age-matched control groups. Slow-twitch SOL had a significantly higher [OH-Pro] than fast-twitch GAST (P < 0.05). Although aging had no effect on [OH-Pro] in GAST, in SOL a significant increase with age was seen (P < 0.02). In sedentary rats both GAST and SOL [HP] increased with age, with this increase being more pronounced for SOL. Additionally, although training had no effect on the aging-associated increase in GAST [HP], it prevented the rise seen in SOL. The observed training-induced reduction in SOL [HP] presumably reflects exercise recruitment and subsequent stimulation of collagen synthesis and degradation rates in this muscle. We conclude that both aging and training affect the extracellular matrix in rodent limb skeletal muscle.

Aging↗

PubMed1993

Mechanisms and physiological implications of leucocyte chemoattraction into periovulatory ovine follicles.

Leucotactic polypeptide was isolated from follicular-conditioned incubation media by ultrafiltration and reverse-phase high performance liquid chromatography. The bioactive fraction was subjected to amino acid analysis and shown to be abundant in glycine, proline and hydroxyproline. These amino acid residues are common to repetitive sequences of alpha collagens. Synthetic collagen-like peptides composed of repeating triplets of glycine, proline and hydroxyproline (GPH x 3, GPH x 9) were active in attracting white blood cells as measured in vitro using a linear under-agarose assay. Accumulation of leucocytes within extravascular spaces of the theca interna was induced by intrafollicular injection of GPH x 3 or bacterial collagenase. Intrafollicular administration of affinity-purified GPH x 3 antibodies during the immediate preovulatory period inhibited thecal extravasation of leucocytes, but did not negate follicular rupture. However, serum concentrations of progesterone were depressed in antibody-treated animals throughout the ensuing luteal phase. This luteal defect was counteracted by injecting leucocytes into the preovulatory follicle. It is concluded that periovulatory follicles of the sheep secrete collagen-like leucotactic peptides. Once drawn into the follicle, resident inflammatory cells are apparently involved in transforming it into a fully functional corpus luteum.

Animals↗

PubMed1993

Growth and slaughter characteristics of ram and wether lambs implanted with zeranol.

Forty-nine Columbia ram and wether lambs born in April 1990 and 46 born in April 1991 were studied to determine the effects of zeranol implants on growth, difficulty of pelt removal, and carcass characteristics. Implanting ram and wether lambs once (1990) or twice (1991) with 12 mg of zeranol did not change live weight or ADG but gain/feed decreased (P < .05) in ram lambs slaughtered at approximately 50 kg. Testes weight was reduced approximately 50% by implanting. Two implants reduced (P < .05) the force needed to pull the pelt from the hind legs of ram lambs, but implanting tended to increase the force required to pull the pelt from wether lambs. Data for pelt weight, force required to pull the pelt, percentage of the carcass in the shoulder or splenius muscle, and Warner-Bratzler shear values showed that zeranol implants resulted in ram lambs becoming more like wethers and wether lambs becoming more like rams. Implanting with zeranol did not affect closure of the metacarpal growth plate in ram or in wether lambs. Difficulty of pelt removal can be reduced by implanting ram lambs with 12 mg of zeranol at approximately 114 d of age and reimplanting zeranol 28 d later.

Age Factors↗

PubMed1993

Intramuscular collagen characteristics of ram, wether, and zeranol-implanted ram lambs.

Eighteen spring-born Columbia ram, wether, and zeranol-implanted ram lambs were studied to determine the influence of castration or zeranol implants on intramuscular collagen (IMC) properties and muscle shear force values. Warner-Bratzler shear force values for longissimus muscle were greatest for ram lambs, intermediate for implanted rams, and least for wethers (P < .05). Nonreducible collagen crosslink concentration was greater in IMC of rams and implanted rams (P < .05). The IMC from rams compared with that from wethers contained proportionately more Type III than Type I collagen (P < .05); values for implanted rams were intermediate. Heat-soluble muscle collagen concentration was greater for rams and implanted rams than for wethers (P < .05); however, insoluble collagen concentration did not differ by treatment. Muscle collagen concentrations were not different for rams, wethers, or implanted rams. Increased shear force values in rams were associated with elevated collagen crosslink concentration and increased proportion of Type III collagen. Greater concentration of soluble collagen in ram IMC neither diminished nor diluted IMC crosslinking. The proportion of heat-labile collagen in the fractions did not reflect the IMC crosslinking profile for ram and wether lambs. Zeranol implantation modified IMC characteristics of rams such that shear force values and some collagen properties were similar to those of wethers.

Adipose Tissue↗

PubMed1992

Sequence analysis of leukocyte chemoattractant peptides secreted by periovulatory ovine follicles.

