Association of neuronal pp60c-src with growth cone glycoproteins of rat brain

Tyrosine phosphorylation and protein tyrosine kinase (PTK) activity in the growth cone membrane-associated glycoprotein (GCGP) fraction of 1-day-old rat brain were examined. Using immunoblotting and immunoprecipitation techniques, pp60^c-src was identified as one of the major PTKs associated with GCGPs. Furthermore, only GCGP-associated src that was also tyrosine phosphorylated was active. Immunoprecipitation experiments using various src antibodies revealed that pp60^c-src contributed partially to the PTK activity detected in GCGPs, and that it is associated with several proteins of Mr 140 K, 120 K, 85 K and 50 K. This association of src protein with GCGPs was specific, and another src family member p59^fyn, which is also abundant in the brain, did not exhibit such an association. In addition to pp60^c-src, the GCGP fraction contained several major phosphotryosine-containing proteins of Mr 140 K, and a 97/90 K doublet that corresponded to the beta subunits of IGF-I/ insulin receptors. These studies show that pp60^c-src associated with GCGPs is an active PTK that could be involved in neuronal growth and development, transmembrane signalling, and in recognition and/or adhesive events. © 1992 John Wiley & Sons, Inc.     

Analysis of pp60c-src tyrosine kinase activity and phosphotyrosyl phosphatase activity in human colon carcinoma and normal human colon mucosal cells

We have compared the level of phosphotyrosyl phosphatase activity in lysates from normal human colon mucosal cells and human colon carcinoma cells and analyzed the effect of incubating these cells with sodium orthovanadate, an inhibitor of phosphotyrosyl phosphatase activity, on the relative abundance of acid-stable phosphotyrosine and on in vitro protein kinase activity of pp60c-src. Additionally, we compared the effect of lysing these cells in buffer containing only nonionic detergents with RIPA buffer, which contains both sodium dodecyl sulfate and deoxycholate, on the in vitro kinase activity of pp60c-src. Our results show that the level of detectable phosphotyrosyl phosphatase activity in lysates derived from normal colon cells and colon carcinoma cells is very similar. Additionally, the abundance of acid-stable phosphotyrosine in these cells cultured in the absence or presence of vanadate is not significantly different. However, incubation of these cells with vanadate significantly stimulates the activity of pp60c-src derived from the normal colon cells in immune-complex kinase assays, while having no detectable effect on the activity of pp60c-src from the colon tumor cells. The in vitro protein kinase activity of pp60c-src derived from RIPA buffer lysates of colon carcinoma cells was found to be elevated five- to sevenfold when compared with pp60c-src from these same cells lysed in buffer containing only Nonidet-P 40 as a detergent. The type of lysis buffer did not effect the activity of pp60c-src from normal colon mucosal cells. These results provide additional evidence that the activity of pp60c-src may be regulated differently in colon carcinoma and normal colon mucosal cells.     

Stimulation of pp60c-src tyrosyl kinase activity in polyoma virus-infected mouse cells is closely associated with polyoma middle tumor antigen synthesis

We have examined the effect of polyoma virus infection of primary mouse embryo cells on the tyrosyl kinase activity associated with the cellular src gene product, pp60c-src. The results of our studies demonstrate that infection of mouse cells with wild-type polyoma virus or viral mutants capable of transforming rodent cells in culture and inducing tumors in animals results in the stimulation of pp60c-src tyrosyl kinase activity. The level of pp60c-src kinase stimulation in infected cells was found to be proportional to both the oncogenic potential of the virus strain used for infection and the characteristic phenotype of rodent cells transformed by the various strains of polyoma virus. Stimulation of pp60c-src kinase activity was not observed in mouse cells infected with transformation-defective strains of polyoma virus. In examining the kinetics of pp60c-src kinase stimulation in mouse cells at various times following wild-type polyoma virus infection, we found that the level of pp60c-src kinase activity correlated directly with the synthesis of polyoma virus-encoded tumor antigens. By comparing wild-type polyoma virus with other viral mutants in these experiments, we conclude that the stimulation of pp60c-src kinase activity in mouse cells following polyoma virus infection is associated with the synthesis of middle tumor antigen.     

