Biomedical subjects

D C Flynn

Publications and source records attributed to D C Flynn.

24 records · Page 2Linked to original sources

PubMed1993

Identification and sequence analysis of cDNAs encoding a 110-kilodalton actin filament-associated pp60src substrate.

Transformation of chicken embryo cells by oncogenic forms of pp60src (e.g., pp60v-src or pp60527F) is linked with a concomitant increase in the steady-state levels of tyrosine-phosphorylated cellular proteins. Activated forms of the Src protein-tyrosine kinase stably associate with tyrosine-phosphorylated proteins, including a protein of 110 kDa, pp110. Previous reports have established that stable complex formation between pp110 and pp60src requires the structural integrity of the Src SH2 and SH3 domains, whereas tyrosine phosphorylation of pp110 requires only the structural integrity of the SH3 domain. In normal chicken embryo cells, pp110 colocalizes with actin stress filaments, and in Src-transformed cells, pp110 is found associated with podosomes (rosettes). Here, we report the identification and characterization of cDNAs encoding pp110. The predicted open reading frame encodes a polypeptide of 635 amino acids which exhibits little sequence similarity with other protein sequences present in the available sequence data bases. Thus, pp110 is a distinctive cytoskeleton-associated protein. On the basis of its association with actin stress filaments, we propose the term AFAP-110, for actin filament-associated protein of 110 kDa. In vitro analysis of AFAP-110 binding to bacterium-encoded glutathione S-transferase (GST) fusion proteins revealed that AFAP-110 present in normal cell extracts binds efficiently to Src SH3/SH2-containing fusion proteins, less efficiently to Src SH3-containing proteins, and poorly to SH2-containing fusion proteins. In contrast, AFAP-110 in Src-transformed cell extracts bound to GST-SH3/SH2 and GST-SH2 fusion proteins. Analysis of AFAP-110 cDNA sequences revealed the presence of sequence motifs predicted to bind to SH2 and SH3 domains, respectively. We suggest that AFAP-110 may represent a cellular protein capable of interacting with SH3-containing proteins and, upon tyrosine phosphorylation, binds tightly to SH2-containing proteins, such as pp60src or pp59fyn. The potential roles of AFAP-110 as an SH3/SH2 cytoskeletal binding protein are discussed.

Actins↗

PubMed1992

Tyrosine phosphorylation of a 120,000 dalton membrane-associated protein by the neural form of pp60c-src, pp60c-src+.

The c-src proto-oncogene encodes a 60,000 dalton tyrosine kinase, pp60c-src, which is the prototype member of the family of non-receptor tyrosine kinases. A neural-specific form of pp60c-src, pp60c-src+, is detected only in neurons of the central nervous system. pp60c-src+ contains a six amino acid insert (neural insert) in the SH3 region that is generated by alternative splicing. Previous reports indicate that the profiles of proteins phosphorylated on tyrosine in chick embryo fibroblast (CEF) cells by pp60c-src+ or pp60c-src are equivalent. In this report, the activities of pp60c-src+ and pp60c-src, as well as the activated variants, pp60(527F+) and pp60(527F), were compared in CEF cells by examining the steady-state levels of tyrosine phosphorylation of several known pp60src substrates. Most substrates examined were phosphorylated on tyrosine to equivalent levels in CEF cells expressing either the neural- or fibroblast-specific src gene products. However, the relative extent of tyrosine phosphorylation of a 120 kDa protein (p120) was increased in cells expressing the neuronal forms of either c-src or c-src527F. The increased tyrosine phosphorylation of p120 did not appear to be caused by the neural insert facilitating a specific interaction between pp60c-src+ and p120. These data indicate that preferential phosphorylation of p120 in neural cells may contribute to the specialized function of pp60c-src+ in neural cells.

Animals↗

PubMed1990

A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction.

A conformational change in the structure of Sindbis (SB) virus was detected after virion attachment to baby hamster kidney cells but before internalization. The alteration was manifested as increased virion binding of certain glycoprotein E1 and E2 monoclonal antibodies (MAbs) that recognized transitional epitopes. These epitopes were inaccessible to MAb on native virions but became accessible to their cognate MAbs in the early stages of infection. Transit of virions through a low-pH compartment apparently was not required for the conformational change. Exposure of transitional epitopes was unaffected by treatment of BHK cells with NH4Cl and occurred normally in Chinese hamster ovary cells temperature sensitive for endosomal acidification. However, the rearrangement was correlated with both the time course and temperature dependence of SB virus penetration, and the rearrangement occurred earlier with an SB virus mutant having an accelerated penetration phenotype. In addition, MAb to a transitional epitope, a probe specific for rearranged particles, retarded penetration of infectious virions. These results suggested that the SB virus E1/E2 glycoprotein spike undergoes a structural rearrangement as a consequence of virion interaction with the cell surface and that this altered virion form may be an important early intermediate in an entry pathway leading to productive infection.

