Role of three-body recombination for charge reduction in MALDI process.

Ions in Matrix-Assisted Laser Desorption/Ionization (MALDI) are predominantly singly charged for small analyte molecules. With the estimated high number density and low temperature of electrons, the three-body recombination mechanism is attractive and should be considered as an important cause for the charge reduction in the plume. Theoretical calculations indicate that the rate coefficient of the three-body recombination is about 50 times higher than that of the two-body recombination if the analyte molecule has insufficient degrees of freedom. Experimental results show that, for small analyte molecules, the ratio of AH2(2+)/AH(+) is close to the theoretical 5% value from the three-body recombination modeling and this ratio declines with the increasing electron and matrix molecule number density caused by greater laser irradiance. The ratio of [A + 2](+)/[A + 1](+) is higher than the theoretical isotopic value, and the excess [A + 2](+) could exclusively result from the three-body recombination collisions. Further evidence demonstrates that [A + 2](+)/[A + 1](+) increases with electron number density, which is in correspondence with the model. All of these theoretical and experimental results indicate that three-body recombination is an essential charge reduction mechanism for small molecules in the MALDI plume.

Src-catalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins.

The protooncogene c-Cbl has recently emerged as an E3 ubiquitin ligase for activated receptor tyrosine kinases. We report here that c-Cbl also mediates the ubiquitination of another protooncogene, the non-receptor tyrosine kinase c-Src, as well as of itself. The c-Cbl-dependent ubiquitination of Src and c-Cbl requires c-Cbl's RING finger, Src kinase activity, and c-Cbl's tyrosine phosphorylation, probably on Tyr-371. In vitro, c-Cbl forms a stable complex with the ubiquitin-conjugating enzyme UbcH7, but active Src destabilizes this interaction. In contrast, Src inhibition stabilizes the c-Cbl. UbcH7.Src complex. Finally, c-Cbl reduces v-Src protein levels and suppresses v-Src-induced STAT3 activation. Thus, in addition to mediating the ubiquitination of activated receptor tyrosine kinases, c-Cbl also acts as a ubiquitin ligase for the non-receptor tyrosine kinase Src, thereby down-regulating Src.

Mitogenic phospholipase D activity is restricted to caveolin-enriched membrane microdomains.

Phospholipase D (PLD) activity is elevated in response to the oncogenic stimulus of several signaling oncogenes. PLD activity is also elevated in response to peptide growth factors, indicating that PLD likely plays an important role in mitogenic signaling. Many proteins that mediate mitogenic signaling are localized in caveolin-enriched membrane microdomains (CEMMs). We report here that the elevated PLD activity in NIH 3T3 cells transformed by activated oncogenic forms of Src, Ras, and Raf is largely restricted to the CEMMs. Likewise, the PLD activity stimulated by epidermal growth factor is also restricted to the CEMMs. Although both PLD1 and PLD2 were found in CEMMs, neither was particularly enriched in the CEMMs of the transformed relative to the parental cells, indicating that it is the specific activity of PLD that is increased in the CEMMs. An apparent PLD substrate specificity in transformed cells for phosphatidylcholine lacking arachidonate acyl groups is also explained by the localization of activity in the CEMMs where [(3)H]arachidonate-labeled PC was excluded. These data indicate that mitogenic signals through PLD are initiated in CEMMs where many signaling molecules colocalize.

Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas.

p130Cas (Cas), the protein encoded by the Crkas gene (also known as Cas), is an adaptor molecule with a unique structure that contains a Src homology (SH)-3 domain followed by multiple YXXP motifs and a proline-rich region. Cas was originally cloned as a highly tyrosine-phosphorylated protein in cells transformed by v-Src (refs 2,3) or v-Crk (ref. 4) and has subsequently been implicated in a variety of biological processes including cell adhesion, cell migration, growth factor stimulation, cytokine receptor engagement and bacterial infection. To determine its role in vivo, we generated mice lacking Cas. Cas-deficient embryos died in utero showing marked systemic congestion and growth retardation. Histologically, the heart was poorly developed and blood vessels were prominently dilated. Electron microscopic analysis of the heart revealed disorganization of myofibrils and disruption of Z-disks. In addition, actin stress fiber formation was severely impaired in Cas-deficient primary fibroblasts. Moreover, expression of activated Src in Cas-deficient primary fibroblasts did not induce a fully transformed phenotype, possibly owing to insufficient accumulation of actin cytoskeleton in podosomes. These findings have defined Cas function in cardiovascular development, actin filament assembly and Src-induced transformation.

