Relationship between T cell receptors and antigen-binding factors. II. Common antigenic determinants and epitope recognition shared by T cell receptors and antigen-binding factors

The T cell hybridomas 231F1 and 12H5 constitutively secrete glycosylation-inhibiting factor (GIF) and glycosylation-enhancing factor (GEF), respectively, which lack affinity for OVA-coupled Sepharose. When the 231F1 and 12H5 cells were stimulated by OVA-pulsed syngeneic macrophages, however, GIF and GEF produced by the cells had affinity for OVA. Both the OVA-binding GIF from the 231F1 cells and OVA-binding GEF from the 12H5 cells bound to a mAb against TCR-alpha beta and a mAb against TCR-alpha, suggesting a serologic relationship between TCR and OVA-binding factors. However, the OVA-binding GIF and GEF bound to mAb 14-12 and 14-30, respectively. Because these mAb do not bind TcR alpha beta-chains, it appears that the Ag-binding factors are different from TCR itself. The OVA-binding factors from both 12H5 cells and 231F1 cells do not bind to urea-denatured OVA. The binding of the factors to OVA Sepharose was inhibited by a peptide corresponding to residues 307-317 (P307-317) in the native OVA, but not by the peptide corresponding to residues 323-339 (P323-339). Furthermore, the OVA-binding factors bound to P306-319-coupled Sepharose but not to P323-339-coupled Sepharose, and were recovered by elution of the former Sepharose at acid pH. The binding of OVA to anti-OVA antibodies was not inhibited by either peptide. Inasmuch as the 231F1 cells and 12H5 cells can be stimulated by P307-317 in the context of a MHC product, it appears that the Ag-binding factors and TCR-alpha beta on the cell sources of the factors may recognize the same epitope in the OVA molecules. The results also showed that Ag-binding factors and antibodies recognize distinct epitopes in the Ag molecules.     

Glycosaminoglycan Composition of PC 12 Pheochromocytoma Cells: A Comparison with PC12D Cells, a New Subline of PC12 Cells

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

Turnover of proteoglycans by chick lens epithelial cells in cell culture and intact lens

Chick lens epithelial cells were cultured on plastic and type IV collagen substrata, and the confluent cultures were labeled continuously with [35S]sulfate for 20 h. Intact lenses were also labeled in the same way. 35S-Proteoglycans isolated from those cultures were compared for their molecular sizes and glycosaminoglycan compositions. The results have shown that: 1) Proteoglycans synthesized by cells on type IV collagen were significantly smaller than those by cells on plastic. 2) Proteoglycans of intact lens showed a broad distribution of molecular size and contained a high proportion of chondroitin sulfate in the medium fraction compared to those of the two cell cultures. In order to explain such differences between proteoglycans from cultures, label-chase experiments with [35S]sulfate were done for proteoglycans synthesized. 35S-Proteoglycans isolated at each chase time 0, 2.5, and 17 h) were compared and the following results were found: 1) The cell layers of both "plastic" and "type IV collagen" cultures contained glycosaminoglycan species predominantly at each chase time rather than proteoglycans. 2) Changes in the glycosaminoglycan compositions of medium fractions of cell cultures were observed during the chase period; in medium of the "plastic" culture, proteoheparan sulfate increased with chase time, whereas in medium of the "type IV collagen" culture, chondroitin sulfate glycosaminoglycan (not proteoglycan) increased with chase time. 3) In intact lens culture, lens capsule fraction at every chase time contained a proteoglycan unique in molecular size, which was not found in cell culture fractions. 4) All fractions from intact lens cultures contained a higher content of chondroitin sulfate at every chase time than the respective fractions from cell cultures. These results suggest that adhesion of the cells to type IV collagen or lens capsule influences the degradation and secretion of proteoglycans. In addition, they can account partially for the above-described differences in molecular sizes and glycosaminoglycan compositions between 35S-proteoglycans from various cultures continuously labeled with [35S]sulfate.     

