Biomedical subjects

S A Maxwell

Publications and source records attributed to S A Maxwell.

44 records · Page 3Linked to original sources

PubMed1985

Recognition of mos-related proteins with an antiserum to a peptide of the v-mos gene product.

A mos-specific antiserum was generated by injection of rabbits with a peptide predicted from the sequence of the v-mos gene of Moloney murine sarcoma virus (MuSV) strain 124. The peptide is composed of amino acids 37-55 (cyclized at the cysteine residues) conjugated to keyhole limpet haemocyanin. This serum [anti-mos(37-55c)] specifically recognized p37mos in MuSV-124 acutely infected NIH-3T3 cells, P85gag-mos in 6m2 cells, an NRK clone infected with the temperature-sensitive mutant (ts110) of Moloney MuSV, and P100gag-mos in 54-5A4 cells, an NRK clone infected with a spontaneous revertant of ts110. An additional protein of Mr 55000 from uninfected cells was recognized by this serum. Reactivity of the serum toward the v-mos-containing proteins and the 55K protein was completely inhibited by prior incubation with free peptide. The 55K protein was not recognized by antisera made from synthetic peptides prepared from the C-terminal eight or 12 amino acids of v-mos.

Animals↗

PubMed1985

Use of site-specific antipeptide antibodies to perturb the serine kinase catalytic activity of p37mos.

The mos oncogene of Moloney murine sarcoma virus encodes a protein of approximately 37,000 daltons (designated p37mos). We have detected a serine protein kinase activity which is closely associated with p37mos in immune complexes obtained with antibodies [anti-mos(37-55) serum] that were generated with a peptide containing amino acids 37 through 55 of the v-mos protein (S. A. Maxwell and R. B. Arlinghaus, Virology 143:321-333, 1985). Immune complexes that were derived with antibodies generated against peptides representing the C-terminal 8 or 12 amino acids of v-mos (anti-C2 and anti-C3 serum, respectively) exhibited very little kinase activity capable of phosphorylating p37mos. Treatment of anti-mos(37-55) complexes containing active v-mos kinase with anti-C3 or anti-C2 serum resulted in a dramatic reduction of the in vitro phosphorylation of p37mos. Antiserum blocked with the appropriate C-terminal peptide had no inhibitory effect on the phosphorylation of p37mos in anti-mos(37-55) complexes which indicated that the inhibition of v-mos kinase activity was a specific effect of these antibodies. The specific inhibition of the in vitro phosphorylation of p37mos by antibodies directed against the C terminus of the v-mos protein provides strong evidence that the v-mos gene encodes a serine protein kinase. In addition, the extreme C terminus of p37mos may be critical for an active v-mos kinase.

Antibodies↗

PubMed1984

Further characterization of the P85gag-mos -associated protein kinase activity.

The gag-mos hybrid protein encoded by ts110 MoMuSV was shown to have an associated protein kinase activity which phosphorylated both P85gag-mos and P58gag when [gamma-32P]ATP and a manganese cofactor were added to an immune complex containing P85gag-mos. Immunoprecipitation and removal of P85gag-mos from the reaction mixture by either an anti-mos or anti-gag serum resulted in a subsequent elimination of in vitro P85gag-mos and P58gag phosphorylation. This kinase activity was shown to be either an intrinsic property of P85gag-mos or else a tightly bound cellular enzyme activity resistant to elution with 2.0 M NaCl, 0.5% deoxycholate, and 0.1% SDS. A correlation was made between the amount of kinase activity and the concentration of P85gag-mos. Viral gag antisera were also used to show immune complex phosphorylation of another gag-mos hybrid protein termed P100gag-mos, derived from a revertant of ts110. In vitro phosphorylation experiments derived from v-mos transformed MuSV 124 cells using viral gag antisera were completely negative which shows that the gag-mos kinase in 6m2 cells is not merely a gag-associated kinase that phosphorylates MuSV coded gag gene products. When shifting 6m2 cells from a permissive temperature to the nonpermissive temperature of 39 degrees for 2-4 hr, a noticeable change toward a more normal morphology occurs. NRK 54-5A4 cells infected with a revertant of ts110 with wild-type phenotype, showed little change in morphology between permissive and nonpermissive temperatures. In addition to the ts defect affecting P85gag-mos production previously reported, a second ts defect in ts110 is reported here which is functional in nature; it can be detected within 5 min after shift to 39 degrees by the heat lability of the P85-associated kinase activity. The P100gag-mos protein kinase from the wild-type revertant cells did not exhibit this heat sensitivity under similar conditions. The thermal inactivation of the P85 kinase was shown to precede events that occur as cells are shifted to the restricted temperature including morphological reversion to the normal phenotype, and the decrease in P85gag-mos concentration. Based on all of these observations, it is suggested that the P85-associated kinase activity is not merely an adherent cellular kinase, but actually a function of the gag-mos gene product.

