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S A Maxwell

Publications and source records attributed to S A Maxwell.

At least 37 records · Page 2Linked to original sources
PubMed1993

Sequence-specific interaction of a conformational domain of p53 with DNA.

Mutations within a conserved "conformational" domain of the p53 protein have frequently been observed in a wide variety of human cancers. A hybrid protein containing the wild-type conformational domain of p53 fused to protein A bound to calf thymus DNA and a specific p53 DNA-binding motif. Hybrid proteins containing mutations in p53 bound to DNA less efficiently than wild-type hybrid protein. In addition, competition experiments showed that mutated p53 DNA-binding motif failed to interact with p53 hybrid proteins. The DNA-binding activity of wild-type p53 hybrid protein was inhibited by the metal chelator 1,10-phenanthroline. These results demonstrate that DNA-binding activity resides in the conformational domain of p53, providing a structural model for disruption of DNA binding by mutation. Furthermore, metal ions may regulate binding of p53 to DNA by modulating its conformation.

Animals↗
PubMed1993

Mental health services for medical students: perceptions of students, student affairs deans, and mental health providers.

PURPOSE: To examine (1) whether there is any consistency among medical schools in mental health services provided and (2) how these services are perceived by student affairs deans, mental health service providers, and the students themselves. METHOD: Questionnaires were sent in October 1991 to the student affairs dean (or director), the individual responsible for student mental health services, and a student representative in each of the 126 U.S. and Canadian medical schools. Data were sought regarding personnel, individuals served, location, hours, administration, funding, confidentiality, administrative referrals, and respondents' suggestions for improvement. Possible differences among the three groups of respondents were tested by chi-square. RESULTS: Responses were received from 75 student affairs deans, 53 mental health providers, and 30 students. There was much diversity among schools in services provided, especially in the areas of administration and funding. Although perceptions of the three respondent groups were often the same, they differed significantly in a number of areas. Suggestions for improvement of services involved funding, personnel, hours, confidentiality and privacy, specialty services, preventive and support programs, and visibility. The suggestion most frequently made by the students was for increased information and visibility. CONCLUSION: The differences among schools coupled with the differing perceptions within schools indicate a need for a comprehensive consideration of what kinds of mental health services are needed and how they can best be made accessible to a diverse body of students experiencing a variety of academic and personal challenges.

Canada↗
PubMed1993

Binding of cellular proteins to a conformational domain of tumor suppressor protein p53.

The genes regulated by p53, as well as the factors modulating its function, need to be identified before the mechanism of action of p53 in control of cell growth can be adequately understood. Binding of the SV40 large T-antigen protein to an evolutionally conserved (conformational) domain of p53 inhibits p53's DNA-binding and transcription activation activities. Cellular proteins might also bind to this same region of p53 to regulate its function. A hybrid protein composed of protein A fused to the conformational domain (amino acids 115-295) of p53 was expressed in Escherichia coli and used as an affinity probe for binding proteins in detergent lysates of non-small cell lung carcinoma (NSCLC) cells. The wild-type p53 hybrid protein associated with several major proteins of molecular weights 45 K, 56 K, and 70 K, as well as other minor species ranging in molecular weight from 30 K to 90 K. These proteins bound specifically to the p53 sequence of the hybrid protein. Protein A did not associate with these proteins and the two p53 hybrid proteins containing missense mutations at codons 273 and 175 exhibited a 40-80% weaker association. In addition, T antigen competed with the cellular proteins for binding to the conformational domain. The conditions of cell growth had a profound effect on the expression of the p53 binding proteins. Considerably more p53 binding proteins were expressed in actively growing cells than in cultures maintained under conditions for slow growth. Quantitative differences in expression of p53-binding proteins were observed among different NSCLC cell lines. The expression of p53-binding proteins was not restricted to NSCLC cell lines; detergent extracts of an osteosarcoma cell line yielded similar p53-binding proteins.

Antigens, Polyomavirus Transforming↗
PubMed1993

Analysis of data.

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Adolescent↗
PubMed1991

Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells.

The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000-60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the Mr 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the beta subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperature-dependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoelectron microscopy. Treatment of mKSA cells with 10 microM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control.

Amino Acid Sequence↗
PubMed1989

Epidermal growth factor receptor protein-tyrosine kinase activity in human cell lines established from squamous carcinomas of the head and neck.

