Alloantigen presentation by B cells: two types of alloreactive T cell hybridomas, B cell-reactive and B cell-nonreactive

T cell-depleted, Sephadex G-10-passed unstimulated splenic B cells from C57BL/6 mice stimulated splenic T cells from CKB mice to produce IL 2 and to proliferate. The stimulatory ability of the unstimulated B cells was eliminated by 4000 rad irradiation of the unstimulated stimulator B cells. LPS-activated B cells could stimulate responder T cells more efficiently than unstimulated B cells. For further analysis of allostimulation by B cells, we established a series of alloreactive T cell hybridomas. Forty-five percent of these alloreactive T cell hybridomas could be stimulated to produce IL 2 by either macrophage-dendritic cells or unstimulated B cells. Fifty-five percent of these alloreactive T cell hybridomas could be stimulated by macrophage-dendritic cells but not by unstimulated B cells. T cell hybridomas that were not reactive with unstimulated B cells were also nonreactive to LPS-activated B cells. Analysis of two representative I-Ab-reactive T cell hybridoma clones, B cell-reactive clone CB-11.4 and B cell-nonreactive clone HTB-9.3, revealed again that the stimulatory ability of unstimulated B cells was sensitive to 4000 rad irradiation in the activation of CB-11.4 clone and that CB-11.4 could be stimulated more efficiently by LPS-activated B cells than by unstimulated B cells, but HTB-9.3 could not be stimulated by LPS-activated B cells. Thus, there may be two distinct types of T cells in the alloreaction: B-cell-reactive and B cell-nonreactive.     

Nitroarenes in Suimon River sediment

Mutagenic activity was observed in sediments of the Suimon River bed with and without S9 mix. The direct-acting mutagens in the sediment were investigated. The sediment was extracted with methanol and fractionated on a Silica gel column. The benzene fraction from the Silica gel column exhibited mutagenic activity without S9 mix in strain TA98, while it failed to show mutagenic activity in nitroreductase-deficient strain TA98NR. This observation led to the suspicion that nitro compounds were the direct-acting mutagens of these samples. The benzene fraction was treated by heptafluorobutyric anhydride (HFBA) and investigated with gas chromatography equipped with an electron capture detector (GC-ECD). 2-Nitrofluorene, 4,4'-dinitrobiphenyl, 2,7-dinitrofluorene and 1-nitropyrene were detected and measured quantitatively. The mutagenic activity of a mixture of these compounds was compared with that of the original fraction and the direct-acting mutagenicity of Suimon River sediment can be explained by these nitroarenes, especially 1-nitropyrene.     

Xyloglucan in the Cell Walls of Suspension-Cultured Rice Cells

The xyloglucan present in the 24% KOH extract of the cell wallsof suspension-cultured rice cells was characterized by fragmentationanalysis with Trichoderma viride cellulase and Aspergillus oryzaeß-D-glucosidase. The xyloglucan is composed mainlyof the following oligosaccharide units: Results showed that the xyloglucan of suspension-cultured ricecells is more extensively branched than is that of rice seedlings.Another structural characteristic of the former xyloglucan isthe presence of D-galactosyl-D-xylosyl side chains that arenot found in the latter. (Received June 15, 1984; Accepted January 11, 1985)     

Distribution of vasopressin and oxytocin in rat brain

Arginine-vasopressin and oxytocin in various portions of rat brain were determined by radioimmunoassays. The hormones were extracted from tissue samples into 0.1 N HCl and then purified partially with acetone-petroleum ether extraction. The non-equilibration method was used for the assays. In this method recovery rates of arginine-vasopressin and oxytocin were 73.0 +/- 4.4% and 75.0 +/- 3.8%, respectively. Sensitivities of the assays were 1 pg of arginine-vasopressin and 0.75 pg (0.3 microU) of oxytocin per assay tube. The higher concentrations of arginine-vasopressin and oxytocin were confirmed in the hypothalamo-neurohypophyseal system, where these hormones are synthesized, transported and stored. Relatively high concentrations of these hormones, especially oxytocin, were detected in spinal cord. Amygdala, hippocampus, limbic forebrain and pineal body contained a certain amount of arginine-vasopressin (2-20 pg/mg protein). Oxytocin (1-7 pg/mg protein) was also detected in amygdala, pons and medulla oblongata, pineal body and midbrain. The low concentrations of these hormones were also found in cerebral cortex and cerebellum.     

Kinetic studies on surface-mediated activation of bovine factor XII and prekallikrein. Effects of kaolin and high-Mr kininogen on the activation reactions

The kaolin-mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high-Mr kininogen and kaolin surface on the kinetics of these activation reactions were studied. High-Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high-Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 X 10(7) s-1 X M-1, respectively. The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high-Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 microM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 X 10(4) to 1.1 X 10(6) s-1 X M-1). These results suggest that high-Mr kininogen accelerates the surface-mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.     

Ovine prolactin potentiates the action of adrenocorticotropic hormone on the secretion of dehydroepiandrosterone sulfate and dehydroepiandrosterone from cultured bovine adrenal cells

To determine the direct effect of prolactin on adrenal androgen secretion, the daily secretions of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), androstenedione and cortisol were determined in monolayer culture of bovine adrenal cells in the presence or absence of adrenocorticotropic hormone (ACTH) and/or prolactin. In the absence of ACTH ovine prolactin alone had no effect on steroid secretion during seven-day culture. Ovine prolactin, when administered in combination with ACTH, significantly potentiated the stimulatory effect of ACTH on DHEA-S and DHEA but not androstenedione secretion on the seventh day in culture. On the first day in culture prolactin showed no synergistic effect with ACTH on DHEA and DHEA-S secretion, although ACTH significantly increased DHEA and cortisol secretion. DHEA-S secretion increased as a function of prolactin concentration in the presence of ACTH. These results indicated that long-term treatment by ovine prolactin with ACTH caused the increase in adrenal androgen secretion from bovine adrenal cells. The site of action of prolactin was suggested to be the partial inhibition of adrenal 3 beta-hydroxysteroid dehydrogenase by the result of increases in DHEA-S and DHEA but not androstenedione secretion.     

Rat plasma high-molecular-weight kininogen. A simple method for purification and its characterization

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.     

Central inhibitory action of TRH on prolactin secretion in the rat

Intravenous (iv) injection of FK33-824 [( D-Ala2, MePhe4, Met-(O)5-ol]-enkephalin, 8 and 16 nmole/100 g body wt), a potent Met5-enkephalin analog, and domperidone (1.2, 2.4, and 24 nmole/100 g body wt), a dopamine antagonist, resulted in a dose-related increase in plasma prolactin (PRL) levels in urethane-anesthetized male rats. PRL release induced by FK33-824 (16 nmole/100 g body wt, iv) was inhibited by intraventricular (icv) injection of TRH (0.6 nmole/rat). DN-1417 (gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate, 0.6 nmole/rat, icv), a TRH analog, also blunted PRL release induced by FK33-824. PRL release induced by a smaller dose of domperidone (1.2 nmole/100 g body wt, iv) was blunted by TRH and DN-1417, whereas both peptides failed to suppress elevated PRL levels induced by larger doses of domperidone. These results suggest that TRH not only stimulates PRL secretion by acting directly at the pituitary, but has an inhibitory action on PRL release through activation of the central dopaminergic mechanism.