Inhibition of rat parotid gland growth response induced by chronic isoproterenol following treatment with quinolone antibiotics.

While antibiotics are broadly used in dental and medical therapy, little attention has been directed towards the potential toxic side effects of antibiotics on tissue regeneration. Here we examined the effect of a quinolone antibiotic, pefloxacin (Rhone Poulenc) on rat parotid gland responses to chronic isoproterenol treatment. Groups of rats received injections of isoproterenol to induce glandular growth, saline (controls), pefloxacin, or isoproterenol and pefloxacin in combination. Parotid gland weight decreased significantly after pefloxacin treatment for 7 days as well as inhibiting glandular enlargement provoked by isoproterenol. The same trend was observed for the rates of DNA synthesis, with the incorporation of [3H]-thymidine in isoproterenol/pefloxacin-treated rats reduced to 49% of isoproterenol treatment alone levels. Saline-treated animals were 42% of the rate of [3H]-thymidine incorporation into DNA observed in isoproterenol treated rats. While isoproterenol treatment increased steady-state mRNA levels for fos, jun, myc, src, c-erbB-2, ras and topo II, inclusion of pefloxacin with the isoproterenol regimen blocked these increases. Pefloxacin treatment by itself did not alter proto-oncogene mRNA levels in the parotid gland. Glandular amylase activity was decreased in the pefloxacin treated group, while the combination of isoproterenol with pefloxacin did not decrease glandular amylase levels to the extent of that observed with beta-agonist treatment alone. In acute experiments, pefloxacin significantly decreased the volume of saliva secreted by the parotid gland. These results suggest that quinolone-based antibiotics disturb the secretory function of the parotid gland and can inhibit cell proliferation and regeneration.

Expression of V-SRC and chromosome analysis of a newly established cell line from rat sarcoma induced by an avian retroviral strain.

In the newly established rat sarcoma cell line LSR-SF (SR) expression of pp60v-src was detected. Karyotype analyses revealed various chromosome aberrations during prolonged passaging of the tumor cells in vitro. Polyploidy was found to be a characteristic feature of the line studied. A large metacentric chromosome persistently present in the cells was accepted as a line marker.

p80/85 cortactin associates with the Src SH2 domain and colocalizes with v-Src in transformed cells.

Expression of oncogenic variants of pp60src leads to dramatic changes in cytoskeletal organization characteristic of transformation. Activated Src associates with the cytoskeletal matrix, resulting in tyrosine phosphorylation of specific cytoskeletal substrates. We have previously shown that stable association of Src with the cytoskeletal matrix is mediated by the Src SH2 domain in a phosphotyrosine-dependent interaction. In this report, we demonstrate that one of the cytoskeletal binding partners of Src is p80/85 cortactin. The association was observed in lysates of transformed cells but was not seen in normal fibroblasts. The interaction could be reconstituted in vitro using transformed cell extracts and a glutathione S-transferase (GST) fusion protein containing the Src SH2 domain but not with GST-Src SH3 or with GST-Src SH2 containing a point mutation in the FLVRES sequence. Confocal microscopy revealed that cortactin redistributed and colocalized with v-Src and a Src SH3 deletion mutant in transformed cells. However, in cells expressing a Src SH2 deletion mutant, the redistribution of cortactin and colocalization with Src did not occur. Furthermore, biochemical fractionation of transformed cells indicated that a significant increase in cortactin distribution to the cytoskeletal fraction occurred, which correlated with a shift in the tyrosine-phosphorylated form of the protein. Cortactin fractionated from cells expressing kinase-defective or myristylation-defective Src mutants did not exhibit this shift. These data suggest a molecular mechanism by which tyrosine phosphorylation of cortactin and association with the Src SH2 domain influence the cytoskeletal reorganization induced in Src-transformed cells.