Two cell lines established from tumors of the head and neck area at different clinical stages were found to differ in the expression and in the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Cell line 183A was derived from an early-stage tumor and cell line 1483 was derived from a tumor that had metastasized to lymph nodes. The 1483 cells displayed a higher plating efficiency and clonogenicity in soft agar, suggesting a more tumorigenic phenotype over the 183A cells. Analyses of EGF receptor levels by using R1 anti-EGF receptor serum indicated that the 1483 cells expressed 5-fold more receptor than did the 183A cells. EGF receptors isolated from each cell line were active for kinase activity in an immune complex kinase assay, using monoclonal R1 anti-EGF receptor antibody. The autophosphorylation activity of both receptors was stimulated by addition of EGF to isolated membrane preparations and intact cells, although the EGF receptor of the 1483 cells was much less responsive to EGF than the receptor from 183A cells. In addition, the 1483 receptor consistently incorporated about twice as much phosphate as did the 183A receptor in an immune complex kinase assay. These data suggest that the basal tyrosine kinase activity of the EGF receptor from 1483 cells may be more active than the EGF receptor kinase from 183 cells.

Adenosine Triphosphate↗
PubMed1989

Solubilization of SV40 plasma-membrane-associated large tumor antigen using single-phase concentrations of 1-butanol.

The nature of the interaction of the simian virus 40 (SV40) transforming protein, large tumor antigen (T-ag), with the plasma membrane of transformed cells is not well understood. We report here that SV40 plasma-membrane-associated large tumor antigen (pmT-ag) can be solubilized by using single-phase concentrations of 1-butanol. Purified plasma membranes from SV40-transformed mouse cells yielded T-ag when treated with 2.5% butanol; solubilization of T-ag from the purified membranes in butanol was temperature dependent, with approximately 10-fold more T-ag extracted at 37 degrees C than at 22 degrees C; and application of 2.5% butanol to mKSA cells after cellular surface proteins had been radiolabeled with 125I resulted in the release of iodinated T-ag. Butanol-extracted pmT-ag coprecipitated with p53 and several cellular proteins ranging in size from 35 to 60 kDa. One cellular component migrated at a mobility similar to that of tubulin (56 kDa), and a monoclonal antibody against the alpha subunit of tubulin coprecipitated T-ag. Immunoblotting of proteins immunoprecipitated with monoclonal antibodies against T-ag or p53 from butanol extracts with a monoclonal antibody against the beta subunit of tubulin revealed specific coprecipitation of tubulin with T-ag and p53. This suggests that complexes composed of tubulin, T-ag, and p53 exist in butanol extracts. Control experiments eliminated the possibility of an artifactual association of tubulin with T-ag and p53 induced by butanol. Two-dimensional gel analyses revealed that 2.5% butanol at 37 degrees C extracted a subset of membrane-associated proteins and some cytosolic proteins, as well as a number of proteins that were not soluble in either high salt or detergent. Thus, the butanol extraction conditions employed in this study recovered a species of pmT-ag that appears to complex with tubulin. As butanol reportedly is less deleterious to native protein structures than other agents, including high salts and detergents, this extraction procedure may be useful for studying the structure and function of other membrane-associated proteins.

1-Butanol↗
PubMed1989

SV40 T-antigen as a dual oncogene: structure and function of the plasma membrane-associated population.

SV40 T-antigen (T-ag) is localized in both the nucleus (nT-ag) and plasma membrane (pmT-ag) of cells and provides multiple functions necessary for cell transformation. The pmT-ag population is structurally very similar to the nT-ag. Transport to the cell surface is by an unknown mechanism that does not involve the secretory pathway. The disposition of T-ag in the membrane exposes both the amino and the carboxyl terminus on the exterior of the cell. Nuclear-transport-defective mutants of T-ag can transform established cells in culture, but not primary cells, suggesting that non-nuclear forms of T-ag may mediate some transformation-related process(es). A non-cytolytic protein extraction technique utilizing 1-butanol solubilized from SV40-transformed cells a multimeric complex composed of pmT-ag and at least five cellular proteins ranging in size from 35,000 (35K) to 60K M. Both amino- and carboxylterminal T-ag-specific monoclonal antibodies co-precipitated T-ag and the 35-60K Mr proteins, but antibodies against the internal portion of T-ag precipitated only uncomplexed T-ag. The growth state of the cells markedly influenced the expression of the T-ag-containing surface complexes; more complexes were recovered from actively dividing cells than from confluent cell cultures, and suspension cells yielded more complexes than cells on a substratum. The complex exhibited a highly dynamic association with the cell membrane, as demonstrated by pulse-chase analysis. The characteristics of growth-dependent expression and rapid turnover rate suggest a functional role for the membrane complex. The identities of the cellular proteins in the complex with pmT-ag are unknown, although one member (56K) is recognized by p53-specific monoclonal antibodies.

