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.     

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.     

Mechanism of copper transport from plasma to hepatocytes

The effects of plasma components on the kinetics of copper transport by rat hepatocytes were examined in an attempt to determine how copper is mobilized from plasma for uptake by the liver. Specific protein-facilitated transport was indicated by saturation kinetics, competition by related substrates, and similar kinetic parameters for uptake and efflux. For copper uptake, Km = 11 +/- 0.6 microM and Vmax = 2.7 +/- 0.6 nmol Cu/(min X mg protein). Zinc is a competitive inhibitor of copper uptake, and copper competes for zinc uptake. Copper efflux from preloaded cells is biphasic. The kinetic parameters for the initial rapid phase are similar to the parameters for uptake. Copper transport by hepatocytes is strictly passive. A variety of metabolic inhibitors have no effect on uptake and initial rates are solely dependent on extracellular-intracellular concentration gradients. Albumin markedly inhibits copper uptake by a substrate removal mechanism, and histidine facilitates albumin-inhibited copper uptake. The active species that delivers copper to hepatocytes under conditions of excess albumin and excess histidine is the His2Cu complex. Experiments with [3H]His2 64Cu showed that the transported species is free ionic copper. The kinetic parameters of copper transport by hepatocytes isolated from the brindled mouse model of Menkes' disease are normal. However, these cells show a decreased capacity to accumulate copper on prolonged incubation. An intracellular metabolic defect seems to be involved.     

Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes

Experiments were performed to probe the role of exocytotic and endocytotic processes in the regulation of the human granulocyte O-2-generating system. Analytical subcellular fractionation studies indicated that 25-30% of the total cellular b-cytochrome and 8-10% of the flavin co-sedimented with plasma membrane markers, irrespective of stimulation of the cells by the chemoattractants N-formyl-Met-Leu-Phe (FMLP) or C5a. Phorbol myristate acetate stimulation resulted in significant translocation of b-cytochrome but not flavin from the specific granule/Golgi to the plasma membrane-enriched fractions. These results indicated that approximately 3.1 X 10(5) flavin and 0.8-1 X 10(6) b-cytochrome molecules are present in the plasma membrane of an isolated unstimulated human granulocyte and that these levels are invariant upon stimulation with chemoattractants. Maximal instantaneous rates of O-2 generation by cells in these preparations, however, were equivalent for all the stimuli. Since stimulation of granulocytes by phorbol myristate acetate, FMLP, or C5a results in exocytosis and/or endocytosis, then the role of these processes in regulating stimulated O-2 production by controlling the content of plasma membrane redox enzymes is questionable. This conclusion was supported by observations made with cytoplasts, which do not have an intracellular reserve of granules. Cytoplasts prepared from granulocytes produced O-2 at equivalent rates as their parent cells on a per unit surface area basis. These results suggest: 1) that stimulation of granulocytes with chemotactic peptides leads to full generation of O-2 at the cell surface without exocytotic recruitment of additional b-cytochrome and flavoprotein from the cytoplasmic compartment; 2) that these redox enzymes are not internalized along with chemoattractant receptors; and 3) that traffic of these redox enzymes between endo- and plasma membranes is not involved in the regulation of O-2 production in suspensions of human granulocytes stimulated by chemoattractants.     

Immunoregulation in disseminated murine histoplasmosis: disturbances in the production of interleukins 1 and 2

Production of IL 1 and IL 2 by splenocytes from C57BL/6 mice was measured at wk 1, 3, 8, and 14 after i.v. inoculation with 6 X 10(5) Histoplasma capsulatum (Hc) yeasts. As compared with age-matched controls, IL 1 production by splenocytes from Hc-infected mice was reduced severely at wk 1 and 3 of infection, greater than normal at wk 8, and within normal range at wk 14. IL 2 production was also reduced at wk 1 and 3 of infection; it was normal at wk 8 and was elevated at wk 14. Indomethacin and catalase failed to restore IL 1 production by splenocytes from infected mice, and exogenous IL 1 did not augment IL 2 production by these cells. A factor capable of suppressing the activity of IL 2 was detected in supernatants of concanavalin A-stimulated splenocytes from infected animals at wk 1 and 3 of infection, respectively. No factor capable of suppressing IL 1 activity was detected. Thus, the deficits of cell-mediated immunity in mice with systemic Hc infection may derive, in part, from impaired amplification of the immune response consequent to abnormal generation of IL 1 and IL 2.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

Definition of three resolvase binding sites at the res loci of Tn21 and Tn1721.

The dual functions of resolvase, site-specific recombination and the regulation of its own expression from tnpR, both require the interaction of this protein with the DNA sequence at res, but the specificity of this interaction differs between groups of Tn3-like elements. In this study, DNA fragments that contained res from Tn21 or Tn1721 were subjected to either cleavage by DNase I or methylation by dimethyl sulphate in the presence of the purified resolvase from Tn21 or Tn1721. These experiments showed that each resolvase bound to the same three sites (I, II and III) within res from Tn1721 and to an equivalent series of three sites on Tn21: the differences in the amino acid sequences of the two proteins did not affect their interaction with either DNA. The DNA sequences at each site had some similarities and, in conjunction with data from the related transposon Tn501, a consensus was established. However, the three sites are functionally distinct: site I (tnpR-distal) spans the recombination cross-over point and sites II and III (tnpR-proximal) overlap the promoter of tnpR. The binding sites on these transposons were compared with those in the gamma delta/Tn3 system: the similarities between the two groups of transposons revealed some general features of resolvase-DNA interactions while the differences in fine structure elucidated the specificity of each resolvase.     

Inhibition of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation).

The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase.