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Biomedical subjects

G D Green

Publications and source records attributed to G D Green.

At least 19 recordsLinked to original sources
PubMed1992

Determination of hydroxyproline by high pressure liquid chromatography.

A rapid, precise, and simple HPLC method provides an assay of hydroxyproline from tissue extracts or solutions of collagen. Samples are hydrolyzed with 6 N HCl, derivatized with phenyl isothiocyanate, and chromatographed on a small, C18 reverse-phase HPLC column. Hydroxyproline (Hyp) is separated from other amino acids and detected by absorption at 254 nm. The method detects 0.40 to 36 micrograms of Hyp with a linear response. Separation requires a total of 6 min, including column cleanup and reequilibration. All components are commercially available, making this a convenient method for routine measurement of collagen concentration.

Chromatography, High Pressure Liquid↗
PubMed1991

Recipes for reconstituting skin.

Reconstituted Living Skin Equivalent (LSE) is made up of a dermal equivalent (DE) on which keratinocytes are plated where they give rise to a multilayered differentiated epidermis. The dermal equivalent develops through interactions between fibroblasts and collagen fibrils that begin to form after the cell-matrix precursor is cast. The gel that forms as a result of collagen polymerization and fluid trapping is contracted uniformly in all dimensions. By securing it at ends and edges in the mold in which it is cast, the final dimensions, strength and morphology of the forming tissue are altered. The same phenomena are seen in casting tubular tissues for the fabrication of small caliber blood vessel equivalents. The cells of the dermal equivalent are biosynthetically active and enrich the matrix to different degrees with secretory products, depending on how the cells are stimulated and on the presence or absence of an epidermis. Collagen biosynthesis by dermal cells in the DE is sensitive to growth factors, ascorbate concentrations and amino acid pools. Both ascorbate and TGF beta 1 increase total collagen biosynthesis at least two-fold by one week after tissue formation. With TGF beta 1 present, the capacity of cells in the DE to synthesize collagen increases with time, over a two-week period. If ascorbate (200 micrograms/ml) is added just after the tissue is cast and daily thereafter, contraction lattice is blocked, and collagen biosynthesis is enhanced relative to contracted controls that had received 200 micrograms/ml ascorbate once. The increase was nearly an order of magnitude over that of controls and was coordinate with a comparable increase in hyaluronate and sulfated glycosaminoglycan (GAG) production as shown by TCA-precipitable glucosamine in the intercellular matrix of the DE. Both the LSE and the Living Dermal Equivalent (LDE) exhibit complex responses to UV radiation and to various chemicals that are greatly different from responses given by monolayered cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1988

Intravitreal clearance of tissue plasminogen activator in the rabbit.

The clearance of tissue plasminogen activator (t-PA) injected into the midvitreous cavity was studied in the phakic, vitrectomized rabbit eye with and without intravitreal fibrin clots. The quantity and activity of t-PA in the vitreous, serum, and aqueous were determined at ten minutes and at 3, 6, 15, 24, and 48 hours after initial injection by an enzyme-linked immunosorbent assay (ELISA) and a spectrophotometric solid-phase fibrin assay (SOFIA). In eyes without an intravitreal fibrin clot, the estimated half-life for t-PA was 4.3 hours by SOFIA and 5.8 hours by ELISA. In eyes containing a vitreal fibrin clot, the half-life increased to 9.8 hours by SOFIA and 11.9 hours by ELISA. Both of these half-lives were significantly greater than the half-life for eyes without fibrin. Regardless of the presence of fibrin, intravitreal t-PA activity was significantly less than t-PA quantity, suggesting the presence of a t-PA inhibitor. A peak in aqueous t-PA occurred before six hours, indicating that t-PA was cleared in part through the anterior chamber. There was no measurable serum t-PA at any of the sampling times.

Animals↗
PubMed1988

Treatment of postvitrectomy fibrin formation with intraocular tissue plasminogen activator.