Engagement of the osteoclast integrin alpha v beta 3 by osteopontin stimulates phosphatidylinositol 3-hydroxyl kinase activity.

Osteoclast adhesion via integrin alpha v beta 3 to the bone matrix protein osteopontin (OPN) results in stimulation of bone resorption. We characterized an immediate signaling event that takes place upon osteoclast interaction with OPN. OPN binding to alpha v beta 3 results in the rapid production of the phosphoinositides (PtdInsP), including phosphatidylinositol triphosphate (PtdIns 3,4,5P3). Stimulation of 3,4,5-PtdInsP3 production by OPN was produced by increased activity of PtdInsP 3-OH kinase, which was found in immunoprecipitates formed by antibodies to integrin alpha v beta 3. The association of PtdIns 3-OH kinase with integrin may have been through association with c-src. The latter was present in immunoprecipitates formed by the antibodies to integrin alpha v beta 3, and src kinase was activated by OPN. These findings demonstrate a mechanism for rapid generation of cell signals upon matrix protein binding to alpha v beta 3, which resembles the mechanisms used by tyrosine kinase growth factor receptors.

The expression profile of alternatively spliced neuronal c-src RNA distinguishes between human tumours of the sympatho-adrenal lineage.

Human neuronal and neuroendocrine tumour specimens and cell lines were analysed regarding proteins and transcripts coded by the proto-oncogene c-src. At the protein level, most of the neuroblastomas and phaeochromocytomas expressed the neuronal c-src form, pp60c-srcN. None of the other neuroendocrine tumours, i.e. paragangliomas, neuroendocrine pancreatic tumours, or carcinoid tumours and small-cell lung carcinomas of different types, appeared to express the neuronal form. In the brain, c-src is transcribed into 3 differently spliced mRNA variants, c-src, c-srcNI, and c-srcNI+NII. The expression of these transcripts was analysed by PCR amplification of fragments covering the mini-exons I and NII of the corresponding cDNAs. The PCR products were analysed by Southern hybridization and characterized by determination of their sequences. Neuroblastomas, paragangliomas, retinoblastomas and the phaeochromocytomas expressed neuronal c-src splice variants. However, whereas neuroblastomas and retinoblastomas contained all 3 transcripts, the phaeochromocytomas and paragangliomas expressed, with 2 exceptions, only the c-src and the c-srcNI+NII mRNA species. To assess whether neuroblastomas display adrenal chromaffin characteristics, they were analysed regarding expression of the chromaffin marker enzyme, phenylethanolamine-N-methyl transferase. Whereas phaeochromocytomas were positive, all neuroblastomas were immuno-chemically negative for this enzyme. These results and the c-src expression profile suggest that neuroblastomas, including those with an adrenal location, do not originate from the adrenal chromaffin differentiation lineage. The data further suggest neuronal c-srcNI mRNA as a marker for sympathetic neuronal cells of the sympatho-adrenal lineage.

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures.

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage-independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage-independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.

Diminished tyrosine protein kinase activity in T cells unresponsive to TCR stimulation.

Tyrosine phosphorylation is thought to be one of the earliest steps in antigenic activation of T cells. Three nonreceptor tyrosine kinases, p56lck, p60fyn, and ZAP-70, are known to be involved in T cell receptor (TCR) signaling, albeit their functional roles appear to be different. Whereas p60fyn and ZAP-70 are functionally associated with the T cell antigen receptor, p56lck is essential for TCR signaling without being directly coupled to the TCR. We have studied a mutant variant of the Jurkat T cell line (J32-3.2), in which basal activities of p56lck and p60fyn are 2- to 2.5-fold reduced relative to those in its parental line (J32) while basal activity of ZAP-70 remains unchanged, and compared responses of J32-3.2 and J32 to TCR stimulation. We have demonstrated that tyrosine phosphorylation following CD3 cross-linking in J32-3.2 cells was extremely short-lived and thus insufficient for the induction of subsequent physiological responses. This was at least partially due to the diminished tyrosine kinase activity in these cells. A decrease in the activity of src-related kinases was caused primarily by their lower expression, whereas expression of ZAP-70 was unchanged but its response to CD3 cross-linking was diminished, correlating with the deficient tyrosine phosphorylation of the CD3 zeta-chain, recently observed in J32-3.2. These data are consistent with the idea that src-related kinases phosphorylate the zeta-chain, which in turn recruits ZAP-70 required to sustain the signal.

