Joseph R. Merkel

Type I and Type III Collagen Content of Healing Wounds in Fetal and Adult Rats

Abstract Full-thickness, dermal wounds were surgically created on the dorsa of fetal rats on the 17th day of gestation. The granulation tissue which developed after 2 days (19 days of gestation) was harvested from six to nine animals and pooled and the collagen was extracted with 0.5 M acetic acid and acetic acid plus pepsin. The ratio of type III:type I collagen was estimated from densitometer scans of electrophoretically separated α-chains. Full-thickness (to fascia depth) wounds were also produced on the dorsa of adult rats and granulation tissue which had developed for different periods of time up to 30 days was excised. Relative proportions of type III and type I collagen were assessed in normal and granulation tissues taken from the adult rats. Both fetal and adult granulation tissues have elevated type III collagen content but normal fetal tissue has a much higher content of type III than does normal adult tissue.

Analysis of Collagen Content in the Fetal Wound

The absence of apparent scar formation following the creation of surgical wounds in utero appears to be a phenomenon peculiarly privileged as a sequela of fetal wound healing. Little information exists to explain this disparity from our knowledge of adult wound healing. Therefore, following creation of surgical wounds in fetal rats, at different intervals the healing wounds were harvested and analyzed for collagen content and types. The average proportion of type III collagen was elevated in normal (26.5%) as well as wounded fetal skin (33.8%) when compared with normal levels for the adult (15%). The total collagen content was markedly diminished in the fetal wound. Although embryonal collagen synthesis apparently does exist in fetal reparative processes, the relationship to the lack of gross scarring remains undetermined.

Purification and some properties of sn-glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

Abstract An NAD-dependent glycerol-3-phosphate dehydrogenase ( sn -glycerol-3-phosphate: NAD + oxidoreductase, EC 1.1.1.8) has been isolated and purified from Saccharomyces cerevisiae by affinity and exclusion chromatography. The enzyme was purified 5100-fold to a specific activity of 158. It has a molecular weight of approximately 31,000, a pH optimum between 6.8 and 7.2, and is sensitive to high-ionic-strength salt solutions. The enzyme is most strongly inhibited by phosphate and chloride ions.

The ontogenetic transition of collagen deposition in rat skin

Minimizing the morbidity of in utero surgery or, perhaps more important, capturing the unique characteristics of scarless remodeling, as is the fetal response to injury, demands better elucidation of the observed variations from adults in whom the normal progression of wound healing leads to fibrosis. Species-dependent fetal phlogistic responses and wound scar formation represent a temporal continuum before the onset of adult patterns. We have analyzed skin collagen synthesis and content in Sprague-Dawley rats as one possible factor in this evolution showing that the fetal characteristics of a high percentage of type III collagen relative to type I and low total collagen content are maintained as long as the first 10 to 15 days postpartum. Although extrapolation of such a crucial “golden period” to justify the delay of human surgical procedures while still capturing the benefits of the fetal milieu remains speculative, anecdotal observations of minimal scar formation lend some credibility for performing less invasive maneuvers in the neonate.

A dimeric, extracellular, heat-stable aminopeptidase produced by a marine pseudomonad

An extracellular aminopeptidase (Alteromonas aminopeptidase III) of a marine pseudomonad, designated Alteromonas B-207, was purified to homogeneity. The enzyme was found to have a native molecular weight of 63 000 by exclusion chromatography and a subunit weight of 33 000 by SDS-polyacrylamide gel electrophoresis. Cross-linking with dimethyladipimidate prevented dissociation of the dimer. The enzyme has a pH optimum of 9.3, a temperature optimum of 70°C and is stable at 60°C for approx. 1 h. It has high specificity for l-leucyl-β-naphthylamide and, of the peptides tested, it shows a preference for l-Leu, Gly and l-Pro over l-α-Asp and l-Lys. The enzyme is inhibited by 1,10-phenanthroline, added to the enzyme-substrate reaction mixture, and by EDTA when the enzyme is dialyzed against 10−3 M soln. Activity can be partially restored to EDTA-dialyzed enzyme by removal of the EDTA and incubation of the enzyme with Zn2+. Atomic absorption data also implicate zinc as a required metal. Sulfhydryl- and serine- inhibitors have no effect on the enzyme.