Amino Acid Sequence↗

PubMed1983

P85gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity.

A protein identified as P85(gag-mos) was shown to be phosphorylated when immunoprecipitates from ts110 Moloney murine sarcoma virus transformed nonproducer cells (clone 6m2) were incubated with [gamma-(32)P]ATP. The in vitro-labeled 85,000-dalton phosphoprotein comigrated on NaDodSO(4)/polyacrylamide gels with authentic phosphorylated P85(gag-mos). Immunoprecipitates obtained with antisera prepared against Rauscher murine leukemia virus core protein p30 were active in the immune complex kinase assay but anti-murine leukemia virus p10 precipitates were not. Previous studies have shown that anti-p30 but not anti-p10 antisera recognize P85(gag-mos). The 6m2 clone has been shown to express P85(gag-mos) at 33 degrees C but not at 39 degrees C. Anti-p30 immune complexes from 6m2 cells maintained at 39 degrees C failed to phosphorylate the 85,000-dalton protein. Furthermore, the in vitro phosphorylated 85,000-dalton protein gave the same pattern of V8 protease-generated cleavage products as in vivo(32)P-labeled P85(gag-mos). We conclude from these results that P85(gag-mos) is phosphorylated in anti-p30 immune complex kinase reactions. Phosphoamino acid analyses indicated that the in vitro phosphorylated P85(gag-mos) contained phosphoserine and phosphothreonine. Our findings indicate that incubation of anti-p30 immunoprecipitates at 39 degrees C drastically reduced, in a specific way, the kinase activity associated with P85(gag-mos). This result and other data suggest that the kinase is virus-encoded. Because P85(gag-mos), but not Pr65(gag) is phosphorylated in anti-p30 immunoprecipitates from MuLV-MuSV ts110 producer cells, the kinase enzyme is associated with P85(gag-mos) and not gag gene products. A second major polypeptide of the size of P58(gag) was also phosphorylated in anti-p30 immunoprecipitates from cells maintained at 33 degrees C but not at 39 degrees C. Since 6m2 cells at 39 degrees C contain P58(gag), this is also consistent with the kinase activity being associated with P85(gag-mos).

Animals↗

PubMed1982

Electrophysiologic studies of cutaneous nerves of the forelimb of the cat.

The cutaneous innervation of the forelimb was investigated in 20 barbiturate-anesthetized cats by using electrophysiological techniques. The cutaneous area (CA) innervated by each cutaneous nerve was delineated in at least six cats by brushing the hair in the CA with a small watercolor brush while recording from the nerve. Mapping of adjacent CA revealed larger overlap zones (OZ) than were noted in the dog. Remarkable findings were that the brachiocephalic nerve arose from the axillary nerve and the CA comparable to that supplied by the cutaneous branch of the brachiocephalic nerve in the dog was supplied by a cutaneous branch of the suprascapular nerve. The CA supplied by the communicating branch from the musculocutaneous to the median nerve was similar in both species except that the communicating branch arose proximal to any other branches of the musculocutaneous nerve in the cat, whereas it was a terminal branch in the dog. The superficial branch of the radial nerve gave off cutaneous brachial branches in the cat proximal to the lateral cutaneous antebrachial nerve. The CA of the palmar branches of the ulnar nerve did not completely overlap the CA of the palmar branches of the median nerve as occurred in the dog; thus an autonomous zone (AZ) for the CA of the palmar branches of the median nerve is present in the cat, whereas no AZ existed for the CA of this nerve in the dog.

Animals↗

PubMedDate not supplied

Selective compartmentalization of different mdm2 proteins within the nucleus.

