Distribution and molecular form of immunoreactive big endothelin-1 in porcine tissue

In the present study a specific and sensitive radioimmunoassay for big endothelin-1 was developed. Half maximal inhibition of binding of radioiodinated big endothelin-1 was observed at 58 pg/tube and big endothelin-1 was detectable as low as 2 pg/tube. With this assay, the regional distribution of big endothelin-1 was determined in porcine tissue and compared to the distribution of an immunoreactive endothelin. Considerable amount of immunoreactive big endothelin-1 was observed in the aortic intima (0.84 +/- 0.094 pg/mg wet tissue; mean +/- S.D.) and the lung (0.47 +/- 0.055), but there was a low concentration in other tissue including the kidney inner medulla, which has been shown to be abundant in immunoreactive endothelin. Furthermore the molecular form of immunoreactive big endothelin-1 in aortic intima was found to be big ET-1[1-39], but the molecular form of major immunoreactive big endothelin-1 in the lung is big endothelin-1[22-39] with big endothelin-1[1-39] being minor.     

Glutamate Inhibits Adenylate Cyclase Activity in Dispersed Rat Hippocampal Cells Directly via an N-Methyl-d-Aspartate-Like Metabotropic Receptor

Three major subtypes of glutamate receptors that are coupled to cation channels--N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors--are known as ionotropic receptors in the mammalian CNS. Recently, an additional subtype that is coupled to GTP binding proteins and stimulates (or inhibits) metabolism of phosphoinositides has been proposed as a metabotropic receptor. Incubation of dispersed hippocampal cells from adult rats with glutamate or NMDA decreased forskolin-stimulated cyclic AMP (cAMP) accumulation; half-maximal effects were obtained with 5.6 +/- 2.2 and 6.4 +/- 2.3 microM, respectively. Kainate and quisqualate were less potent. The effect of glutamate was antagonized by 2,3-diaminopropionate and 2-amino-5-phosphonovalerate, NMDA/glutamate receptor antagonists, but not by 0.5 microM Joro spider toxin, a specific blocker of the AMPA receptor. The inhibitory effect of glutamate on cAMP formation was not blocked by 2 microM tetrodotoxin or by the absence of Ca2+. In hippocampal membranes, glutamate, similar to carbachol, inhibited adenylate cyclase activity in a GTP-dependent manner. These findings suggest that the glutamate inhibition of adenylate cyclase is direct and is not due to a result of the release of other neurotransmitters. The effect of glutamate on cAMP accumulation was observed in an assay medium containing 0.7 mM MgCl2, which is known to inhibit both ionotropic NMDA receptor/channels in the hippocampus and metabotropic NMDA receptors in the cerebellum. The inhibitory effect of glutamate was abolished by pertussis toxin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation.

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 5 (IL-5) are composed of two distinct subunits, alpha and beta c. The alpha subunits are specific for each cytokine, whereas the beta subunit (beta c) is shared by the three receptors and is an essential component of signal transduction. We have made a series of mutant beta c cDNAs that delete various regions of the cytoplasmic domain and examined the function of these mutants by coexpressing them with the alpha subunit of the human GM-CSF receptor (hGMR) in an IL-3-dependent mouse pro-B cell line BaF3. Two domains in the membrane-proximal portion of beta c were found to be important for transducing the hGM-CSF-mediated growth signals: one domain between Arg456 and Phe487 appears to be essential for proliferation, and the second domain between Val518 and Asp544 enhances the response to GM-CSF, but is not absolutely required for proliferation. The region between Val518 and Leu626 was responsible for major tyrosine phosphorylation of 95 and 60 kDa proteins. Thus, beta c-mediated major tyrosine phosphorylation of these proteins was apparently separated from proliferation. However, the beta 517 mutant lacking residues downstream of Val518 transmitted a herbimycin-sensitive proliferation signal, suggesting that beta 517 still activates a tyrosine kinase(s). We also evaluated the role of the cytoplasmic domain of the GMR alpha subunit and the results suggest that it is involved in the hGM-CSF-mediated signal transduction, but is not essential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristic induction of 70 000 Da-heat shock protein and metallothionein by zinc in HeLa cells