Influence of salts on the vibriostatic action of 2,4-diamino-6,7-diisopropyl pteridine

SummaryIt was found that the vibriostatic agent, 2,4-diamino-6,7-diisopropyl pteridine, was ineffective against Vibrio parahaemolyticus and two marine Vibrio species when the agent was tested on media prepared with natural or synthetic seawater. The action of the vibriostatic agent was also abolished when magnesium or sodium chloride was added to a basal medium prepared with distilled water.

Direct detection of proteolytic enzymes that have been separated by zone electrophoresis

Abstract A method is described for the direct detection of proteolytic enzymes that have been separated by zone electrophoresis. The method employs an algal chromoprotein medium on which cellulose acetate electrophoresis strips are placed, and proteolytic activity is detected by observing decolorization of the chromoprotein in the area of the active zones. The method has been used for identification of active proteins and the determination of enzyme purity and activity.

Improved Purification and Some Molecular and Kinetic Properties of sn-Glycerol-3-Phosphate Dehydrogenase from Saccharomyces cerevisiae

The purification procedure for isolating sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from Saccharomyces cerevisiae was improved by the introduction of an ion-exchange step. Enzyme yields were doubled and the specific activity was increased as compared to the original procedure. A new value of 42,000 was obtained for the molecular weight by several denaturing methods. By native gel chromatography the molecular weight appears to be 31,000 as reported earlier. Michaelis constants were found to be 0.37 mM with dihydroxyacetone phosphate as the variable substrate and 0.018 mM for NADH as the variable substrate.

Marine bacterial proteases. I. Characterization of an endopeptidase produced by Vibrio B-30.

Abstract A purification procedure is described for a highly active endopeptidase produced by a marine bacterium ( Vibrio B-30). The purified enzyme was shown to be homogeneous by ion-exchange chromatography, gel filtration, and electrophoresis. A crystalline preparation was obtained. The pH optimum of the enzyme was about 7.0, and it was stable in the pH range of 5.0–8.5. Using hemoglobin as the substrate, a K m of 0.095 mm was obtained. The temperature optimum of the enzyme was 40 ° in the absence of calcium and about 50 ° in the presence of 10 −3 m calcium. Calcium both activated and stabilized the enzyme against thermal denaturation. The enzyme was shown to be a serine protease which was irreversibly inhibited by certain metal-complexing agents. Gel filtration studies revealed that Vibrio B-30 endopeptidase had a molecular weight of 49,000 ± 5,000 but it rapidly autolyzed during the second and third passage through a gel column. Removal of a metal ion (probably calcium) resulted in the formation of a high-molecular-weight, enzymatically inactive component and a low-molecular-weight, partially active component.

Marine bacterial proteases: II. Peptide specificity of an endopeptidase produced by Vibrio B-30☆☆☆

Abstract The peptide specificity of an endopeptidase isolated from the culture filtrate of a new marine bacterium, Vibrio B-30, was determined. The enzyme hydrolyzed hemoglobin, chromoproteins, casein, ribonuclease, and the B-chain of oxidized insulin but failed to hydrolyze di- or tripeptides or their CBZ-derivatives to any appreciable extent unless both NH 2 - and COOH-terminal amino acids were blocked with bulky radicals. From detailed studies on the oxidized B-chain of insulin and selected synthetic peptides, it was concluded that the endopeptidase acts rapidly on those peptide bonds in which an aromatic amino acid (tyrosine, phenylalanine) provides the amino group, and a neutral amino acid with a bulky side chain forms the acyl portion of the peptide bond.