Synthetic activities of mass cultures and clones of human gingival fibroblasts

The percentage of synthesis dedicated to collagen is elevated in low-density cultures of human gingival fibroblasts, as is per-cell total protein synthetic activity and glycosaminoglycan accumulation. These observations can be explained, in part, by a decrease in membrane transport of precursor substance in high-density cultures. Synthetic activity by human fibroblasts can be reliably assayed in vitro using as few as 500 cells sparsely seeded. Such low-cell number assay is essential for study of single-cell clones, where replicative life span is limited.     

Effects of the antiprotease Trasylol® on peripheral blood leucocytes

Summary Binding of the antiprotease Trasylol^® to human peripheral blood lymphocytes and polymorphonuclear leucocytes (PMNs) was demonstrated at the ultrastructural level using an indirect immunoperoxidase technique. This also revealed endocytosis of membrane bound Trasylol by PMNs. Trasylol inhibited PHA- and Con A-induced lymphocyte stimulation, and was cytotoxic to unstimulated cells.One of us (D.J.T.) is supported by Bayer Pharmaceuticals Ltd., from whom we also acknowledge a generous supply of Trasylol.     

Effects of the antiprotease Trasylol on peripheral blood leucocytes.

Binding of the antiprotease Trasylol to human peripheral blood lymphocytes and polymorphonuclear leucocytes (PMNs) was demonstrated at the ultrastructural level using an indirect immunoperoxidase technique. This also revealed endocytosis of membrane bound Trasylol by PMNs. Trasylol inhibited PHA- and ConA-induced lymphocyte stimulation, and was cytotoxic to unstimulated cells.     

Capping of concanavalin A- or ricin-binding sites does not influence phagocytosis in polymorphonuclear leukocytes

Summary Rabbit polymorphonuclear leukocytes (PMNs) were capped with territin-conjugated concanavalin A or ricin, and then allowed to phagocytose yeast cells. Phagocytic activity and lectin distribution were determined by ultrastructural morphometry. Capped PMNs were found to phagocytose as efficiently as control PMNs, and always to ingest the particles with a lectin-free portion of their plasma membrane. This clearly indicates that concanavalin A- and ricin-binding sites of the PMN membrane are not involved in the recognition and phagocytosis of yeast particles.     

Stimulation of macromolecular synthesis by endotoxin-treated 3T6 fibroblasts

The interaction of endotoxin with cultured fibroblasts resulted in a depression of cellular proliferation and an increased synthesis of macromolecules, namely collagenous and non-collagenous proteins. The collagen salt-soluble fraction was increased at the expense of the insoluble fraction, and both the salt-soluble fraction and collagen secreted into the medium was underhydroxylated.     

Stimulation of macromolecular synthesis by endotoxin-treated 3T6 fibroblasts

Summary The interaction of endotoxin with cultured fibroblasts resulted in a depression of cellular proliferation and an increased synthesis of macromolecules, namely collagenous and non-collagenous proteins. The collagen salt-soluble fraction was increased at the expense of the insoluble fraction, and both the salt-soluble fraction and collagen secreted into the medium was underhydroxylated.     

Differential distribution of TASK-1, TASK-2 and TASK-3 immunoreactivities in the rat and human cerebellum

In this work, the distributions of some acid-sensitive two-pore-domain K⁺ channels (TASK-1, TASK-2 and TASK-3) were investigated in the rat and human cerebellum. Astrocytes situated in rat cerebellar tissue sections were positive for TASK-2 channels. Purkinje cells were strongly stained and granule cells and astrocytes were moderately positive for TASK-3. Astrocytes isolated from the hippocampus, cerebellum and cochlear nucleus expressed TASK channels in a primary tissue culture. Our results suggest that TASK channel expression may be significant in the endoplasmic reticulum of the astrocytes. The human cerebellum showed weak TASK-2 immunolabelling. The pia mater, astrocytes, Purkinje and granule cells demonstrated strong TASK-1 and TASK-3 positivities. The TASK-3 labelling was stronger in general, but it was particularly intense in the Purkinje cells and pia mater.Received 25 February 2004; received after revision 19 April 2004; accepted 28 April 2004     

The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata

Summary Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25–2.0 Mrads is optimal for cell growth.     

The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata

Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25-2.0 Mrads is optimal for cell growth.     

Evaluation of the ability of intact platelets to accumulate acridine orange

A new approach to the evaluation of the uptake of fluorescent probes by intact cells is described. Acridine orange (AO) was used because it can be selectively accumulated by serotonin-containing granules of platelets. Analysis of the fluorescence signal allows the estimation of the relative volume of the granules and the equilibrium coefficients for AO transport across the cytoplasm and granule membranes. The following results were obtained for human and rabbit platelets: the relative volumes of the granules were 14 +/- 1% and 29 +/- 2%, the ratios of intragranular-extracellular probe concentration were 2260 +/- 382 and 30,000 +/- 5550, and the cytoplasm-extracellular medium concentration ratios were 375 +/- 60 and 225 +/- 60, respectively.     

Collagen of the calcified layer of human articular cartilage

The distribution patterns of collagen types I, II and III were studied using immunofluorescent staining techniques in human articular cartilage, including the calcified layer. Tissue taken from femoral heads was stained with the appropriate antiserum. Adjacent sections were stained with von Kossa or Alizarin red to determine the distribution of calcium salts. Results indicate that endochondral ossification at this site occurs by calcium being deposited initially within a matrix of type II collagen.     