Cells of the white blood series infiltrate ovarian follicles during the ovulatory process. A segment of the wall of periovulatory ovine follicles was incubated and conditioned media subjected to ultrafiltration. Leukocyte chemoattractant activity of media was measured using a linear under-agarose migration assay. Bioactivity was recovered following filtration through a 3000 molecular weight cut-off membrane. Filtrate was then fractionated by reverse phase high performance liquid chromatography. Peptides recovered from two fractions with significant chemoattractant activity were sequenced. One fraction contained 16 amino acid residues with repeating triplets of Gly-X-Y, where X and Y were often proline and hydroxyproline, respectively. Because this motif is characteristic of alpha collagens, and since thecal collagen is degraded during the mechanics of ovulation, it appears that this chemoattractant is derived from the connective tissue matrix of the follicle. Peptide isolated from the other bioactive chromatographic fraction was 15 amino acids in length, and rich in glycine, but did not contain imino acids. To our knowledge this is the first report of purification of leukocyte chemoattractants of reproductive tissue origin. Resident follicular granulocytes and mononuclear cells are capable of secreting a broad spectrum of potent chemicals that could be involved in the mechanisms of ovulation and luteinization.

Amino Acid Sequence↗

PubMed1992

Enzymatic and nonenzymatic cross-linking of collagen and elastin.

Knowledge regarding the steps and mechanisms related to the intra- and interchain cross-linking of collagen and elastin has evolved steadily during the past 30 years. Recently, effort has been directed at identifying the location and types of cross-links that are found in collagen and elastin. There are two major groups of cross-links: those initiated by the enzyme lysyl oxidase and those derived from nonenzymatically glycated lysine and hydroxylysine residues. The formation of enzymatic cross-links depends on specific enzymes, amino acid sequences, and quaternary structural arrangements. The cross-links that are derived nonenzymatically occur more adventitiously and are important to pathobiological processes. Considerable progress has been made in elucidating the pathways of synthesis for several of the enzymatically mediated cross-links, as well as possible mechanisms regulating the specificity of cross-linking. Although less is known about the chemistry of cross-links arising from nonenzymatically glycated residues, recent progress has also been made in understanding possible biosynthetic pathways and control mechanisms. This review focuses on such progress and hopes to underscore the biological importance of collagen and elastin cross-linking.

Animals↗

PubMed1992

Enhanced degradation of collagen within apical vs. basal wall of ovulatory ovine follicle.

Electron microscopic examination of ovulatory ovine follicles indicated that dissolution of thecal collagen was enhanced along the follicular circumference adjacent to the ovarian serosa. Concentrations of hydroxyproline were correspondingly lower within apical than basal tissues. Collagenolytic activity of tissue homogenates and extracts was assessed by monitoring radioactive peptide release from cocultured collagen fibrils. Collagenase inhibitors were separated from enzyme by sequential extraction of follicular homogenates with detergent and heating. Enzymatic activities of homogenates and heat extracts of apical and basal tissues increased toward ovulation. A distinction between apex and base was not attributed to significant differences in homogenates; however, collagenolytic activity of heat extracts of apical tissue was selectively elevated (i.e., extraction was evidently required to effectively dissociate collagenase from thecal fibrils, making more enzyme available for substrate digestion). Activity of detergent extracts was restored by dithiothreitol and iodoacetamide (which inactivate tissue inhibitors of metalloproteinases), and collagenolysis induced by heat extracts was augmented by aminophenylmercuric acetate (a stimulator of latent collagenases). Nonetheless, variations between apex and base invoked by these compounds were relative. It therefore appears that active collagenase is more tightly bound within the extracellular matrix of the follicular apex than enzyme associated with basal tissue. This phenomenon might serve to preferentially favor enzymatic catabolism of collagen in that region, thereby dictating the ovarian site of follicular rupture.

Animals↗

PubMed1992

Aging- and training-induced alterations in collagen characteristics of rat left ventricle and papillary muscle.

We evaluated the single and interactive effects of aging and exercise training on selected parameters of myocardial interstitium in both the left ventricle (LV) and LV papillary muscle of female Fischer 344 specific pathogen-free rats. Ten weeks of treadmill running resulted in significant LV hypertrophy as well as elevated plantaris muscle citrate synthase activity in both young adult (5-mo-old) and senescent (23-mo-old) trained animals (YT, young trained; OT, old trained) compared with age-matched sedentary controls (YC, young control; OC, old control). Proline and hydroxyproline pools were significantly higher (both P less than 0.05) in 23-mo-old vs. 5-mo-old papillary muscles. Degree of maturation (nonreducible cross-linking) of LV collagen was evaluated by measurement of hydroxylysylpyridinoline concentration ([HP]). In a comparison of YC with OC rats, ventricular [HP] increased approximately fivefold from 0.059 +/- 0.007 to 0.285 +/- 0.018 (SE) mol HP/mol collagen (P less than 0.001). Whereas training had no effect on ventricular [HP] in young adult rats, it significantly reduced LV collagen cross-linking in OT rats (0.131 +/- 0.027) so that HP values in this group were less than one-half of those observed in OC rats. Because both collagen concentration and degree of cross-linking are thought to affect muscle stiffness characteristics, we conclude that the observed changes should be considered in any explanation for aging- and training-induced alterations in LV and papillary muscle contractile indexes.

Aging↗