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Biomedical subjects

E Harper

Publications and source records attributed to E Harper.

At least 19 recordsLinked to original sources
PubMed1994

Characterization of a satellite DNA from Antilocapra americana.

The nucleotide sequence of a cloned satellite DNA from Antilocapra americana (American pronghorn antelope) is presented. The 1477-bp satellite is composed of degenerate 31-bp sub-repeats which are very similar in sequence to those of the major satellite DNAs from cattle and sheep. The sub-repeat sequence is more degenerate and variable in pronghorn than it is in cattle or sheep. The sequence is organized in the pronghorn genome in multicopy tandem arrays.

Animals↗
PubMed1993

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco.

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3' end of the element on germinal excision frequencies. Alteration of three bases to create a Bgl II site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.

Agrobacterium tumefaciens↗
PubMed1993

Collagen characterization in volume-overload- and pressure-overload-induced cardiac hypertrophy in minipigs.

Previous studies in several different species have shown reduced extractability of collagens in some types of cardiac hypertrophy (volume overload) but not others (pressure overload). The objective of the present study was to examine collagen proteins from the same species (minipigs) with both pressure-overload- and volume-overload-induced cardiac hypertrophy. Hypertrophy was induced by two methods: thoracic banding of the aorta to create pressure overload and arteriovenous shunt to cause volume overload in a porcine model. Collagen types I, III, IV, and V were isolated by pepsin digestion from normal and hypertrophied pig left ventricle tissues. Types I and III collagens from normal and hypertrophied samples, when separated from types IV and V, were digested with cyanogen bromide (CB), and the peptides were examined. Collagen concentration was increased in myocardium removed from hearts subjected to volume overload and unchanged in hearts subjected to pressure overload. The extractability of total collagen was unaffected in pressure-overloaded left ventricles but lower in samples from volume-overloaded hearts. CB digestion cleaved all of the types I and III collagens into similar smaller CB peptides with the exception of a 100-kDa peptide that was observed in both control and hypertrophied hearts. This peptide corresponds to one of the high-molecular-weight peptides found in canine heart tissue. The mature collagen cross-link hydroxylysylpyridinoline (HP) was identified in normal and hypertrophied types I and III collagen from porcine sources. Pressure-overload- and volume-overload-induced cardiac hypertrophy in the pig produced different alterations in the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1993

The presence of an inhibitor of human skin collagenase in the roots of healthy and periodontally diseased teeth: changes that occur with age.

The amount of anti-collagenase in the root has been reported in a group 18 to 35 years of age. Healthy roots had more than the diseased; and apical more than the cervical. The purpose of the present study is to determine the effect of age on these values. In a group 50 to 92 years of age, all values were markedly higher than in the younger group. In addition, all relationships between values were preserved. These include location and presence of disease. The higher levels might be due to an aging process or a protective defense against collagenase in the gingival crevice.

Aged↗
PubMed1992

Inhibitors of collagenase in ligaments and tendons of rabbits immobilized for 4 weeks.

Our laboratories have previously measured the capacity for collagenase production by normal and immobilized rabbit periarticular connective tissues (PCT), i.e., anterior cruciate ligaments (ACL), medial collateral ligament (MCL) and patellar tendon (PT). Rabbit limbs were immobilized for 4 weeks and then the tissues were cultured for 4 days. Collagenase and inhibitor (TIMP) were measured in the medium. Collagenase activity was significantly decreased in the medium from all experimental tissues, relative to the levels in contralateral control tissues. On the other hand there was no significant change in inhibitor levels in the three experimental tissues. These results support the idea that collagenase is decreased due to decreased enzyme expression and not to increased inhibitor production.

Animals↗
PubMed1989

Collagenases from periarticular ligaments and tendon: enzyme levels during the development of joint contracture.