Overexpression of the mdm2 protooncogene protein, which can lead to the inactivation of normal p53, has been observed in some human cancers. The mdm2 gene is positively regulated by p53, providing for a feedback loop in the control of both p53 and mdm2 activity. The expression of the mdm2 and p53 proteins in different non-small cell lung carcinoma (NSCLC) cell types harboring wild-type or mutant p53, or lacking p53 altogether, were investigated to determine whether a correlation exists between the expression of these two proteins. The mdm2 protein was expressed at very low levels in all NSCLC lines examined, regardless of the p53 status. To determine whether mdm2 could be induced by p53 in NSCLC, NSCLC cells were transfected with a recombinant adenovirus expressing high levels of wild-type p53. The highest levels of exogenous wild-type p53 were observed in p53-null H358 and H1299 cells and in H226b cells expressing endogenous wild-type p53 were observed in p53-null H358 and H1299 cells and in H226b cells expressing endogenous wild-type p53. In these cells, wild-type p53 induced the expression of 90/92K M(r) mdm2 proteins, as well as several faster-migrating mdm2-related species exhibiting relative mobilities of 76/78K, 57/59K, 46K, 28K, and 12K. Northern analyses of H358 and H1299 cells transfected with wild-type p53 showed that these cells expressed three species of mdm2 mRNA of 5.5, 4.6-3.8, and 2.1 Kb in size. Subcellular fractionation revealed that the 90/92K M(r) mdm2 protein species was localized to both the crude plasma membrane/cytoplasmic and nuclear fractions, and that the smaller mdm2 proteins associated selectively with different nuclear substructures. The 76/78K, 57/59K, and 46K Mr(r) mdm2 proteins may be derived by differential splicing of the 5.5 Kb mRNA, and their differential compartmentalization within the nucleus suggests that each has a distinct function, potentially in the regulation of p53 and other gene products.

Carcinoma, Non-Small-Cell Lung↗

PubMedDate not supplied

Two-dimensional gel analysis of p53-mediated changes in protein expression.

The p53 gene product suppresses both tumor cell growth and the cellular transformation process promoted by oncogenes. Although several genes are known to be positively regulated by p53, it is unclear how many gene expression events are involved in p53-mediated growth arrest and apoptosis. Two-dimensional gel electrophoresis was employed to investigate changes in whole-cell protein expression in p53-null H1299 cells that were transfected with a recombinant adenovirus expressing wild-type p53 at high efficiency. The two-dimensional gel analysis was restricted to proteins ranging in mass from 12,000 to 80,000 daltons. A total of 17 proteins were induced and one was repressed within 16 h of expression of exogenous wild-type p53 in H1299 cells. These results indicate that p53-mediated growth suppression involves a complex array of gene expression events.

Adenoviruses, Human↗

PubMedDate not supplied

Retinoblastoma protein in non-small cell lung carcinoma, cells arrested for growth by retinoic acid.

The growth of non-small cell lung carcinoma (NSCLC) cells was inhibited by retinoic acid after 16 h of treatment. However, the growth of all cell lines except one became refractory to retinoic acid after 48 h of exposure. The expression of the hyperphosphorylated retinoblastoma protein species (RB p110) was observed to be increased fivefold to tenfold in NSCLC cells within 16 h of exposure to retinoic acid. In the H460a and H226b cell lines, p110 showed some conversion to the underphosphorylated p105 form after 24 h of retinoic acid treatment. After 48 h, p105 became the predominant form, along with a 60K M(r) species. After 72 h, expression of all RB protein species became almost undetectable in H460a and H226b cells, concomitant with increases in cell growth rates. A different pattern of RB expression was observed in the H322j and H358 cell lines. In these cells, both p110 and p105 were induced within 8 h of retinoic acid treatment. Furthermore, the elevated levels and the phosphorylation state of RB in retinoic acid-treated H322j and H358 cells remained essentially unchanged for up to 72 h and only low amounts of the 60K M(r) species were detected. I believe that a post-translational mechanism is responsible for the retinoic acid-mediated induction of RB, since levels of RB mRNA remained relatively unchanged during the time course of retinoic acid exposure. I conclude that retinoic acid inhibited the growth of NSCLC cells by inducing high levels of RB and that increases in RB levels occurred as a result of either an increase in stability of the protein or by downregulation of an RB-specific protease. The inhibition of growth by retinoic acid must involve other molecular events, since the H596b cell line, which lacks RB protein, exhibited growth properties in the presence of retinoic acid similar to those of cells in which RB expression was induced.

Blotting, Northern↗