Synthesis of stromal glycosaminoglycans in response to injury

Our goal is to examine the synthesis and deposition of corneal glycosaminoglycans (GAGs) in response to a wound created by the insertion of porous discs into stromal interlamellar pockets. The disc and the surrounding stromal tissue were assayed and compared to contralateral control stroma and to sham operated corneas at 14,42, and 84 days. The tissue and/or discs were removed and labeled with ³⁵S-sulfate for 18 h; GAGs were extracted with 4 M guanidine–HCl. Extracts were chromatographed on Q-Sepharose columns, bound proteoglycans were eluted with a linear salt gradient, and radioactive fractions were analyzed. Total GAG content was determined colorimetrically, using dimethylmethylene blue. Specific GAGs were determined using enzymatic digestion with selective polysaccharide lyases and protein cores were examined using SDS–PAGE. The nonbound fractions from the chromatography were assayed for TGF-β using Western blot analysis and for hyaluronic acid using an ¹²⁵I-radiometric assay. Specific GAGs were localized 42 days after the disc had been implanted in the stroma. The placement of the discs into the stroma resulted in a decrease in the total amount of GAG. However, the ratio of dermatan-chondroitin sulfate and heparan sulfate to keratan sulfate increased in the surrounding tissue and disc. Hyaluronic acid was elevated at day 14 in the surrounding tissue, and not until day 84 in the disc. Western blot analysis of surrounding tissue extracts revealed forms of TGF-β that migrated with an apparent molecular mass of 63 and 43 kDa. The results indicate that the insertion of discs into interlamellar pockets causes changes in the sulfation and proportion of the glycosaminoglycans in the surrounding tissue and the disc. These changes are coincident with the appearance of TGF-β. After 84 days, the population of glycosaminoglycans in the disc begins to resemble the surrounding stroma. This model will allow us to examine further the synthesis and deposition of proteins following an extensive wound in which cells must migrate to the wound site and then undergo extensive remodeling. © 1995 Wiley-Liss, Inc.     

Propylene Oxide as Sterilizing Agent

Liquid propylene oxide added to a variety of disinfectant solutions resulted in a significant reduction of the bacterial spore content of these solutions.     

Conjugation of metallothionein to a murine monoclonal antibody

A method of conjugation of the metal-binding protein, metallothionein, to an anticarcinoma murine monoclonal antibody, B72.3, and its F(ab')2 fragment has been developed utilizing the heterobifunctional crosslinking reagent, succinimidyl 4-(N-maleimidomethyl)-cyclohexane 1-carboxylate. This crosslinking reagent is first reacted with the free amines on the immunoglobulin. After removal of unreacted crosslinker, conjugation is affected through a sulfhydryl group on metallothionein. Under the conditions employed all immunoglobulin aggregates contained metallothionein. The degree of undesired aggregation is directly proportional to the number of metallothioneins attached to the immunoglobulin. This aggregation can be controlled by the amount of crosslinker and metallothionein presented to the immunoglobulin. The immunoglobulin conjugate retains full immunoreactivity and can be readily purified from the unreacted metallothionein and high molecular weight aggregates. The metallothionein-B72.3 conjugate functions as an efficient and stable chelator of radiometals. Thus metallothionein-monoclonal antibody conjugates have potential utility in cancer diagnosis and therapy.     

Comparison of the effects of treatment of acute myocardial infarction in a coronary unit and on a general medical ward

During 1968, 400 cases of proven acute myocardial infarction were admitted to the Toronto General Hospital (mortality 25.0%).Approximately half the patients who survived their stay in the Emergency Department were admitted to the Coronary Unit (mortality 15.6%) while the other half, because of lack of beds in the Coronary Unit, were treated on a general medical ward (mortality 26.5%). More elderly patients (> 70) were admitted to the medical wards and contributed to the higher mortality.The frequency of successful resuscitation following cardiac arrest was twice as great in the Coronary Unit as on the medical wards. Antiarrhythmic drugs given in the Coronary Unit with the assistance of the electrocardiographic monitor did not influence the early mortality. Digitalis was used more frequently on the medical wards, but did not appear to exert an unfavourable effect on survival.     

Sodium dodecyl sulphate--polyacrylamide gel electrophoresis of cell-surface proteins as an aid to the identification of the Bacteroides fragilis group.

The outer-membrane complexes from 40 strains of Bacteroides, representing eight of the species included in the Bacteroides fragilis group, were released by EDTA treatment. The component polypeptides were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on slab gels. Within a species (biotype) the patterns produced indicated marked similarities in the structures of the surface proteins among the strains examined. The patterns produced by strains belonging to different species, however, showed fewer similarities. An unknown organism could therefore be identified to species level using this technique and a few selected biochemical tests.