Collagen of the calcified layer of human articular cartilage

Summary The distribution patterns of collagen types I, II and III were studied using immunofluorescent staining techniques in human articular cartilage, including the calcified layer. Tissue taken from femoral heads was stained with the appropriate antiserum. Adjacent sections were stained with von Kossa or Alizarin red to determine the distribution of calcium salts. Results indicate that endochondral ossification at this site occurs by calcium being deposited initially within a matrix of type II collagen.     

The adhesion receptor GPR56 is activated by extracellular matrix collagen III to improve β-cell function

Aims

G-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on β-cell function.

Methods

GPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on β-cell proliferation, apoptosis, intracellular calcium ([Ca²⁺]_i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56^?/? mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56^?/? mice.

Results

Immunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet β-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected β-cells from cytokine-induced apoptosis, triggered increases in [Ca²⁺]_i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56^?/? islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation.

Conclusion

We have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.

     

Differential roles of multiple adhesion molecules in cell migration: granule cell migration in cerebellum

The migration of cerebellar granule cells from the external granular layer to the internal granular layer is mediated by the radical Bergmann glial fiber. Recent works have shown that cell adhesion molecules, extra-cellular matrix proteins and proteolytic enzymes or their activators are involved in this process. Immuno-localization studies showed differential temporal and spatial expression patterns of different adhesion molecules, their isoforms, and post-translational modification during different stages of granule cell migration. Functional perturbation experiments using cerebellar explant cultures demonstrated that several adhesion molecules as well as plasminogen activator are involved in granule cell migration and are required in different stages. Other systems used to study granule cell migration including dissociated microwell cultures and granule cell deficient mouse mutants are discussed in the context of adhesion molecules. The results accumulated so far suggest that the migration of granule cells is a complex process in which the cooperation of a group of molecules with different functions, some for adhesion some for de-adhesion, are required to fulfill the different needs during the migratory course.     

Isolation of a growth-stimulating agent from human skin fibroblast cultures

Summary Cell-free supernatants were harvested from cultures of human skin fibroblasts, were applied on to DEAE-cellulose columns, and the first fraction eluted with phosphate-buffered saline contained the growth-stimulating agent. The eluted fraction was then passed through a series of amicon membranes. After passing through PM-10, the filtrate stimulated growth of bovine vascular endothelial, canine myocardial, and human mammary carcinoma cells.     

Differential roles of multiple adhesion molecules in cell migration: Granule cell migration in cerebellum

Summary The migration of cerebellar granule cells from the external granular layer to the internal granular layer is mediated by the radial Bergmann glial fiber. Recent works have shown that cell adhesion molecules, extra-cellular matrix proteins and proteolytic enzymes or their activators are involved in this process. Immuno-localization studies showed differential temporal and spatial expression patterns of different adhesion molecules, their isoforms, and post-translational modification during different stages of granule cell migration. Functional perturbation experiments using cerebellar explant cultures demonstrated that several adhesion molecules as well as plasminogen activator are involved in granule cell migration and are required in different stages. Other systems used to study granule cell migration including dissociated microwell cultures and granule cell deficient mouse mutants are discussed in the context of adhesion molecules. The results accumulated so far suggest that the migration of granule cells is a complex process in which the cooperation of a group of molecules with different functions, some for adhesion some for de-adhesion, are required to fulfill the different needs during the migratory course.     

Collagen synthesis of cultured fibroblast from Werner's syndromes of premature aging

Summary The difference of collagen producibility between 2 groups of skin fibroblasts from patients with Werner's syndrome with skin change and with normal skin, and the difference of collagen accumulation to cell layer between skin fibroblast from Werner's syndrome and controls were studied.Acknowledgments. We are grateful to Dr M. Ohkido and Dr I. Matsuo of the Department of Dermatology of Tokai University for their supply of materials and generous advice and Mr K. Takeichi of the Department of Pathology of Tokai University for his technical assistance.     

Stabilisation of collagen by betel nut polyphenols as a mechanism in oral submucous fibrosis

Treatment of reconstituted collagen fibrils and pieces of rat dermis with the crude extract, purified tannins or (+)-catechin from betel nut (Areca catechu) increases their resistance to both human and bacterial collagenases in a concentration-dependent manner. These tanning agents may stabilise collagen in vivo following damage to the oral epithelium, and promote the sub-epithelial fibrosis which occurs in betel nut chewers.     

Stabilisation of collagen by betel nut polyphenols as a mechanism in oral submucous fibrosis

Summary Treatment of reconstituted collagen fibrils and pieces of rat dermis with the crude extract, purified tannins or (+)-catechin from betel nut (Areca catechu) increases their resistance to both human and bacterial collagenases in a concentration-dependent manner. These tanning agents may stabilise collagen in vivo following damage to the oral epithelium and promote the sub-epithelial fibrosis which occurs in betel nut chewers.     

P2X7, NMDA and BDNF receptors converge on GSK3 phosphorylation and cooperate to promote survival in cerebellar granule neurons

Glycogen synthase kinase-3 (GSK3) is a key player in the regulation of neuronal survival. Herein, we report evidence of an interaction between P2X7 receptors with NMDA and BDNF receptors at the level of GSK3 signalling and neuroprotection. The activation of these receptors in granule neurons led to a sustained pattern of GSK3 phosphorylation that was mainly PKC-dependent. BDNF was the most potent at inducing GSK3 phosphorylation, which was also dependent on PI3K. The P2X7 agonist, BzATP, exhibited additive effects with both NMDA and BDNF to rescue granule neurons from cell death induced by PI3K inhibition. This survival effect was mediated by the PKC-dependent GSK3 pathway. In addition, ERK1/2 proteins were also involved in BDNF protective effect. These results show the function of ATP in amplifying neuroprotective actions of glutamate and neurotrophins, and support the role of GSK3 as an important convergence point for these survival promoting factors in granule neurons.