Myristoylation-regulated direct interaction between calcium-bound calmodulin and N-terminal region of pp60v-src

pp60v-src tyrosine protein kinase was suggested to interact with Ca2+-bound calmodulin (Ca2+/CaM) through the N-terminal region based on its structural similarities to CAP-23/NAP-22, a myristoylated neuron-specific protein, whose myristoyl group is essential for interaction with Ca2+/CaM; (1) the N terminus of pp60v-src is myristoylated like CAP-23/NAP-22; (2) both lysine residues are required for the myristoylation-dependent interaction and serine residues that are thought to regulate the interaction through the phosphorylations located in the N-terminal region of pp60v-src. To verify this possibility, we investigated the direct interaction between pp60v-src and Ca2+/CaM using a myristoylated peptide corresponding to the N-terminal region of pp60v-src. The binding assay indicated that only the myristoylated peptide binds to Ca2+/CaM, and the non-myristoylated peptide is not able to bind to Ca2+/CaM. Analyses of the binding kinetics revealed two independent reactions with the dissociation constants (KD) of 2.07 x 10(-9)M (KD1) and 3.93 x 10(-6)M (KD2), respectively. Two serine residues near the myristoyl moiety of the peptide (Ser2, Ser11) were phosphorylated by protein kinase C in vitro, and the phosphorylation drastically reduced the interaction. NMR experiments indicated that two molecules of the myristoylated peptide were bound around the hydrophobic clefts of a Ca2+/CaM molecule. The small-angle X-ray scattering analyses showed that the size of the peptide-Ca2+/CaM complex is 2-3A smaller than that of the known Ca2+/CaM-target molecule complexes. These results demonstrate clearly the direct interaction between pp60v-src and Ca2+/CaM in a novel manner different from that of known Ca2+/CaM, the target molecules, interactions.     

Effects of ionizing radiation (60Co gamma rays) on growth and morphogenesis ofHaworthia mirabilis,haw. Callus tissue

Summary The effects of γ-radiation on growth and morphogenesis ofHaworthia callus in vitro were determined. The doses ranged from 100 to 5000 rads. Survival, growth pattern, growth rate, and differentiation of vegetative buds and roots in both irradiated and nonirradiated callus were compared. Growth data up to 24 weeks for irradiated and control cultures were analyzed. The dose range between 800 to 2500 rads produced compact callus as compared to the controls which were friable. After 12 weeks all control cultures differentiated vegetative buds with roots, whereas callus exposed to 800 to 2500 rads continued to grow with little or no organogenesis. However, it was observed that the wet and dry weights of callus receiving 1000 to 1500 rads ultimately exceeded those of nonirradiated controls.     

Intercellular communication and the control of growth: X. Alteration of junctional permeability by thesrc gene. A study with temperature-sensitive mutant Rous sarcoma virus

Summary To study changes of junctional membrane permeability associated with transformation, the junctions and the nonjunctional membranes of quail embryo-, chick embryo- and mouse-3T3 cell cultures, infected with temperature-sensitive mutant Rous sarcoma virus, were probed with fluorescent-labelled glutamate. Junctional permeability fell in the transformed state. In the quail cells, the fall was detectable within 25 min of shifting the temperature down to the level (permissive) at which tyrosine-phosphorylation by the viralsrc gene product is expressed. This reduction of junctional permeability is one of the earliest manifestations of viral transformation. Normal permeability was restored within 30 min of raising the temperature to the nonpermissive level, a reversibility that could be displayed several times during the span of a cell generation. The reversal seems to reflect a reopening of cell-to-cell channels rather than a synthesis of new ones; it is not blocked by protein-synthesis inhibition. Treatments with cyclic AMP and phosphodiesterase inhibitor or with forskolin, which stimulate serine and threonine phosphorylation—the type of phosphorylation on which normal junctional permeability depends (Wiener & Loewenstein, 1983,Nature 305433)—did not abolish, in general, the junctional effect of the virus;src tyrosine-phosphorylation apparently overrides the junctional upregulation mediated by cyclic AMP. Nonjunctional membrane permeability was not sensibly affected by the virus. It was affected, however, by temperature: lowering the temperature from the nonpermissive to the permissive level caused the nonjunctional permeability to fall, andvice versa. This change was unrelated to transformation. Its secondary effect on junctional transfer is in the opposite direction to that produced by the temperature-activated viral transformation.     