Our laboratories have previously demonstrated that normal rabbit periarticular ligaments, anterior cruciate ligaments (ACL), medial collateral ligaments (MCL) and patellar tendon (PT) secrete collagenase. In this current study we examined these connective tissues following an immobilization period of 4 weeks. In the ligaments producing collagenase, activity was expressed only in the control, not in the immobilized joint. Control and experimental patellar tendon samples produce collagenolytic activity, suggesting that the expression of enzyme is less affected in tendons as compared to ligaments. Characterization of these collagenases was carried out using an antiserum directed against rabbit synovial collagenase. We demonstrated that ligament (ACL) and tendon (PT) collagenases cross react with this antibody in a double immunodiffusion assay. Protein blots of PT, ACL and MCL collagenases identified one major species (Mr = 45,000) and a minor species (Mr = 50,000) of immunoreactive proteins in all three connective tissues. Differences between control and experimental enzyme levels appear to be due to less collagenase protein being produced by immobilized ligaments.

Animals↗
PubMed1988

Increase in cross-linking of type I and type III collagens associated with volume-overload hypertrophy.

Types I, III, IV, and V collagen were isolated and characterized from eight normal dog hearts and seven with volume-overload hypertrophy. Animals with volume-overload hypertrophy were killed at a time when left ventricular end-diastolic pressure and stiffness were increased. The collagens were characterized by solubility properties, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and enzyme-linked immunosorbent assay. The percentage of collagen obtained from canine left ventricles was decreased from 32.4% in normal hearts to 15.0% in hypertrophied hearts. We attribute this to a diminution in the extractability of types I and III collagen, which fell from 199.5 mg type I/g collagen and 76.4 mg type III/g collagen in normal hearts to 83.5 mg type I/g collagen and 26.4 mg type III/g collagen in hypertrophied hearts. The amount of types IV and V collagen isolated remained constant in both the control and arteriovenous shunt hearts averaging 15.7 mg type IV/g collagen and 32.1 mg type V/g collagen in control hearts and 12.5 mg type IV/g collagen and 28.9 mg type V/g collagen in hypertrophied hearts. The reduction in quantity of types I and III collagen probably reflects a greater degree of cross-linking in these two types of collagen. Cyanogen bromide peptide analysis confirmed that there was an increase of high molecular weight cross-linked peptides from 3.96% in normal samples to 8.88% in hypertrophied samples. We conclude that cross-linking of types I and III collagen increases in volume-overload hypertrophy and that this is associated with a rise in diastolic stiffness.

Animals↗
PubMed1988

Collagenase production by rabbit ligaments and tendon.

Three periarticular connective tissues from normal rabbits were examined for collagenolytic activity. Enzyme activity was secreted by cultures of anterior cruciate ligament (ACL), medial collateral ligament (MCL) and patellar tendon (PT). A lag period of six days or more was often observed prior to the detection of active collagenase. We attributed this to the presence of an excess of inhibitor in the early days of culture. We quantitated the amount of enzyme and inhibitor produced in 13 days. The levels of collagenase in the ACL and MCL were comparable. The PT, however, consistently secreted more enzyme than the two periarticular (ACL and MCL) ligaments. The reaction products were analyzed for all three collagenases and compared to those generated by the rabbit skin enzyme. We observed the characteristic TCA and TCB collagen fragments for MCL and PT enzymes. Collagen cleavage by the ACL cultures resulted in a product with a molecular weight intermediate between the alpha 2 chain and the TCA piece. These data suggest that quantitative and qualitative differences exist in the ability of these similar connective tissues to degrade collagen.

Animals↗
PubMed1987

Insulin stimulates secretion of a collagenase inhibitor by Swarm rat chondrosarcoma chondrocytes.