The synthesis of a 70 000 dalton-heat shock protein (hsp70) is one of several heat shock proteins induced in HeLa cells during the incubation in medium containing zinc sulphate. The synthesis of hsp70 was increased in the presence of 200 M zinc sulphate and above, but not at 100 M zinc sulphate. On the other hand, the synthesis of metallothionein was activated in the presence of 100 M zinc sulphate and above. Uptake of zinc into the cells depended on the concentration of zinc sulphate in the medium. The separation of intracellular zinc into three fractions by gel filtration chromatography; high molecular, metallothionein, and low molecular fractions, showed that zinc in the low molecular weight and metallothionein fractions was elevated in the presence of 100 M zinc sulphate in the medium, whereas increase in the zinc content of the high molecular weight fraction occurred at 200 M zinc sulphate and above. Inhibition of cell growth and cellular protein synthesis was also observed at 200 M zinc sulphate and above, but not at 100 M. From these findings, since the induction of hsp70 synthesis and inhibition of cell growth occurred concomitantly with the increase of zinc in the high and low molecular weight fractions, hsp70 seemed not to function in the detoxification of zinc, but it may participate in the repair of zinc-induced damage.     

Uptake and intracellular transport of cationic ferritin in the bronchiolar and alveolar epithelia of the rat

Summary Cationic ferritin was used as a marker to reveal the processes of endocytosis and intracellular transport in bronchiolar and alveolar epithelia. The marker was injected into the lung via the trachea, and ultrastructural observation of the distribution of ferritin particles in bronchiolar and alveolar epithelial cells was carried out at intervals of 5, 15, 30 and 60 min after the injection. The luminal surface of the airway and the alveolar epithelium showed diffuse labeling with cationic ferritin. In general, ferritin particles were observed in vesicles and vacuoles of the bronchiolar and alveolar epithelial cells within 5 min of injection; they appeared in multivesicular bodies within 15 min. Multivesicular bodies and secondary lysosomes containing ferritin particles, some of which showed a positive reaction for acid phosphatase, were seen in the basal cytoplasm within 30 min; ferritin particles appeared in the basal lamina below the Clara cells, ciliated cells and type 2 alveolar cells within 30 min. Ferritin particles were seen in ovoid granules of some Clara cells and in lamellar inclusion bodies of many type 2 alveolar cells. Brush cells and type 1 alveolar cells took up only a small quantity of ferritin particles.     

Isolation and characterization of peptides which act on rat platelets, from a pheochromocytoma.

An increase in the cellular concentration of cAMP leads to the inhibition of platelet aggregation. We have been investigating endogenous peptides which inhibit platelet function, using an assay which detects increase in platelet cAMP. Compared with the human adrenal medulla, a pheochromocytoma (PC) contained abundant peptides that elevate platelet cAMP. About 90% of the activity was found in the SP-III fraction which contained strongly basic peptides. From the SP-III fraction, peptides P-1, P-2 and P-3 were purified to homogeneity as endogenous peptides which elevated platelet cAMP. A gas phase sequencer was used to identify these peptides as follows: P-1 = vasoactive intestinal peptide (VIP); P-2 = calcitonin gene related peptide-I (CGRP-I); P-3 = CGRP-II. It is well known these peptides are potent vasorelaxants. VIP and CGRP-I significantly increased platelet cAMP levels 15- and 6-fold, respectively. These results suggest that VIP and CGRP-I and -II act upon platelets as well as upon vascular tissue.     

Differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In two recent papers, we reported the effects of several point mutations on the thermodynamics of the thermal unfolding of the lysozyme of phage T4 as determined by differential scanning calorimetry. The mutants studied were R96H [Kitamura, S., & Sturtevant, J.M. (1989) Biochemistry 28, 3788-3792] and T157 replaced by A, E, I, L, N, R, and V [Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J.M. (1991) Biochemistry 30, 1887-1891]. Here we report the results of a similar study of the single mutations A82P, A93P, and G113A and the double mutation C54T:C97A. The three single mutants all show small apparent stabilization at pH 2.5 and 46.2 degrees C (the denaturational temperature of the wild-type protein), amounting to -0.5 +/- 0.4 kcal mol-1 in free energy, whereas the double mutant shows a weak apparent destabilization, +0.8 +/- 0.4 kcal mol-1. As in all our previous studies of mutant proteins, the enthalpy changes produced by these mutations are in general of much larger magnitude than the corresponding free energy changes and frequently of opposite sign.     

Possible involvement of different GTP-binding proteins in noradrenaline- and thrombin-stimulated release of arachidonic acid in rabbit platelets.

Noradrenaline (NA) stimulated the release of arachidonic acid (AA) from the [3H]AA-labelled rabbit platelets via alpha 2-adrenergic receptors, since the effect of NA was inhibited by yohimbine. The stimulatory effect of NA in digitonin-permeabilized platelets was completely dependent on the simultaneous presence of GTP and Ca2+. The NA- and thrombin-stimulated releases of AA were markedly decreased by the prior ADP-ribosylation of the permeabilized platelets with pertussis toxin. Antiserum directed against the pig brain Go (a GTP-binding protein of unknown function), recognizing both alpha 39 and beta 35,36 subunits, but not alpha 41, of pig brain, reacted with 41 kDa and 40 kDa bands, with not one of 39 kDa, in rabbit platelet membranes. Anti-Go antiserum inhibited guanosine 5'-[gamma-thio]triphosphate-, A1F4(-)-, NA- and thrombin-stimulated AA releases in the membranes. Although the effect of thrombin was inhibited by low concentrations of anti-Go antiserum, high concentrations of the antiserum was needed for inhibition of the NA effect. Antiserum directed against the pig brain G1 (inhibitory G-protein), recognizing both alpha 41 and beta 35,36 subunits, but not alpha 39, of pig brain, reacted with the 41 kDa band in platelets. Anti-G1 antiserum inhibited only the effect of NA. Reconstitution of the platelet membranes ADP-ribosylated by pertussis toxin with Go, not Gi, purified from pig brain restored the thrombin-stimulated release of AA. In contrast, reconstitution of those membranes with Gi, not Go, restored the NA-stimulated release of AA. These results indicate that different GTP-binding proteins, Gi- and Go-like proteins, may be involved in the mechanism of signal transduction from alpha 2-adrenergic receptors and thrombin receptors to phospholipase A2 in rabbit platelets.     

Effects of the brachyury (T) mutation on mitotic activity in the neural tube.

Mitotic activity in the neural tube was examined in +/+, $\text{T}\text{+}$ and $\text{T}\text{T}$ mouse embryos. The number of mitotic figures per unit area of ependymal layer (area index) was higher in $\text{T}\text{+}$ embryos than in +/+ embryos. In +/+ embryos, area index was higher in the dorsal part of the neural tube than in the ventral part, whereas this pattern is disordered in $\text{T}\text{+}$ embryos. In $\text{T}\text{T}$ embryos, these values fluctuate. The process of nuclear migration accompanying cell division seems to be normal in the neural tube of $\text{T}\text{+}$ and $\text{T}\text{T}$ embryos.     

Metabolism ofL-sorbose by enzymes fromGluconobacter melanogenus IFO 3293

Summary Studies on the metabolism ofL-sorbose by cell-free extracts fromGluconobacter melanogenus IFO 3293 grown inL-sorbose — containing media confirm by different methodology the observations by Makover et al. that the sequence in this bacterium isL-sorbose L-sorbosone 2-keto-L-gulonic acid L-idonic acid.Dedicated to the memory of Prof. Dr. Konrad Bernhauer. For preceeding paper in this series see Kitamura & Perlman (1975).