Morphological studies on endocytosis of chondroitin sulphate proteoglycan by rat liver endothelial cells

Summary Endocytosis via the hyaluronic acid/chondroitin sulphate receptor of rat liver endothelial cells was studied ultrastructurally, by use of a probe consisting of chondroitin sulphate proteoglycan attached to 15-nm gold particles. The probe bound to the surface of the cells exclusively in coated regions of the plasma membrane. Internalization at 37° C took place in less than one minute during which time interval the bound probe was transferred to coated vesicles. Further transfer to lysosomes was delayed in association with an accumulation of probes in a prelysosomal compartment consisting of large vacuoles in which probes lined the inner aspect of the membrane. Transport to lysosomes occurred only after a lag phase of at least 40–60 min at 37° C.Abbreviations CS chondroitin sulphate - CSPG chondroitin sulphate proteoglycan - CSPG-Au CSPG-gold complex - EM electronmicroscopical or electron microscopy - HA hyaluronic acid - KC Kuppfer cells - LEC liver endothelial cells - PC parenchymal cells - RES reticuloendothelial system     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells

The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.     

Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor

The binding of iodinated basic fibroblast growth factor (bFGF) to low-density heparan sulfate proteoglycan purified from the Engelbreth Holm Swarm (EHS) sarcoma was investigated using different techniques. The tumor clearly contained bFGF, the level being comparable to that found in other tissues such as human or bovine brain. 125I bFGF strongly bound to the basement membrane-like matrix of EHS frozen sections as revealed by autoradiography. Iodinated bFGF bound to purified heparan sulfate proteoglycan but not to laminin or collagen type IV, three components isolated from the same tumor. In contrast, acidic fibroblast growth factor (aFGF) displayed negligible binding to heparan sulfate proteoglycan. Binding of bFGF to frozen sections and to purified proteoglycan could be strongly inhibited by heparin and was displaced by an excess of unlabeled factor and completely suppressed after heparitinase and heparinase treatments. Binding was a function of the salt concentration and was abolished at 0.6 M NaCl. Scatchard analysis indicated the affinity site had a Kd of about 30 nM, a value 10-15 higher than that recently reported by Moscatelli (J. Cell. Physiol., 131:123-130, 1987) in the case of the low-affinity binding sites present on the surface of baby hamster kidney (BHK) cells.     

Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation

Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.     

Alport syndrome: a genetic study of 31 families

Thirty one families with Alport syndrome including 3 families with associated syndromes were studied. The location of the COL4A5 gene, responsible for the Alport syndrome, was determined by linkage analysis with eight probes of the Xq arm and by a radiation hybrid panel. Concordant data indicated the localization of the Alport gene between DXS17 and DXS11. Four deletions and one single base mutation of the COL4A5 gene were detected. Homogeneity tests failed to show any evidence of genetic heterogeneity superimposed on clinical heterogeneity for ophthalmic signs and end-stage renal disease age.     

Recognition of HLA-B27 by mouse cytotoxic T-cell clones: a transgenic mouse

As a basis for the characterization of mouse T cells involved in the recognition of xenogeneic HLA molecules, a panel of HLA-B27-reactive cytotoxic T-cell clones was generated upon stimulation by cells from HLA-B27-transgenic mice. The HLA-B27-induced T-cell response was found to comprise two categories of clones: some recognizing HLA-B27 independent of H-2 molecules expressed by the target cells (unrestricted clones), others recognizing HLA-B27 in an H-2 restricted manner. The unrestricted clones exhibited diverse specificities, as judged from their various cross-reactivities with other xenogeneic (HLA) or allogeneic (H-2) molecules. In addition, although most of the unrestricted clones were able to react with both mouse and human HLA-B27-transgenic mice. The HLA-B27 induced T-cell which reacted only with HLA-B27-positive mouse, and not human cells. These findings illustrate that both H-2-restricted and unrestricted T cells with diverse species contribute to HLA-B27-xenorecognition.     

F.9 embryonal carcinoma cell calcitonin autocrine system: correlation between immunoreactive calcitonin secretion and calcitonin receptor number

Mouse teratocarcinoma cells in culture offer an in vitro system to study the initial steps of embryogenesis. It has been suggested that, at such early stages, cell functions are regulated by an autocrine process in which embryonic cells produce factors that in turn act on themselves. F.9 cells possess specific membrane receptors for calcitonin (CT) (120 fmol/mg of protein, Ka, = 3.5 X 10(8) M-1). These cells produce CT detected by heterologous radioimmunoassay in serum-free culture-conditioned medium (75 pg/10(7) cells/12 h). When F.9 cells are incubated in serum-free medium, CT binding and secretion concomitantly drop by 50% within the first 2 h, then increase progressively to an upper plateau after the sixth hour. Preincubation with 10(-6) M CT leads to disappearance of CT receptors and CT secretion in the culture medium up to 6 h. Avoiding accumulation of CT in the medium by a continuous flow rate for 6 h leads to a progressive decrease of CT receptors. In addition, retinoic acid treatment of cells induces a parallel progressive decrease of CT receptor number and of total CT synthesis. These results suggest a reciprocal regulation of CT receptors and CT secretion, or a close relationship between their regulations.