Swarm rat chondrosarcoma chondrocytes produce an inhibitor of collagenase similar to that found in bovine articular chondrocytes and extracts of bovine scapular cartilage. These cells synthesize normal levels of cartilage type proteoglycans when cultured in serum free medium with insulin. Collagen synthesis is also increased when insulin is added to chondrosarcoma chondrocytes. We have demonstrated that insulin stimulates collagenase inhibitor production by these chondrocytes. Enhancement of inhibitory activity occurs over the range of 10 to 1000 ng/ml. A 3.2 fold stimulation was observed at a concentration of 1 microgram/ml. There was a lag period of 24 to 48 hours before the insulin effect became evident. Latent or active collagenase was not detectable under these conditions. These results suggest that the hormone insulin controls the levels of collagen in this tumor by stimulating synthesis of collagen and inhibitors of collagenase.

Animals↗
PubMed1987

Adaptation to exercise-induced muscle damage.

1. Serum creatine kinase (CK) and muscle soreness, common indicators of muscle damage, were assessed in 16 subjects after performance of two bouts of eccentric exercise spaced 7 days apart. The purpose was (1) to examine rapid muscle adaptation when the ipsilateral limb was exercised on the second bout and (2) to investigate possible central adaptations when the contralateral limb was exercised on the second bout. 2. The LSL group (n = 7) and the LOL group (n = 9) performed the exercise with the ipsilateral and the contralateral limb, respectively. Careful attention was paid to stabilization of the subject so that the contralateral limb was not active in isometric stabilization. 3. For the LSL group, a lower CK and soreness response was found on about 2 compared with bout 1. For the LOL group, no significant difference in CK response and soreness was found between bout 1 and bout 2. 4. No repeat bout effect was found when the contralateral limb was exercised; therefore central adaptation from performance of the first exercise was minimal. Because a lower repeated bout effect was found after bout 2 using the ipsilateral limb, it was concluded that an experimental design using ipsilateral muscle groups should provide a good model to study rapid muscle adaptation to exercise damage.

Adaptation, Physiological↗
PubMed1985

The effect of pepsin solubilization on platelet aggregation by types I and III collagens.

The effect of pepsin solubilization on the platelet aggregating activity of type I collagen and type III collagen was examined. Pepsin-digested type I collagen was unable to initiate platelet aggregation in either soluble form or as pre-formed fibrils. In contrast, pepsin-digested type III collagen was active in soluble form or as preformed fibrils. Mixtures of type I and type III collagen were assayed for platelet aggregating activity. In soluble form, these mixtures demonstrated elevated activity with increasing type III concentration. When the mixtures were tested as pre-formed fibrils, the rate of aggregation was relatively constant with the combination 75% type III and 25% type I manifesting the highest activity. The lag time for onset of aggregation was also minimized for this same type III/type I ratio. The combinations of the two collagen types formed fibrils which reflected the amounts of types I and III collagens in solution.

Collagen↗
PubMed1985

Purification of nonspecific protease-free collagenase from Clostridium histolyticum.

The collagenase (EC 3.4.24.3) produced by the bacterium Clostridium histolyticum has been purified free from nonspecific protease contaminants by a two-step procedure. The crude culture medium is chromatographed over heparin-Sepharose and Sephacryl S-200, and the resulting preparation has no activity versus noncollagenous proteins or N alpha-benzoyl-L-arginine ethyl ester, yet cleaves native thermally reconstituted collagen fibrils quite efficiently (specific activity, 3000 units/mg). The purification described may be useful for those investigators requiring substantially purified collagenase for applications such as cell culture or collagen quantitation in protein mixtures.

Arginine↗
PubMed1985

Chemical and kinetic characterization of tadpole back skin collagenase.

