Morphological appearance of epidermal cells cultured on fibroblast-reorganized collagen gels

Summary Human foreskin fibroblasts were used to reorganize hydrated collagen gels into a dermal-like matrix, after which freshly isolated keratinocytes isolated from rabbit ear skin were placed on the surfaces of the matrices and cultured for up to 12 days. Transmission electron microscopy revealed 8–12 cell layers of epidermal cells organized in three distinct strata. The basal stratum consisted of cuboidal to columnar cells with typical complement of organelles, oval nuclei, and prominent tonofilaments inserting into desmosomes. Mitotic cells often were found at this level. There was no well-defined basement membrane region; rather, many of the cells appeared to be in close contact with collagen fibrils. The intermediate stratum of suprabasal cells consisted of elongated cells that had reduced organelles, but still were connected to each other by desmosomes. Finally, the superficial stratum of suprabasal cells contained cells that were completely flattened and often appeared to be sloughing off the apical surfaces of the cultures. Indirect immunofluorescence studies carried out on frozen sections revealed bullous pemphigoid antigen associated with basal epidermal cells; pemphigus vulgaris antigen around the epidermal cells of all strata, and keratin present in the epidermal cells of all strata. Filaggrin was observed in punctate and fibrillar arrangements in suprabasal cells. Fibronectin was found in a linear deposit at the dermal-epidermal junction and around the fibroblasts in the reorganized collagen gels. Type-IV collagen and laminin, however, were not detected.     

Polyphenoloxidases immobilized in organic gels: Properties and applications in the detoxification of aromatic compounds

Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4 degrees C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno-biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. (c) 1995 John Wiley & Sons, Inc.     

Fractionation techniques in a hydro-organic environment. II. Acryloyl-morpholine polymers as a matrix for electrophoresis in hydro-organic solvents

The properties of gels prepared either from acryloyl-morpholine (ACM) or from its mixtures with acrylamide and crosslinked either with bisacrylylpiperazine or with methylenebisacrylamide have been described. ACM-containing gels are compatible with organic solvents. If polymerized in water and dried, they are able to reswell, e.g., in dimethyl sulfoxide or dimethylformamide. If polymerized in presence of dimethylformamide, they form perfectly clear gels, whose mechanical properties are by far superior than those of similar plain polyacrylamide formulations.     

Magnetic separation in biotechnology

New developments in magnetic labelling techniques for cells and microspheres have extended the useful range of magnetic separation, particularly high gradient magnetic separation, into biotechnical areas. The basic magnetic principles involved are reviewed and representative samples of labelling techniques and results drawn from the past three years are presented. Illustrative examples of large scale operation in other industries are also presented, demonstrating the potential of the biotechnological applications.     

Characterization of the subunit composition of HGPRTase from human erythrocytes and cultured fibroblasts

Hypoxanthine-guanine phosphoribosyltransferase is a ubiquitous human enzyme, the inherited deficiency of which leads to a specific metabolic-neurological syndrome. Native acrylamide isoelectric focusing revealed that the human enzyme consists of different numbers of isoenzymes depending on the tissue of origin. The erythrocytic enzyme has the most isoenzymes while the enzyme from cultured fibroblasts has only a single isoenzyme. The isoenzyme pattern of the erythrocytic enzyme changes on storage of the crude hemolysate at 4 C. Treatment of the stored crude hemolysate with 4.5 m urea and 0.35 mm -mercaptoethanol results in an isoenzyme pattern similar to that of the fresh crude extract. Thus the additional isoenzymes are generated on storage not by covalent modification of the enzyme but probably by binding of small molecules to the enzyme or to association of the enzyme molecules. Hypoxanthine-guanine phosphoribosyltransferase has been purified to 80% homogeneity in three steps, DEAE Sephadex chromatography, heat treatment at 85 C for 5 min, and hydroxylapatite chromatography. Denaturing two-dimensional gel electrophoresis of the erythrocytic enzyme revealed that the erythrocytic enzyme is composed of three major types of subunits (1–3) with the same molecular weight but different isoelectric points. In contrast, the fibroblast enzyme is composed of only a single type of subunit, which comigrates with subunit 1 of the erythrocytic enzyme. Since there is a single genetic locus in humans for HGPRTase (the enzyme is X linked) (Nyhan et al., 1967), the observed subunit modification of the erythrocyte enzyme appears to be the result of posttranslational modification. These findings provide a simple explanation for the observed electrophoretic properties of human HGPRTase. A patient with 0.5% of HGPRTase activity in his erythrocytes was found to have small amounts (> 0.5% but < 5% of normal) of the erythrocytic HGPRTase subunits.This work was supported by a grant from NIAMDD, National Institutes of Health, United States Public Health Service. L. J. G. was supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1–4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8, respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6.Purine nucleoside phosphorylase has been purified at least a hundredfold from ³⁵S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of ³⁵S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.This work was supported by a grant from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. L. J. G. is supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.     

Localized synthesis of specific proteins during oogenesis and early embryogenesis inDrosophila melanogaster

Summary Protein synthesis in egg follicles and blastoderm embryos ofDrosophila melanogaster has been studied by means of two-dimensional gel electrophoresis. Up to 400 polypeptide spots have been resolved on autoradiographs. Stage 10 follicles (for stages see King, 1970) were labelled in vitro for 10 to 60 min with³⁵S-methionine and cut with tungsten needles into an anterior fragment containing the nurse cells and a posterior fragment containing the oocyte and follicle cells. The nurse cells were found to synthesize a complex pattern of proteins. At least two proteins were detected only in nurse cells but not in the oocyte even after a one hour labelling period. Nurse cells isolated from stages 9, 10 and 12 follicles were shown to synthesize stage specific patterns of proteins. Several proteins are synthesized in posterior fragments of stage 10 follicles but not in anterior fragments. These proteins are only found in follicle cells. No oocyte specific proteins have been detected. Striking differences between the protein patterns of anterior and posterior fragments persist until the nurse cells degenerate. In mature stage 14 follicles, labelled in vivo, no significant differences in the protein patterns of isolated anterior and posterior fragments could be detected; this may be due to technical limitations. At the blastoderm stage localized synthesis of specific proteins becomes detectable again. When blastoderm embryos, labelled in vivo, are cut with tungsten needles and the cells are isolated from anterior and posterior halves, differences become apparent. The pole cells located at the posterior pole are highly active in protein synthesis and contribute several specific proteins which are found exclusively in the posterior region of the embryo. In this study synthesis of specific proteins could only be demonstrated at those developmental stages which are characterized by the presence of different cell types within the egg chamber, while no differences were detected when stage 14 follicles were cut and anterior and posterior fragments analyzed separately. The differences in the pattern of protein synthesis by pole cells and blastoderm cells indicate that even the earliest stages of determination are reflected by marked changes at the biochemical level.     

Gel-forming algal polysaccharides: structural characterisation and technological applications

Abstract

Some aspects of both the chemical and physico-chemical properties of gelling algal polysaccharides are reviewed in connection with their technological and biotechnological uses. In fact, besides the traditional applications, which are mainly in the field of food industry, recent sophisticated biotechnological manipulations opened the way for the exploitation of these molecules for very advanced applications like the formulation of micro spheres for cell incubation. Some aspects of the structure of alginate gel beads used for cell immobilisation are illustrated.