The effects of hydrated C(60) fullerene on gene expression profile of TRPM2 and TRPM7 in hyperhomocysteinemic mice

Abstract

Background: Hyperhomocysteinemia (HHcy) is associated with neurodegenerative diseases. Transient receptor potential melastatin (TRPM2) and TRPM7 channels may be activated by oxidative stress. Hydrated C(60) fullerene (C(60)HyFn) have recently gained considerable attention as promising candidates for neurodegenerative states. We aimed to examine the effects on TRPM2 and TRPM7 gene expression of C(60)HyFn due to marked antioxidant activity in HHcy mice. Methods: C57BL/6 J. mice were divided into four groups: (1) Control group, (2) HHcy, (3) HHcy?+?C(60)HyFn-treated group and (4) C(60)HyFn-treated group. TRPM2 and TRPM7 gene expression in brains of mice were detected by real-time PCR, Western blotting and immunohistochemistry. Apoptosis in brain were assessed by TUNEL staining. Results: mRNA expression levels of TRPM2 were significantly increased in HHcy group compared to the control group. C(60)HyFn administration significantly decreased serum levels of homocysteine and TRPM2 mRNA levels in HHcy?+?C(60)HyFn group. Whereas, HHcy-treatment and C(60)HyFn administration did not change the expression of TRPM7. Conclusion: Administration of C(60)HyFn in HHcy mice significantly reduces serum homocysteine level, neuronal apoptosis and expression level of TRPM2 gene. Increased expression level of TRPM2 induced by oxidative stress might be involved in the ethiopathogenesis of HHcy related neurologic diseases.     

Roles of the insulin-like growth factor I receptor C-terminus in cellular radioresistance

Available evidence suggests that insulin-like growth factor I receptor (IGF-IR) expression leads to increased cellular radioresistance. The most direct explanation of these findings predicts that IGF-IR is the source of survival signals in resistant cells. Mutational analysis revealed that protein truncated at amino acid 1245 in the C-terminus retained the ability of IGF-IR to confer radioresistance whereas point mutations at both Tyr-1250 and Tyr-1251 abrogated this effect using IGF-IR-deficient mouse embryo fibroblasts (R-) as a recipient. In cells expressing the latter mutant receptors, both phosphatidylinositol-3(') kinase (PI3-K) and mitogen-activated protein kinase (MAPK) signaling pathways remained intact, and addition of exogenous IGF-I could not change the radiosensitivity of these cells. Further analysis indicated that the abrogation of radioresistance required the presence of His-1293 and Lys-1294. These results suggest a novel regulatory role of the C-terminus of IGF-IR in mediating cellular radioresistance that may be independent of survival signals transmitted through this receptor.     

Betacellulin stimulates growth of the mouse intestinal epithelium and increases adenoma multiplicity in Apc+/Min mice

We employed transgenic mice overexpressing betacellulin (BTC) to study its effects in the gut. BTC stimulated crypt cell proliferation and markedly increased intestinal size, while the crypt-villus architecture was preserved. Introduction of a dominant negative epidermal growth factor receptor (EGFR) completely abolished the intestinal hyperplasia. BTC increased polyp multiplicity but did not change the mean size or the histological quality of intestinal polyps in Apc(+/Min) mice. Analysis of intact and cleaved caspase-3 levels indicated that BTC has anti-apoptotic effects in the intestinal epithelium. We conclude that increased BTC levels support the survival of nascent adenomas in Apc(+/Min) mice, resulting in a larger total polyp number at later stages.     

Control of gene expression during induction of cultured peanut cells: mRNA levels,protein synthesis and enzyme activity of stilbene synthase

Stilbene synthase is an inducible enzyme occurring in a small number of plants. The enzyme is amenable to analysis and biochemical studies only after the cells are subjected to induction. Cell suspension cultures of peanut react very selectively if elicited with biotic inducers. Just as intact peanut plants produce stilbene phytoalexins when attacked by fungi so also do sterile cultured cells when treated with sterilized insoluble fungal cell walls. Both systems react by synthesizing stilbene synthase. The time courses of increase in enzyme activity, protein synthesis and mRNA activity were studied, and their relation to other activities of the cells was elaborated. The results show that, after applying the fungal elicitor, the system responds very quickly and selectively: within 2 hours the synthesis rate of stilbene synthase protein is increased more than 30-fold, the increase being detectable 40 min after induction. The first increase in translatable mRNA for stilbene synthase can be seen 20 min after application of the stimulus. Stilbene synthase synthesized in vivo was compared to stilbene synthase prepared by translation in vitro. There was no difference in size, and limited proteolysis did not indicate significant differences in the peptide structure of the primary translation product and the active enzyme.     

