Analysis of two distinct B cell activation pathways mediated by a monoclonal T helper cell. I. MHC-restricted activation of B cells by an IL 2-dependent pathway

The present study was carried out to determine whether the MHC-restricted and MHC-unrestricted B cell activation pathways mediated by a single cloned Th cell are separable, and whether these two pathways are mediated by distinct mechanisms. It was demonstrated that the two B cell activating functions of a single cloned Th cell could be separated by their sensitivity to irradiation. It was shown that MHC-restricted B cell activation is mediated by a radiosensitive Th cell function, whereas MHC-unrestricted B cell activation is mediated by a radioresistant function of the same Th cell. In addition, it was shown that recombinant IL 2 can restore or replace the radiosensitive component of MHC-restricted cognate helper function.     

Uncoupling of Rat Cerebral Cortical α2-Adrenoceptors from GTP-Binding Proteins by N-Ethylmaleimide

Pretreatment of membranes from rat cerebral cortex with N-ethylmaleimide (NEM) decreased [3H]-clonidine binding in a concentration-dependent manner. The Bmax values of high-affinity sites for [3H]clonidine were reduced by 50 microM NEM treatment. Treatment with 500 microM NEM diminished the sum of Bmax of both high- and low-affinity components. GTP, Na+, and Mn2+ exerted little effect on [3H]clonidine binding in NEM-treated membranes. The addition of purified GTP-binding proteins caused an increase in the binding to the membranes pretreated with 50 microM NEM, but did not increase [3H]-clonidine binding in membranes treated with 500 microM NEM. In contrast, NEM pretreatment inhibited islet activating protein (IAP)-catalyzed ADP ribosylation of membrane-bound (41,000-dalton) and purified (39,000/41,000-dalton) GTP-binding proteins. From these results, it is suggested that two or three categories of essential sulfhydryl groups are involved in the coupling between agonist, alpha 2-adrenoceptor, and GTP-binding protein. One is a highly sensitive site to NEM (a concentration range of 1-50 microM), which is probably a cysteine residue, IAP-catalyzed ADP-ribosylating site on the alpha-subunit of GTP-binding protein. Other sites have low sensitivity to NEM (a concentration range of 0.1-1 mM), and are the binding domain of agonist and/or the coupling domain of GTP-binding protein on the alpha 2-adrenoceptor. In addition, Ki-ras p21 protein may lack the capacity to couple with the alpha 2-adrenoceptor.     

Photosynthetic Characteristics of Photoautotrophically Cultured Cells of Tobacco

The photosynthetic characteristics of photoautotrophically culturedcells of tobacco (Nicotiana tabacum cv. Samsun NN) as well asthose of photomixotrophically cultured cells and green leaveswere investigated. Analyses revealed that on a fresh weightbasis cultured tobacco cells had lower chlorophyll contentsthan cells of green leaves. The chlorophyll content per chloro-plast,however, was almost the same in both types of cell, and thechloroplast number per cell accounted for only small differencesin the cellular chlorophyll content. This indicates that thelarger cell volume of cultured cells is the main factor in thedifference in the chlorophyll content of these cells. Photosynthetic activity measurements also showed differencesin the chloroplasts of cultured and leaf cells. The maximumactivities of photosystem I and the Hill reaction for the culturedcells were about half those for leaf cells on a per unit chlorophyllbasis. Moreover, photo-autotrophic cells had relatively constantphotosystem I and Hill reaction activities during growth; whereas,on a fresh weight basis these activities in leaf cells reflecteddevelopmental changes in the chlorophyll content. Lithium dodecyl sulfate-polyacrylamide gel electrophoresis showedqualitatively similar thylakoid polypeptide compositions forcultured and leaf cells at all stages of growth even thoughthere were quantitative decreases in the contents of severalpolypeptides in the cultured green cells (especially in photomixotrophiccells) in comparison to the polypeptide contents of tobaccoleaves. We speculate that the lower photosynthetic activityof the cultured cells may be caused by this reduction in thecontents of certain thylakoid polypeptides. (Received November 14, 1988; Accepted June 19, 1989)     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

Individual differences in PCA response in the guinea pig

Experiments were carried out to determine the cause of individual differences in the passive cutaneous anaphylaxis (PCA) response in guinea pigs. The intensity of 4-h homologous PCA produced by anti-penicillin G serum was not markedly different among eight reactive sites on the back of a specified animal, whereas considerable individual differences were observed in the PCA response, even at a specified reactive site. PCA was significantly inhibited by an antihistaminic agent, promethazine, and the tissue histamine content was significantly reduced after PCA, suggesting histamine release as a mediator. The intensity of PCA in individual animals was highly correlated with that of the histamine-induced cutaneous reaction elicited at a site adjoining the PCA but was unrelated to skin histamine content. These results suggest that the difference in susceptibility to histamine has a considerable effect on individual differences in the PCA response in guinea pigs.     

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.     

Molecular cloning of cDNA for sarcocystatin A and analysis of the expression of the sarcocystatin A gene during development of Sarcophaga peregrina

Sarcocystatin A is a cysteine proteinase inhibitor purified from the hemolymph of Sarcophaga peregrina larvae [Suzuki, T., & Natori, S. (1985) J. Biol. Chem. 260, 5115-5120]. We isolated a cDNA clone for sarcocystatin A and analyzed the structure and expression of the sarcocystatin A gene. Sarcocystatin A consists of 102 amino acid residues. Significant homology was found between amino acid sequences of sarcocystatin A and other mammalian cystatins, and highly conserved sequences among mammalian cystatins were also found in sarcocystatin A. Using cloned cDNA as a probe, we investigated expression of the sarcocystatin A gene during the development of Sarcophaga. Results showed that this gene was transiently activated in the very early embryonic stage and in the pupal stage, suggesting that sarcocystatin A participates in morphogenesis of larval and adult structures of Sarcophaga.     

Distribution of glucose carbons to metabolic products in the perfused rat hindlimb over a range of glucose concentrations

To determine the distribution of glucose carbons to the major metabolic pathways as a function of glucose and insulin concentrations in fed rat muscle, rat hindlimbs were perfused with and without 100 nM insulin in the presence of 1-20 mM [U-14C]-D-glucose. The incorporation of radioactivity into CO2, anionic metabolites released into the medium and muscle cell fractions were determined. The results indicate that the major metabolic fate of glucose taken up by muscle in the fed rat is conversion to anions released into the medium and that this process is saturable with increasing glucose concentrations. The data suggest that saturation of the glycolytic pathway is the major cause of the previously observed saturation of the glucose disposal rate associated with increasing glucose concentration.     

A toxic substance produced by Nocardia otitidiscaviarum isolated from cutaneous nocardiosis

During our studies on toxic substances from clinically isolated Nocarida, a new isolate identified as Nocardia otitidiscaviarum from cutaneous nocardiosis was found to produce a toxic substance called HS-6 that had strong in vitro as well as in vivo toxicity. The mouse intraperitoneal LD₅₀ value was 1.25 mg/kg and the ED₅₀ value for L1210 cultured cells was 0.3 ng/ml. The structure of HS-6 was determined and found to belong to the 16-membered macrocyclic group with a molecular formula of C₄₃H₆₈O₁₂. HS-6 also showed activity against pathogenic fungi such as Cryptococcus neoformans.