Howard A. Bladen

Degradation of collagen by a human granulocyte collagenolytic system

This report suggests a mechanism for collagen degradation mediated by human granulocytic leukocytes. A specific collagenase, which is extractable from human granulocytes, has been partially purified by DEAE chromatography. This collagenolytic enzyme is operative at physiological pH and is inhibited by EDTA, cysteine, and reduced glutathione but not by human serum. The enzyme cleaves the collagen molecule into two specific products, without loss of helical conformation. Electron micrographs of segment long spacing aggregates indicate that the cleavage occurs one-quarter of the length from the carboxy terminal end of the molecule. Experiments with crude extracts from granulocytes suggest that the specific products of granulocyte collagenase activity are then degraded by other proteases present in the human granulocyte.

The Origin of Collagenase in Periodontal Tissues of Man

Collagenase is produced by both epithelial and connective tissue cells of the human gingiva. Collagenase action was shown through viscometry, acrylamide gel electrophoresis, and electron microscopy. Collagen is cleaved into one-quarter and three-quarter pieces by collagenase derived from human gingiva.

ELECTRON MICROSCOPIC LOCALIZATION OF ENDOTOXIC LIPOPOLYSACCHARIDE IN GRAM‐NEGAGIVE ORGANISMS

Endotoxicity and 0-antigenic specificity has been ascribed to the cell-walls of Gram-negative bacteria. Chemically, the cell walls of Gram-negative bacteria have been separated into an outer lipoprotein coat soluble in phenol, which is separated by a less dense layer from an inner mucopolypeptide layer termed the “R layer” which appeared dense in the electron microscope (Weidel et al., 1960). This latter layer contained the polysaccharide-lipid complex which was water-soluble after phenol extraction of cells, and was thought to be the site of endotoxicity. A phenol-water extract of a human oral strain of a Gram-negative, anaerobic coccus (Veillonella paruula) exhibited biological and immunological activities characteristic of somatic 0-lipopolysaccharides or endotoxins (Mergenhagen et al., 1961; Mergenhagen and Varah, 1963; Mergenhagen, 1965). This high-molecular-weight endotoxin was composed chiefly of lipid bound to a polysaccharide whose monosaccharide constituents were identified as glucose, glucosamine, galactosamine, and methyl pentose. In an electron microscopic investigation of this endotoxin and of the ultrastructure of V. paruula, results were obtained which suggested a morphological correlation of the outer membrane of the cell with particles observed in phenol-water extracts (endotoxin) (Bladen and Mergenhagen, 1964). When extracted with phenol, the outer membrane was removed and the cell retained its shape, owing to an inner solid membrane, which was sensitive to lysozyme. The results of the aforementioned study are summarized in this report in addition to a recent collaborative effort on the use of ferritin-conjugated rabbit anti-lipopolysaccharide globulins to localize endotoxic lipopolysaccharide in this organism.

AN ELECTRON MICROSCOPIC STUDY OF A DNA-RIBOSOME COMPLEX FORMED IN VITRO.

Byrne, Levin, Bladen & Nirenberg (1964) recently reported a DNA-ribosome complex which was formed during in vitro protein synthesis. An electron microscopic study of this system, known to contain rapidly sedimenting DNA, ribosomes, RNA and nascent protein, occasionally revealed clusters of particles aggregated about long, thin (15 to 20 A) fibers. These clusters were usually encircled by a profusion of densely stained spots frequently arranged in strands. The strands appeared to branch off the long 20 A fiber and frequently touched the particles. Individual particles also were seen attached to strands which were oriented perpendicular to the long 20 A fiber. Fractions digested with DNase were lacking the long 20 A fibers and contained numerous particles with short strands attached. Long 20 A fibers were observed in RNase-treated specimens. One interpretation of the micrographs, consistent with the biochemical data presented by Byrne et al . (1964) is that the 20 A fiber is DNA, the particles are 70 s ribosomes and the branches represent RNA linking DNA to ribosomes.

Purification and Reconstitution of the Periodic Fibril and Unit Structure of Human Amyloid

Extraction with alkaline sodium glycinate of the periodic fibril and unit structure of human amyloid and subsequent acidification of the extract provided a method for the purification as well as for the reconstitution of the periodic rod and of the unit structure. Guanidine completely solubilized and irreversibly dissociated both the rod and the unit structure of the human amyloid fibril.