Plasmodium berghei: Isolation and maintenance of an irradiation attenuated strain in the nude mouse

An attenuated strain of malaria causing limited parasitemia in mice was derived from a highly virulent strain of Plasmodium berghei (NK65) which produced 100% lethality in mice. A pool of mouse blood infected with the original highly virulent P. berghei was exposed to 40 Krad irradiation and parasites were inoculated into nude mice as well as into thymus competent normal littermates. Thymus competent mice showed no parasitemia, while one out of the five nude mice inoculated with the irradiated parasites developed a slow and progressive parasitemia. These parasites induced a self-limiting parasitemia in thymus competent mice, even when a large inoculum was administered. Maintenance of the low virulence strain required passage through nude mice. After 50 passages at two weekly intervals, reversion to virulence did not occur. A single vaccination with the attenuated strain induced immunity in mice against a challenge inoculation with the original virulent strain. Specific IgG persisted at high titer for more than 9 weeks in mice receiving a single inoculation of the attenuated strain.     

Solubilization of adenylate cyclase of brain membranes by lipid peroxidation

Adenylate cyclase in the membrane fractions of bovine and rat brains, but not in rat liver plasma membranes, was solubilized by treatment with Fe²⁺ (10 μM) plus dithiothreitol (5 mM). Solubilization of the enzyme by these agents was completely prevented by simultaneous addition of N,N′-diphenyl-p-phenylenediamine (DPPD), an inhibitor of lipid peroxidation. Ascorbic acid also solubilized the enzyme from the brain membranes. Lipid peroxidation of the brain membranes was characterized by a selective loss of phosphatidylethanolamine. Solubilization of membrane-bound enzymes by Fe²⁺ plus dithiothreitol was not specific for adenylate cyclase, because phosphodiesterase, thiaminediphosphatase and many other proteins were also solubilized. Solubilized adenylate cyclase had a high specific activity and was not activated by either NaF, 5′-guanylyl imidodiphosphate (Gpp[NH]p) or calmodulin. These results suggested that lipid peroxidation of the brain membranes significantly solubilized adenylate cyclase of high specific activity.     

Properties of Detergent-Dispersed Adenylate Cyclase from Cerebral Cortex. Presence of an Inhibitor Protein

Abstract: Adenylate cyclase was solubilized from washed paniculate fraction of rabbit cerebral cortex with the nonionic detergent Lubrol 12A9 and subjected to either gel filtration on Ultrogel AcA 34 or chromatography on DEAE Bio-Gel A. By both procedures the enzyme was resolved into two components, one insensitive to guanyl 5'-yl imidodiphosphate [Gpp(NH)p] and NaF but stimulated by Ca²⁺ and calmodulin, and another that was sensitive to Gpp(NH)p and NaF but relatively insensitive to Ca²⁺ and calmodulin. The data support the possibility that two independent forms of adenylate cyclase exist in cerebral cortex, one regulated by guanine nucleotide regulatory protein and another by Ca²⁺-calmodulin. Fractions containing the guanylnucleotide-sensitive activity were found to contain a factor that inhibited basal and Ca²⁺-stimulated adenylate cyclase in the Ca²⁺-sensitive fraction. The inhibitor was inactivated by heating at 60°C and by incubation with trypsin. Inhibition was not time-dependent, and it was not due to destruction of cAMP by phosphodiesterase or of ATP by ATPase. Inhibitory action was not reversed by calmodulin and therefore it does not appear to be a calmodulin binding protein. Sucrose density gradient sedimentation indicated a sedimentation coefficient of 4S for the inhibitor; by this technique it co-sedimented with the adenylate cyclase sensitive to Gpp(NH)p and NaF.     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

Cysteine Sulfinic Acid in the Central Nervous System: Specific Binding of [35S]Cysteic Acid to Cortical Synaptic Membranes-An Investigation of Possible Binding Sites for Cysteine Sulfinic Acid

Abstract: Specific binding sites for cysteine sulfinic acid, an excitatory amino acid, in crude synaptic membrane fractions of rat cerebral cortex were examined, using L-[³⁵S]cysteic acid as a ligand. Two specific binding systems of [³⁵S] cystec acid were found, one Na⁺-dependent and the other Na⁺-independent. The Na⁺-independent specific binding of [³⁵S]Cysteic acid was saturable, with a K_d of 474 n M and B_max of 3.29 pmol/mg protein. The binding was optimal at pH 7.4 and at 37°C. Treatment of the membranes with proteases, concanavalin A, or Triton X-100 markedly reduced the binding. Of various compounds related to cysteic acid, L-cysteine sulfinic acid was the most effective competitor of this binding. These results indicate the existence of an Na⁺-independent specific binding site for cysteic acid in the synaptic membrane of rat cerebral cortex, which may be different from that for glutamate. Possible involvement of cysteine sulfinic acid as an endogenous ligand for this binding site is discussed.