Ammonium Chloride↗

PubMed1988

Antibody-mediated activation of Sindbis virus.

The biological activity of an anti-Sindbis monoclonal antibody (MCAB 49) has been explored. The antibody recognizes an epitope on the E2 glycoprotein of Sindbis virus and, in the presence of complement (C'), neutralizes virus infectivity. In the absence of C', reaction of the antibody with our laboratory strain of Sindbis, SB, increased the number of plaque-forming units (PFU) detected on baby hamster kidney (BHK) cells rather than neutralizing virus infectivity. The elevated titers of SB approached, but never exceeded, the number of virions calculated from the particle:PFU ratio, indicating that the additional PFU might have resulted from activation of normally noninfectious particles. The apparent activation could not be attributed to disaggregation of SB by MCAB 49 as shown by ultraviolet inactivation experiments with antibody-treated and untreated virus preparations. Fc receptors did not appear to be involved in the antibody-mediated activation. Fab' and F(ab')2 fragments of MCAB 49 also increased the number of observed PFU of SB. Control monoclonal antibodies of the same isotype, but specific for the tobacco etch virus capsid protein, were unable to compete for cellular binding sites with the SB/MCAB 49 complex. Rather, the SB/MCAB 49 complex appeared to utilize the same receptor(s) as SB in that SB and the SB/MCAB 49 complexes competed with each other for binding sites on BHK cells. Binding studies with 32P-labeled SB showed that a higher proportion of MCAB 49 activated virions than untreated virions associated with BHK cells. Moreover, activated virions were much less susceptible to elution. These results suggest that reaction which MCAB 49 may facilitate successful attachment of SB to its receptor, or receptors, on BHK cells.

Animals↗

PubMedDate not supplied

Efficacy of taurine based compounds as hydroxyl radical scavengers in silica induced peroxidation.

While it is widely believed that taurine may play an important role in protecting cells against toxic injury by functioning as an antioxidant, there is a lack of evidence to support this hypothesis. In this study, electron spin resonance (ESR) was used to investigate the reaction of taurine and hypotaurine with hydroxyl radicals (.OH). The Fenton reaction (Fe(II) + H2O2-->Fe(III) + .OH + OH-) and the Cr(V)-mediated Fenton-like reaction (Cr(V) + H2O2-->Cr(VI) + .OH + OH-) were used as sources of .OH radicals. The results show that hypotaurine but not taurine effectively scavenges .OH radicals with a reaction rate constant of k = 1.6 x 10(10) M-1s-1. That is comparable with other efficient .OH radical scavengers. The effect of taurine and hypotaurine on silica-induced lipid peroxidation was evaluated using linoleic acid as a model lipid. Hypotaurine, but not taurine, caused a significant inhibition of silica-induced lipid peroxidation. The results show that hypotaurine is an excellent antioxidant and appears to have the potential for being a therapeutic agent against silica-induced lung injury.

Antioxidants↗

PubMedDate not supplied

Vanadium (IV) formation in the reduction of vanadate by glutathione reductase/NADPH and the role of molecular oxygen.

Experimental evidence documenting the formation of a relatively stable V(IV) species appears to be important with regard to the biochemical mechanism of reduction of vanadate by enzymatic systems. The present study demonstrates that a mixture of vanadate and glutathione reductase/nicotinamide-adenine-dinucleotide phosphate (NADPH), in phosphate (pH 7.2) buffer generates V(IV) under ambient conditions. Once formed, V(IV) does not rapidly autoxidize so as to defy detection by electron spin resonance (ESR) spectroscopy. The aerobic environment was guaranteed by preparing reaction mixtures in well stirred, wide mouth, standard test tubes in air over a period of 50 minutes, and by making ESR measurements in nuclear magnetic resonance (NMR) sample tubes as well as oxygen-permeable Teflon tubes. The V(IV) ESR signal intensity was found to increase linearly with time elapsed after reaction initiation. The linear growth of the V(IV) species also shows that this species is fairly stable, over a period of at least 50 minutes. Similar V(IV) stability data were obtained from VOSO4, a model compound as a source of V(IV). The results obtained in the present study demonstrated that V(IV) can be generated in the reduction of V(IV) by glutathione reductase in the presence of NADPH under aerobic condition.

Electron Spin Resonance Spectroscopy↗