Twenty-five micrograms of human recombinant tissue plasminogen activator (tPA) was injected intracamerally into the eyes of three aphakic patients who developed severe intraocular fibrin formation within 24 hours after vitrectomy surgery. Fluid obtained from gas-fluid exchange specimens taken 24 hours after tPA injection was analyzed for tPA by an enzyme-linked immunosorbent assay and a spectrophotometric solid-phase fibrin assay. In each of the three patients, complete fibrin resolution occurred within four hours after the tPA injection. There were no complications associated with the intraocular tPA injections. There was measurable tPA activity 24 hours after the initial injection ranging from 0.23 to 1.4 micrograms. In contrast, tPA was undetectable in gas-fluid exchange specimens obtained from seven patients who did not receive intraocular tPA injections. Intraocular tPA is an effective means of treating postvitrectomy fibrin formation in selected aphakic patients.

Adolescent↗
PubMed1987

Age-related increase in a cathepsin D like protease that degrades brain microtubule-associated proteins.

In microtubules isolated from brains of very old rats, two of the major microtubule-associated proteins, MAP1 and MAP2, are found only in degraded form. MAP1 is present as a piece whose molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is circa 50,000 smaller than the native protein, and MAP2 is extensively fragmented. The native forms of both proteins are present in tissue homogenates but are rapidly degraded during microtubule isolation. The proteolytic activity responsible for this degradation is cathepsin D like, being more active at acid pH than neutral and being completely blocked by pepstatin at 10(-7) M. Fractionation of aged brain supernatant by gel permeation chromatography showed that the MAP1 and MAP2 degrading activity elutes with a single peak of cathepsin D like activity. MAP1 and MAP2 are known to promote microtubule assembly, and their degradation by a protease whose levels increase with age could be related to defective microtubule assembly which is known to occur in age-related degenerative conditions such as Alzheimer's disease.

Aging↗
PubMed1986

Poly-lysines as modifiers of one- and two-chain tissue-type plasminogen activator activity.

Poly-L-lysine and certain mixed polymers of L-lysine and other amino acids modify the activity of one- and two-chain tissue-type plasminogen activator (t-PA) towards its substrates. In particular the rate of plasminogen activation in the presence of optimal poly-L-lysine concentrations, is increased by approximately 100-fold. In contrast, activity towards a small synthetic substrate is inhibited by 85%. These effects are observed with both the one- and two-chain forms of t-PA. The use of poly-L-lysines in a coupled assay system optimised for t-PA and plasmin activities allows the reproducible assay of t-PA at the 10(-12) to 10(-13) molar level.

Enzyme Activation↗
PubMed1986

The inactivation of one- and two-chain forms of tissue plasminogen activator by a series of peptidyl chloromethyl ketones.

The inhibition of one- and two-chain forms of tissue-type plasminogen activator by a series of peptide derivatives of arginine chloromethyl ketone was studied. Both forms of the enzyme were inhibited by such reagents at varying rates, dependent on the peptide sequence of the inhibitor. The order of relative effectiveness of the inhibitors was the same for both one- and two-chain tissue plasminogen activator. However, an approximately ten to twenty-fold higher concentration of reagent was required with the one-chain activator to produce similar rates of inactivation. It appears that the specificity of the active sites of the one and two chain forms of tissue plasminogen activator are very similar. It is suggested that the differences in enzymatic properties of the two forms are due mainly to effects on binding (Ki) rather than on the rate of alkylation and inhibition (k2).

Amino Acid Chloromethyl Ketones↗
PubMed1985

Human liver cathepsin L.

Cathepsin L was purified to apparent homogeneity from human liver obtained post mortem. It was necessary to treat the homogenate at pH 4.2 and 37 degrees C to release active enzyme. The purification procedure involved ion-exchange chromatography on carboxymethyl-Sephadex and the Mono S column of a Pharmacia fast-protein-liquid-chromatography system. The enzyme was found to consist of two polypeptide chains of Mr 25 000 and 5000. The larger chain was shown to contain the active-site cysteine residue. Human cathepsin L proved to be similar to the rat and rabbit enzymes in regard to kinetic constants for the substrate benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide and rates of inactivation by the active-site-directed reagents benzyloxycarbonylphenylalanylphenylalanyldiazomethane and benzyloxycarbonylphenylalanylalanyldiazomethane. Thus clear characteristics of cathepsin L are now emerging, and these should simplify the identification of the enzyme in other tissues and species.