Herbimycin A inhibits the association of p60v-src with the cytoskeletal structure and with phosphatidylinositol 3' kinase.

To search for the biochemical prerequisites for morphological transformation by p60v-src, we examined the effect of herbimycin A, a potent inhibitor of cell transformation, on chicken embryonic fibroblasts transformed by Rous sarcoma virus. A small dose of herbimycin (0.1-0.3 micrograms ml-1) was enough to convert the cell morphology to a normal phenotype with a concomitant reassembly of microfilament bundles. In the cells treated with the drug, the majority of the substrates for p60v-src remained phosphorylated and p60v-src was myristylated, membrane associated and fairly active as a protein kinase. Under the same conditions, however, the association of p60v-src with the cytoskeletal structure and with phosphatidylinositol 3' kinase was strongly inhibited, suggesting that the interactions of p60v-src with the cellular structure and the enzyme were indispensable for morphological transformation.

Transformation of NIH3T3 cells by Rous sarcoma virus occurs with high efficiency in the absence of proviral rearrangements or amplification.

NIH3T3 cells could be transformed by a mammaltropic strain of Rous sarcoma virus (RSV) with an efficiency 10(3) times greater than that observed in Balb/c 3T3 cells or other mammalian cell lines and almost identical to that of chick embryo fibroblasts. In infected NIH3T3 cells a single, properly integrated, provirus was sufficient to induce focus formation; moreover, kinase activity of pp60v-src and tyrosine phosphorylation of cellular proteins could be detected very soon after infection in the majority of cells. On the other hand, in transformed foci from RSV-infected Balb/c 3T3 cells both rearrangements and amplification of proviral sequences were frequently detected. Accordingly, expression of pp60v-src and ensuing tyrosine phosphorylation of cellular proteins occurred, at high levels, only in a minority of the infected cells. Furthermore, by using a murine retrovirus carrying the v-src oncogene and an independent selectable marker, we found that Balb/c 3T3 cells were transformed with a 100-fold lower efficiency than NIH3T3 cells, yet the majority of infected untransformed Balb/c 3T3 cells expressed active pp60v-src. These findings are consistent with the existence in most mammalian cell lines of a major restriction to v-src-induced transformation, operating at the level of proviral expression, that is apparently absent in NIH3T3 cells.

Infection of terminally differentiated myotubes with Rous sarcoma virus: reduced synthesis of env and v-src proteins.

We studied Rous sarcoma virus (RSV) protein synthesis in RSV-infected, terminally differentiated chicken myotubes ('late-infected' myotubes), in which no viral DNA integration takes place but all three viral mRNAs (38S, 28S and 21S) are transcribed normally. With the use of specific anti-RSV protein antisera, we found that only the viral gag and pol proteins were synthesized at levels similar to those synthesized in RSV-transformed fibroblasts; the synthesis of env and v-src proteins was significantly reduced in these infected myotubes. We concluded that the viral RNA transcribed from the unintegrated RSV DNA was functional but that genes at the 3' end of the RSV genome were translated at a lower level. By contrast, when mononucleated replicating chicken myoblasts were infected with a mutant (tsNY68) carrying a temperature-sensitive v-src gene and maintained at the non-permissive temperature for this gene, they developed into myotubes with viral DNA integrated in their chromosomal DNA. These 'early-infected' myotubes expressed all four viral genes (gag, pol, env and v-src) at a level similar to that in infected fibroblasts. This result ruled out the possible presence of specific factor(s) in myotubes that preferentially inhibit the 3' genes of RSV, and suggested other translational control(s) of viral gene expression in late-infected myotubes.

Rapid high resolution western blotting: from gel to image in a single day.