Differential response of the epidermal growth factor receptor tyrosine kinase activity to several plant and mammalian lectins

Biosignalling via lectins may involve modulation of protein kinase activities. This aspect of the biological action of mammalian and plant lectins has been investigated for their effect on the activity of the isolated epidermal growth factor receptor (EGFR). The constitutive tyrosine kinase activity of the epidermal growth factor receptor from rat liver, isolated by calmodulin-affinity chromatography, was activated by concanavalin A (ConA), and wheat germ agglutinin (WGA) to a similar extent as the measured enhancement induced by EGF. In contrast, two mannose-specific lectins, the mannan-binding protein (MBP) and serum amyloid P component (SAP), isolated from human serum, have inhibitory effects, both in the absence and presence of EGF. The differential effects of these lectins were tested using as phosphorylatable substrates a co-polymer of glutamic acid-tyrosine, as well as calmodulin. However, two galactoside-specific lectins, the laminin-binding -galactoside-binding 14 kDa lectin, isolated from bovine heart (14K-BHL), and the /-galactoside-binding lectin, isolated from mistletoe (Viscum album L.) leaves (VAA), do not inhibit the EGFR tyrosine kinase activity. The sugar dependence of the lectin-mediated action was studied by inhibition assays. Mannose and a mannose-containing neoglycoprotein prevent the activating effect of ConA, and N-acetyl-D-glucosamine partially prevents the activation produced by WGA. However, mannose and mannose-containing neoglycoprotein were ineffective to reduce the inhibitory effect of MBP or SAP. Although the response to binding of ConA and WGA was different to that of MBP or SAP with respect to the tyrosine kinase activity of the EGFR, it should be noted that the four lectins inhibited the binding of [¹²⁵I]EGF to its receptor with similar efficiency.Abbreviations EGF epidermal growth factor - EGFR epidermal growth factor receptor - ConA concanavalin A - MBP mannan-binding protein - SAP serum amyloid P component - WGA wheat germ agglutinin - 14K-BHL bovine heart 14 kDa lectin - VAA Viscum album L. (mistletoe) agglutinin - EGTA [ethylenebis(oxyethylenenitrilo)]-tetraacetic acid; poly(Glu:Tyr)-co-polymer of L-glutamic acid and L-tyrosine - Hepes 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - Tris tris(hydroxymethyl)-aminomethane - DSS suberic acid bis(N-hydroxy-succinimide ester) - PMSF phenylmethanesulfonyl fluoride - Man mannose - Gal galactose - BSA bovine serum albumin - Man-BSA neoglycoprotein containing -D-mannose - Lac-BSA neoglycoprotein containing -lactose - Gal-BSA neoglycoprotein containing galactose     

Induction of chitinase activity by exogenous cytokinins in excised dark-grown cucumber cotyledons: involvement of Ca and staurosporine-sensitive protein kinase(s) in cytokinin signaling

Cotyledons were excised from 7-day-old dark-grown cucumber seedlings and treated with water, benzyladenine (BA), kinetin (K), zeatin (Z), or zeatin riboside (ZR) in dark after endogenous cytokinin depletion. We have compared changes in chitinase (EC. 3.2.1.14) activity induced by these cytokinins. We find that the activities of chitinase and its isoforms increase by approximately 3- to 6-fold following BA, Z, and ZR treatments. Among these treatments, Z was more effective. K was totally ineffective in inducing chitinase activity. Immunoblot analysis suggests that the cytokinin Z-induction of enzyme activity is due to the induction of higher chitinase protein levels and not the activation of existing enzyme. Furthermore, the Z-induced chitinase activity and its protein accumulation were completely inhibited by the protein kinase inhibitor staurosporine, whereas the protein phosphatase inhibitor sodium fluoride was not effective in such inhibitions. Treatment of cotyledons with extemal CaCl₂ and calcium ionophore increased the basal chitinase activity by 6- and 5-fold, respectively. Moreover, the effects of staurosporine, sodium fluoride, and Ca²⁺ on Z-induced chitinase activity correlate with their effects on chitinase protein levels. Taken together, our data suggests Ca²⁺ and staurosporine-sensitive protein kinase(s) as components of the cytokinin transduction machinery involving induction of chitinase in cucumber.     