Cathepsin B↗
PubMed1979

Failure of pacemaker electrode leads.

With the application of new power sources to pacemakers, considerable improvement has been achieved in the theoretical implant lifetime of current pulse generators, and equally reliable electrode leads will be necessary to ensure long-term pacing. The durability of the electrodes implanted in the past ten years in this centre has been carefully studied. The findings suggest that the use of a more durable conductor material and development of the concept of a 'fail safe' lead (using multi-filament conductors or a secondary conductive pathway along the electrode) will be necessary to match with the implant lifetime of the newer pulse generators. The diagnosis and management of broken conductors is discussed.

Electrodes, Implanted↗
PubMed1978

Inactivation of trypsin-like proteases by sulfonylation. Variation of positively charged group and inhibitor length.

Attempts to achieve selective inactivation of serine proteases of closely related specificity (trypsin-like) by aryl sulfonylation have been extended. Nitrophenyl esters of benzenesulfonic acid and phenylmethanesulfonic acid containing various positively charged groups have been synthesized and examined as inactivators of trypsin, thrombin, plasmin, plasma kallikrein, and urokinase. Examples of selective inactivation by isothiouronium derivatives were found and attributed to differences among these enzymes in geometry and flexibility of the primary specificity sites.

Chemical Phenomena↗
PubMed1977

Long-term epicardial ventricular pacing from endocardial bipolar pacemaker lead: perforation of right atrial wall.

One of the hazards of endocardial cardiac pacing is that the pacemaker lead may perforate the myocardial wall or interventricular septum although the incidence of such perforations is believed to be small. This paper describes what is believed to be a unique case in which a pacemaker lead perforated the atrial wall at implantation (or possibly shortly afterwards) and yet gave satisfactory right ventricular epicardial pacing for more than five years. The perforation was discovered during a routine postmortem examination but earlier lateral x-ray examinations would probably have identified the abnormal position of the electrodes. Moreover, the present implantation technique would not have allowed perforation of the atrial wall at implantation to go undetected.

Aged↗
PubMed1976

The kinetics of hydrolysis of some extended N-aminoacyl-l-lysine methyl esters.

1. The action of two active forms of bovine trypsin (alpha and beta-trypsin) on a series of specific methyl ester substrates of general formula: N-acetyl-(glycyl)n-L-lysine methyl ester (n = 0, 1, 2) and N2-benzoyl-L-arginine ethyl ester have been investigated. With the L-lysine methyl esters the catalytic rate constant for hydrolysis (kcat) was found to be significantly lower for alpha-trypsin than for beta-trypsin, whereas with N2-benzoyl-L-arginine ethyl ester there was no significant difference for the two enzymes. 2. By measurement of the kinetic constants (kcat and Km) in the presence of a nucleophile, which competes with water in the deacylation process, it has been shown that, in common with the specific ester substrates of trypsin, the rate-determining step for the extended L-lysine methyl esters is decaylation of the enzyme. 3. It has been found that by extending the aminoacyl group of N-acetyl-L-lysine methyl ester by one glycine residue (n = 1), a greatly enhanced deacylation rate constant is observed for both alpha and beta-trypsin. The higher rate constants were maintained at the higher levels by the addition of a further glycine residue (n = 2). These results have been interpreted in terms of the 'induced fit' hypothesis the substrates binding to an enzyme subsite adjacent to the active site. 4. The beta-trypsin-catalysed hydrolysis of the L-lysine substrates was investigated over a range of temperature (15--35 degrees C). The Arrhenius law was obeyed, within experimental error, by all three substrates allowing the estimation of the thermodynamic function of activation (delta S not equal to and deltaH note equal to) for the deacylation reactions. The significantly higher values of deltaS not equal to and deltaH not equal to obtained for the two extended substrates are interpreted in terms of additional hydrogen bonding between the longer aminoacyl chains and the enzyme molecule. The results are compared with those for non-extended specific substrates, which have a possible hydrophobic interaction with the enzyme surface.

Animals↗
PubMed1976

Cardiac pacing.

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Congresses as Topic↗