A streamlined protocol is described that allows high sensitivity antigen detection by Western blotting in a single day. The choice of membrane blotting matrix, as well as blocking reagents, has been optimized in order to allow rapid development of the blot with chemiluminescent reagents. The entire process, from gel to blot to a permanent, hard copy image on x-ray film, can be accomplished within six hours.

Suppression of src transformation by overexpression of full-length GTPase-activating protein (GAP) or of the GAP C terminus.

Overexpression of the full-length GTPase-activating protein (GAP) has recently been shown to suppress c-ras transformation of NIH 3T3 cells but not v-ras transformation (36). Here, we show that focus formation induced by c-src was inhibited by approximately 80% when cotransfected with a plasmid encoding full-length GAP. In a similar assay, focus formation by the activated c-src (Tyr-527 to Phe) gene was inhibited by 33%. Cotransfection of the GAP C terminus coding sequences (which encode the GTPase-accelerating domain) with c-src or c-src527F inhibited transformation more efficiently than did the full-length GAP, while the GAP N terminus coding sequences had no effect on src transformation. When cells transformed by c-ras, c-src, c-src527F, or v-src were transfected with GAP or the GAP C terminus sequence in the presence of a selectable marker, 40 to 85% of the resistant colonies were found to be morphologically revertant. The GAP C terminus induced reversion of each src-transformed cell line more efficiently than the full-length GAP, but this was not the case for reversion of c-ras transformation. Biochemical analysis of v-src revertant subclones showed that the reversion correlated with overexpression of full-length GAP or the GAP C terminus. There was no decrease in the level of pp60src expression or the level of protein-tyrosine phosphorylation in vivo. We conclude that GAP can suppress transformation by src via inhibition of endogenous ras activity, without inhibiting in vivo tyrosine phosphorylation of cellular proteins induced by pp60src, and that src may negatively regulate GAP's inhibitory action on endogenous ras.

Human cellular src gene product: identification of the myristoylated pp60c-src and blockage of its myristoyl acylation with N-fatty acyl compounds resulted in the suppression of colony formation.

A p60K protein in human colon adenocarcinoma tumor cell lines was identified as a myristoylated pp60c-src by fluorography and radioimmunoprecipitation analysis. Prevention of the myristoylation of pp60c-src was determined with N-fatty acyl glycinal compounds. Of the compounds tested, N-myristoyl glycinal diethylacetal, N-lauroyl glycinal diethylacetal, N-myristoyl glycyl glycinal diethylacetal, and N-myristoyl-4-aminobutyl-aldehyde diethylacetal strongly blocked the myristoylation, but N-decanoyl glycinal diethylacetal and N-palmitoyl glycinal diethylacetal did not. The myristoyl blocking compounds depressed colony formation, cell proliferation, and specific localization to the plasma membrane of pp60c-src. The results taken together suggest that myristoylation of the c-src oncogene product may be very important for tumorigenicity of c-src gene expressed cells.

Wing web or intravenous inoculation of chickens with v-src DNA induces visceral sarcomas.

We have previously found that sarcomas localized to visceral organs frequently arise following wing web inoculation of Rous sarcoma virus. This observation prompted an investigation of whether visceral sarcomas are also inducible by wing web inoculation of a subgenomic viral DNA fragment (v-src DNA) that includes v-src but lacks genes encoding viral structural protein. For this analysis, line SC chickens were inoculated with v-src DNA at 1-2 days posthatch and monitored for 9 weeks. Ninety percent of the chickens developed wing web (primary) tumors at the site of inoculation and, of these, about 30% exhibited visceral tumors. All tumors were histologically identifiable as sarcomas, and both the primary and visceral sarcoma cells were specifically reactive with two monoclonal antibodies elicited to different peptide fragments of pp60v-src. In a separate set of experiments, visceral sarcomas were also observed in about half of the line SC chickens that had been inoculated intravenously with v-src DNA. These results indicate that exogenous progeny virus production is not required for v-src-induced, visceral sarcoma formation. In addition, they demonstrate that intravenous inoculation of v-src DNA is a means to achieve rapid and widespread, disseminated sarcoma growth.