Differential effect of PKA on the Ca2+ release kinetics of the type I and III InsP3 receptors

The effects of protein kinase A (PKA) on the inositol 1,4,5-trisphosphate (InsP(3)) receptor isoforms type I and type III were studied. The effects of PKA on the extent and rate constants for InsP(3)-induced Ca(2+) release (IICR) were different for the two isoforms. The effects of PKA on the type I isoform showed a biphasic relationship dependent upon the concentration of PKA used. At low concentrations of PKA (<50U/ml), both the extent and rate constants for IICR increased, while at higher concentrations (>200U/ml) the extent and rate constants decreased. The type III isoform showed only an increase in the extent of IICR and not in the rate constants. The effects of PKA on the type I InsP(3) receptor using single channel electrophysiological studies were also investigated. The stimulatory effect of PKA is due to an increase in conductance levels and not to a change in the mean open time of the channel.     

Intercellular communication and the control of growth: XI. Alteration of junctional permeability by thesrc gene in a revertant cell with normal cytoskeleton

Summary To learn whether the reduction of cell-to-cell communication in transformation is a possible primary effect of pp60^src phosphorylation or secondary to a cytoskeletal alteration, we examined the junctional permeability in transformed cells with normal cytoskeleton. The permeability to fluorescentlabelled mono- and diglutamate was compared in clones of Faras' vole cells—clones transformed by Rous sarcoma virus and reverted from that transformation. One revertant clone (partial revertant), had the high levels of pp60^src kinase activity and tumorigenicity of the fully transformed parent clone, but had lost the cytoskeletal alterations of that clone. Another revertant clone (full revertant) had lost the tumorigenicity and most of the pp60^src kinase activity, in addition (J.F. Nawrocki et al., 1984,Mol. Cell Biol. 4:212). The junctional permeability of thepartial revertant with normal cytoskeleton was similar to that of the fully transformed parent clone with abnormal cytoskeleton. The permeabilities of both were lower than those of thefull revertant and the normal uninfected cell, demonstrating that the junctional change by thesrc gene is independent of the cytoskeletal one.     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

伤害和挥发物对棉花花外蜜的诱导效应

植物花外蜜的分泌是一种植物间接防御反应。为了明确植食性昆虫、机械伤和机械伤诱导的挥发性气体在植物花外蜜诱导分泌中的作用,分析了咀嚼式口器昆虫棉铃虫Helicoverpa armigera(Hübner)、刺吸式口器昆虫棉蚜Aphis gossypii Glover取食、剪刀机械伤、剪刀机械伤+棉铃虫反吐物、针刺机械伤以及机械伤诱导挥发物、顺式-茉莉酮对棉花Gossypium hirsutum L.叶片花外蜜分泌量的影响。结果表明,棉铃虫取食、剪刀机械伤、剪刀机械伤+棉铃虫反吐物处理均显著增加了被处理叶片花外蜜的分泌量。棉花花外蜜的诱导效应在处理叶片上表现明显,并且在较幼嫩的第3片真叶上也有系统性增长。顺式-茉莉酮和机械伤挥发物处理1 d对棉花较幼嫩的第4、5片真叶花外蜜有诱导效应。棉花叶片花外蜜的诱导主要与植物组织损伤有关;不同口器类型的昆虫对棉花叶片花外蜜的诱导量不同,咀嚼式口器的棉铃虫对棉花花外蜜的诱导强度显著高于刺吸式口器的棉蚜;顺式-茉莉酮和机械伤诱导的挥发物能作为棉花植株间交流的信息物质诱导棉花幼嫩叶片花外蜜的分泌。     

Studies on (K + H)-ATPase. V. Chemical composition and molecular weight of the catalytic subunit

(1) A (K⁺ + H⁺)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH₂ terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, n = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

The effects of immobilization on the biochemical,physiological and morphological features of Anabaena azollae

Anabaena azollae, a presumptive isolate from Azolla filiculoides, was immobilized in polyurethane foam, hydrophilic polyvinyl foam and alginate. When viewed by low-temperature scanning electron microscopy a thick mucilage layer covered the surface of both cells and matrix; this closely resembles the mode of attachment of the symbiont Anabaena in the Azolla leaf cavity. The heterocyst frequency of the immobilized A. azollae doubled relative to free-living cells and reached a level of 14–17%. Immobilization induced increases in both hydrogen production via nitrogenase or hydrogenase and in the rates and stabilization of acetylene reduction (N₂-fixation). Ammonia production by immobilized cells with L-methionine-D,L-sulfoximine (MSX) is greater than that of freeliving cells. Immobilized cells without MSX were, however, able to excrete ammonium at lower rates thus emulating the characteristic of the symbiotic cyanobacteria (A. azollae) in the leaf cavity of Azolla.Abbreviations Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-D,L-sulfoximine - SEM scanning electron microscopy - PU polyurethane - PV polyvinyl