Phorbol esters and diacylglycerol inhibit vasopressin-induced increases in cytoplasmic-free Ca and Ca efflux in Swiss 3T3 cells

Vasopressin increased intracellular free calcium concentration [Ca²⁺]_i in quin-2-loaded quiescent Swiss 3T3 cells. This effect of vasopressin was rapidly inhibited by biologically active tumour promoters including phorbol dibutyrate (PBt₂) and by the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG). Prolonged pretreatment of Swiss 3T3 cells with PBt₂ causes a loss of protein kinase C activity (Rodriguez-Pena & Rozengurt, Biochem biophys res commun 120 (1984) 1053) [28]. This pretreatment abolished the inhibition by PBt₂ or OAG of vasopressin-mediated increases in Ca²⁺]_i. Vasopressin also stimulated ⁴⁵Ca²⁺ efflux from cells pre-loaded with the isotope. This effect of the hormone was also inhibited by PBt₂. Prolonged pretreatment with PBt₂ prevented the inhibition of vasopressin-stimulated ⁴⁵Ca²⁺ release by PBt₂. Thus, protein kinase C stimulation inhibits vasopressin-mediated increases in [Ca²⁺]_i and ⁴⁵Ca²⁺ efflux apparently by blocking the increased release of Ca²⁺ from an intracellular store caused by the hormone. These findings suggest that activation of protein kinase C may act as a feedback inhibitor to modulate ligand-mediated increases in [Ca²⁺]_i.     

Functional groups and quaternary structure of chicken liver xanthine dehydrogenase

Xanthine dehydrogenase from chicken liver is a dimeric enzyme, each hemimolecule containing one FAD and two Fe/S groups. Determination of sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuribenzoic acid (PMB) showed a variable number of sulfhydryl groups depending onpH, ionic strength, and nature of the reaction medium and buffer. The number of disulfide bonds was determined with DTNB and reducing conditions. Amino groups were determined with 2,4,6,-trinitrobencensulfonic acid (TNBS). At constant temperature andpH the reaction of DTNB and TNBS with native xanthine dehydrogenase showed an exponential dependence on time. From the obtained parameters the number of available sulfhydryl and amino groups at infinite concentration of enzyme and the rate constant of the equation were determined. The absorption spectrum of the enzyme changed with time when a chaotropic agent (1 M sodium nitrate) was added to the medium. This difference was detected by measuring the absorbance in the range 450–550 nm. The absorption spectrum (between 350 and 600 nm) also changed when a denaturating agent (sodium dodecyl sulfate) was added. This modification increased with time and depended on the medium.     

Lectin binding sites in the vitelline envelope of Bufo arenarum oocytes: Role in fertilization

Incubation of dejellied spawned oocytes from Bufo arenarum with different lectins results in a decrease of oocyte fertility. Concanavalin A was the most effective lectin; phytohem-agglutinin P and wheat germ lectin were less effective. Agglutinin from soybean was scarcely active. These lectin effects could be ascribed to a hindering of specific sites for some proteases, since the same treatment renders the oocyte vitelline envelope insensitive to spermatolysin (an essential requisite for fertilization) and to trypsin. Also in this case concanavalin A was the most effective lectin. Univalent concanavalin A was also effective in blocking the fertility of dejellied oocytes. These results indicate that the residues of α-D-glucose and α-D-mannose present in the vitelline envelope are involved in gamete interactions in Bufo arenarum. This idea is also supported by the finding that dejellied oocytes (fertilizables) have a number of binding sites for concanavalin A that is three or four orders of magnitude higher than coelomic or fertilized oocytes (both not penetrable by spermatozoa).     

Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum

The activity of guanine deaminase (GAH, E.C. 3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C. 3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4), 5-nucleotidase (5N, E.C. 3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system.     

Regulatory interaction of photosynthetic nitrate utilization and carbon dioxide fixation in the cyanobacterium Anacystis nidulans

The rate of photosynthetic nitrate utilization in Anacystis nidulans is strongly influenced by the availability of carbon dioxide. This dependence can be relieved by inhibiting the metabolism of the ammonium derived from nitrate reduction. Nitrate uptake seems to be modulated through a sensitive regulatory system integrating the photosynthetic metabolism of carbon and nitrogen, with CO₂ fixation products antagonizing the inhibitory effect of ammonium derivatives.     

Antioxidant Activity of 5-Hydroxytryptophan, 5-Hydroxyindole, and Dopa Against Microsomal Lipid Peroxidation and Its Dependence on Vitamin E

The antioxidant capacity of 5-hydroxy-tryptophan. 5-hydroxy-indole. and DOPA (3,4-dihydroxy-phenyI-alanine) was tested in the Fe-induced lipid peroxidation of liver microsomes of normal- and vitamin E-deficient rats, using ascorbate as a reductant. Lipid peroxidation was monitored as low-level chemilu-minescence, indicative of generation of electronically-excited states arising from the recombination of secondary lipid peroxyl radicals.     

Four new dinoflagellates

Fragilidium fissile is a new species of this rare genus. It somewhat resemblesF. subglobosum. It differs from the latter in having a slot and a pore in the first apical plate 1′ (the nomenclature of dinoflagellate plate designation follows the Kofoid system). Both species are also distinguishable by noticeable differences in Po, 1″″ and 1‴.Peridinium tyrrhenicum is a small species differing from all the other known species ofPeridinium in its shape, apical channel and several plates, especially some of the sulcal components.Alexandrium foedum somewhat resemblesA. balechii, but it differs from the latter in that its width is greater than its length, and in the characters of all the main sulcal plates. The above listed species were found in a sample from the Tyrrhenian Sea. The fourth species,Alexandrium andersoni, is a small dinoflagellate obtained in coastal waters off Cape Cod. It differs from all the other species of the minutum group in the very typical shapes of both the 6″ and the S. s.Pentapharsodinium daleii Indelicato and Loeblich is transferred toPeridinium.     

Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS,of protein kinase C

Summary Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (I _K) and the M-current (I _M). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E₁ (PGE₁) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: I _k, a small current through a T-like Ca²⁺ channel, and no M-current.Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of I _k observed with cardiac cells, PKC activation produced only a small inhibition of I _k, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of I _k in neuroblastoma cells.Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.We thank Dr. Peter J. Parker for his generous gift of PKC, and Yvonne Vallis for her skillful assistance with the cultures and harvesting of the NG108-15 transfected cells.     

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118–120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.