The purified collagenase from tadpole (Rana catesbiana) back skin was studied with respect to its activation energy using soluble and fibrillar type I collagen, as well as a synthetic peptide substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. The activation energy appeared to be independent of the nature of the substrate, ranging between 28 and 35 kcal/mol. The peptide was cleaved at the Gly-Ile bond and proved to be a poor substrate (kcat/Km, 1.21 h-1 microM-1) when compared with native type I collagen in solution (kcat/Km, 40.6 h-1 microM-1), consistent with the enzyme's low activity versus gelatin [T. A. Bicsak and E. Harper (1984) J. Biol. Chem. 259, 13145]. The amino acid composition of the collagenase was shown to be high in glycine and glutamic acid, and the preparation was shown not to be contaminated with collagen by digestion with bacterial collagenase. The enzyme was not inhibited by iodoacetic acid or 2-hydroxy-5-nitrobenzyl bromide, suggesting the lack of essential cysteinyl and tryptophanyl residues, but was inhibited by micromolar concentrations of ZnCl2, consistent with the presence of essential histidine(s). Ethoxyformic anhydride irreversibly inhibited the collagenase suggesting the presence of essential lysyl residues.

Amino Acids↗
PubMed1985

Inhibition of collagenase activity by extracts of bovine ocular tissues.

Bovine eyes were dissected and separate pools of lens, lens capsule, cornea and vitreous were extracted in guanidine, subjected to ultrafiltration, and examined for their effects on collagenolytic activity. Although lens extract was not inhibitory, the cornea and vitreous both contained inhibitors of collagenase. More inhibition was present in the filtrate of the vitreous than in the retentate, whereas the total amount of inhibition in the cornea was distributed almost equally between the two fractions. The inhibition observed was dose dependent. The partially purified inhibitors from cornea and vitreous blocked the activity of human skin and tadpole back skin collagenases, but they failed to inhibit the bacterial (Clostridium histolyticum) collagenase. The inhibitor was stable to heating to 60 degrees for 30 minutes and to trypsinization.

Animals↗
PubMed1984

Purification and characterization of tadpole back-skin collagenase with low gelatinase activity.

A collagenase secreted by tadpole (Rana catesbiana) back-skin explants in culture has been purified to electrophoretic homogeneity by successive chromatography on sulfopropyl Sephadex, Sephacryl S-200, collagen Sepharose, and heparin Sepharose. The purified enzyme has a molecular weight of approximately 49,000 and an isoelectric pH of 5.0. The enzyme is more active versus soluble collagen than reconstituted fibrils and exhibits very low activity against gelatin (specific activities: Type I collagen, 7660 units/mg; Type I gelatin, 66 units/mg). The collagenase obeys simple Michaelis-Menten kinetics using soluble type I collagen (Km), 0.35 microM; Vm, 1380 units/mg, at 25 degrees C and pH 7.4) and is inhibitable by chelating agents specific for transition metals. Methylene blue catalyzes the photoinactivation of this collagenase, suggesting the presence of essential histidine, tryptophan, tyrosine, or methionine residues.

Animals↗
PubMed1984

Purification and characterization of three forms of collagenase from Clostridium histolyticum.

Three collagenases from Clostridium histolyticum, designated C1, C2, and C3, with apparent molecular weights of 96 000, 92 000, and 76 000 were purified. Peptide maps of the enzymes prepared by digestion with Staphylococcus aureus V-8 protease were found to be similar. Cleavage of native C1 with alpha-chymotrypsin or V-8 protease yielded C2 and C3. This suggested that proteolysis of the Mr 96 000 collagenase may have occurred in vivo, producing the other two lower molecular weight enzymes. Previously prepared antiserum directed against a form of the bacterial enzyme similar by molecular weight and charge to collagenase C3 and Fab' fragments generated from this antiserum inhibited the collagenolytic activity. C1, C2, and C3 were immunologically identical by Ouchterlony double diffusion, and C3 was able to compete with C1 for the antiserum binding site. The ability of each enzyme to bind to antiserum raised against the bacterial collagenase supported the hypothesis that these three proteins were closely related. Zinc analyses of C1 and C3 resulted in a value of 1.14 mol of zinc/mol of C1 and 0.82 mol of zinc/mol of C3. C1 did not contain carbohydrate as measured by gas-liquid chromatography or periodic acid-Schiff staining.

Amino Acids↗