Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.     

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. I. Helper cells have a more restricted specificity repertoire than proliferative cells

The proliferative and helper T cell repertoires were compared in the CBA/J mouse for the response to the large protein antigen, tetrameric beta-galactosidase (GZ = 1021 a.a/monomer). The systems assessed the ability of cyanogen bromide (CB) peptides of GZ to: 1) prime for a T cell proliferative response to GZ; or 2) generate T cell help, measured by the production of anti-FITC PFC in the in vitro response to GZ-FITC. Priming for in vitro proliferation was attempted with 11 CB peptides comprising 70% of the GZ molecule. Strong priming was found with five peptides and intermediate priming was found with four other peptides; two peptides were without effect (CB-20 = a.a 767-862, and CB-4 = a.a. 188-202). Despite this indication of generally dispersed recognition of GZ epitopes, only two CB peptides, CB-2 (a.a. 3-92) and CB-10 (a.a. 378-418) were able to induce a T helper cell response. The surprising dearth of helper T cell-inducing epitopes may be peculiar to the limited fluorescein (FITC) substitution on GZ-FITC (17-25 FITC residues per tetramer) or it may reflect the constraints involved in T cell recognition required for T-B collaboration. Also considered was the possibility that the helper T cell repertoire might be distinct from the proliferative repertoire, the latter reflecting DNA synthesis and recruitment by other functional T cell subpopulations.     

Combined effects of diflunisal and nifedipine on uterine contractility in dysmenorrhoeic patients

In eight nulliparous women with severe primary dysmenorrhoea, intrauterine pressure was recorded on the first day of menstruation before and after administration of diflunisal 1000 mg. Uterine activity was significantly decreased in all patients but abolished in none. Seven women experienced almost complete relief of pain. To four of the patients, including the one who did not become pain-free after diflunisal, nifedipine 30 mg was also given. Uterine activity was abolished in all, but the patient not responding to diflunisal had persistent pains. It is suggested that diflunisal may be used for treatment of pain in primary dysmenorrhoea. Addition of nifedipine can produce a further decrease in uterine activity, but whether combined therapy may offer therapeutic advantages remains to be established.     

Electric field-mediated cell fusion

The use of electrical fields to guide, hold and fuse cells is described. The electrical fusion process consists of two steps: the cells are collected to form pearl-chains between Pt electrodes by the action of dielectrophoresis, then a brief DC pulse is applied, such that the breakdown voltage of the membranes is briefly exceeded and cell-to-cell juncture of the membranes occurs around the pore formed by the pulse. Giant fused cells (diameter up to 100 m) can be formed by the electrically mediated fusion of mouse 3T3 fibroblast cells, provided that pronase is added just before field application.     

Depside as potent inhibitor of prostaglandin biosynthesis: a new active site model for fatty acid cyclooxygenase

Forty depsides and depsidones, the esters of phenolcarboxylic acids, were examined for their inhibitory effect against prostaglandin biosynthesis with rabbit renal microsomes. 4-0-Methylcryptochlorophaeic acid was the most active inhibitor so far tested and its IC50 value was 0.34 muM. Kinetic investigation has shown that this depside acts competitively with respect to arachidonic acid as most of the non - steroidal antiinflammatory drugs. X-Ray analysis has revealed that 4-0-methylcryptochlorophaeic acid maintains its rigid conformation by forming a strong hydrogen bond between the hydroxyl and methoxyl groups. Comparison of CPK models between 4-0-methylcryptochlorophaeic acid and non-steroidal antiinflammatory drugs revealed that the carboxyl group and the two rings of these drugs are almost superimposable to those of the depside. This finding led us to propose a new active site model based on the three dimentional structure of the depside.     

The use of acridine orange for testing blood platelet integrity

Two processes are involved in the accumulation of acridine orange in human blood platelets. One follows a diffusion like kinetics and is independent of the ATP level whereas the second one can be completely abolished by ATP depletion. The acridine orange incorporation rate seems to be a suitable parameter for testing platelet integrity. It reflects very sensitively the influence of the preparation method as well as of anticoagulating substances used on the stability of platelet suspensions. The rates of acridine orange incorporation and of aggregation were measured in platelet-rich plasma and in saline suspended platelets after gel filtration, respectively, over a period of 120 min storage. Both rates are influenced to a different degree by anticoagulating agents such as citrate, heparin and EDTA. When contact with anticoagulating agents during platelet preparation is avoided, platelets show a constant acridine orange incorporation and aggregation during storage and the smallest morphological alteration.     

Reduced cell proliferation in fetal lung after maternal administration of pilocarpine: a scintillation autoradiographic study.

Fetuses were obtained on the 28th gestational day from pregnant New Zealand white rabbits treated daily, on the 24th through the 27th gestational day, with pilocarpine HCl, 5 mg/kg in saline, or saline alone. Lung fragments from these fetuses were incubated for two hours in medium containing 3H-thymidine. Scintillation autoradiography of 1-micrometer-thick sections of these fetal lungs revealed that the lung tissue from pilocarpine-treated fetuses had significantly lower labelled cell indices for both alveolar epithelial cells and interstitial cells. These results indicate that pilocarpine treatment promotes differentiation of immature cells in the fetal lung at the expense of cell proliferation.     

Multiple forms of human phosphoglycerate kinase.

Phosphoglycerate kinase was isolated by affinity chromatography from human skeletal muscle and erythrocytes. As in the tissue extracts, the purified enzyme showed in Cellogel electrophoresis one major and two minor bands with phosphoglycerate kinase activity. The multiple forms were separated by chromatography on CM-Sepharose. From the three separated forms, A, B, and C, the latter was not detectable in electrophoresis of tissue extracts or in the purified unresolved phosphoglycerate kinase. The faintest, most anodically migrating form observed in the tissue extracts could not be isolated in pure form by chromatography on CM-Sepharose. The electrophoretic mobility of the phosphoglycerate kinase forms depended strongly on the buffer systems used. The different forms had identical molecular weight, substrate affinity, and heat stability and were inhibited to the same extent by antibody. They could also not be separated by column affinity chromatography. Small differences were found in thiol group content and in the specific activity, the latter being a consequence of diminished free sulfhydryl residues. Exposure to either reductive or oxidative conditions changed the specific activity, but did not result in interconversion among the pure forms. The multiple forms probably arise as a result of epigenetic factors occurring after the primary polypeptide chain has been synthesized.     

Mechanical stabilization of guard cell protoplasts of Vicia faba

Guard cell protoplasts of Vicia faba were immobilized in cross-linked Ca-alginate. No visible morphological changes were detected under the light microscope over a period of 14 days. The entrapped cells reacted normally to changes of the external osmolarity by shrinking and swelling. Addition of the calcium complexing agent, citrate, led to dissolution of the matrix. After reequilibration with Ca ions the released cells regained their ability to swell and shrink in response to external stress. The released protoplasts could be stained with the vital dye, neutral which was accumulated in the vacuoles. It should also be noted that the protoplasts can be transported when immobilized.