Animals↗
PubMed1988

Potassium inhibition of transforming protein P85gag-mos and reversal of the transformed phenotype in 6m2 cells.

K+ at high concentrations (52-72 mM hypertonic KCl) has been reported to induce reverse transformation in the 6m2 cell, which is a clone of normal rat kidney cells (NRK) infected with a temperature-sensitive transformation virus. When exposed to high K+, 6m2 cells grown at the permissive temperature (33 degrees C) exhibit normal morphology and reduced soft agar growth, characteristics of cells grown at nonpermissive temperature (39 degrees C). In the current study, flattening of cells and rearrangement of surface microvilli were demonstrated by scanning electron microscopy to occur within 6 hr of exposure to high K+, similar to the effect of temperature shift to 39 degrees C. Exposure to K+ resulted in a 90% inhibition of P85gag-mos-associated serine kinase activity within 5 min, with a subsequent reduction of up to 75% of the synthesis of this protein. These alterations in the putative transforming protein were similar to those induced by temperature shift and were considered to be the basis for retrotransformation. The cell microtubular system and F-actin cables were affected more slowly by K+ than by a temperature shift to 39 degrees C. The former did not achieve the fine reticulum network seen in NRK cells until 72 hr later, but the latter remained aberrant. The effect on the enzyme might be mediated by alteration in phosphorylation, but the mechanism by which kinase inactivation induces retrotransformation is not yet known.

Actins↗
PubMed1987

Analysis of P210bcr-abl tyrosine protein kinase activity in various subtypes of Philadelphia chromosome-positive cells from chronic myelogenous leukemia patients.

An altered c-abl gene product (P210bcr-abl) possessing associated tyrosine protein kinase activity was recently been reported in several blast chronic myelogenous leukemia (CML) cell lines. We have examined different morphological types of leukocytes directly obtained from patients at the blast crisis stage of CML for expression of P210bcr-abl tyrosine protein kinase activity. Phosphorylation of P210bcr-abl in an immune complex kinase assay using an anti-v-abl peptide serum was observed in blast cells from four Philadelphia chromosome (Ph1)-positive CML patients in blast crisis. P210bcr-abl protein kinase activity was detected regardless of whether the blast cells were of myeloid, lymphoid, or undifferentiated morphology. P210bcr-abl protein kinase activity was not detected in immune complexes either from leukocytes of four Ph1-negative CML patients in blast crisis, of five acute myelogenous leukemia patients, or in the promyelocytic cell line HL-60. Mature myeloid cells are associated with an inhibitory factor for not only P210bcr-abl protein kinase activity, but also protein kinases in general. Therefore, analyses of Ph1-positive benign phase CML myeloid cells, the majority of which are well differentiated, could not be successfully performed. The inhibition of P210bcr-abl protein kinase activity is not a specific property of mature cells from CML patients since granulocytes from a normal volunteer also demonstrated a similar effect. However, extracts of Ph1-positive cultured B-lymphocytes from a patient in benign phase demonstrated active P210bcr-abl protein indicating that the P210bcr-abl protein is expressed in an enzymatically active form in the earlier phases of CML. In addition to the previously reported P210 and P190 abl-related proteins, a novel Mr 53,000 protein was found to undergo phosphorylation at serine and tyrosine in immune complex kinase assays of two blast crisis CML cell lines (K562 and EM2) and in samples from blast crisis patients in which P210bcr-abl was detected. Peptide mapping by the Cleveland technique suggested that Mr 53,000 protein is unrelated to P210bcr-abl. Immune complex kinase assays of K562 cells with an anti-src serum (GD-11) yielded active c-src kinase and a Mr 50,000 phosphorylated protein, both of which were resistant to alkaline hydrolysis. Peptide mapping suggested that Mr 53,000 protein is related to Mr 50,000 protein which is precipitated with P210bcr-abl as an Mr 300,000 protein complex.

Amino Acids↗
PubMed1987

Skin effects of trichothecenes and their amelioration by decontamination.

The ability of trichothecenes, in particular T2 toxin (T2), to cause irritant effects on the skin has been investigated in laboratory rodents and rabbits. Quantitatively, T2 was found to be highly potent in this respect, causing irritant reactions on rat skin at contamination densities of less than 1 microgram.cm-2. The first appearance of skin effects was delayed for approximately 6 h after application, irrespective of the dose applied. Similarly, variation in solvent or injection into or beneath the skin failed to accelerate the onset of the irritant reaction. An aqueous soap solution was largely effective in removing low doses of T2 from the skin, but was ineffective in removing larger doses. However, washing the contaminated skin with polyethylene glycol 300 was very effective at removing even large doses of T2 from the skin. The macrocyclic trichothecene verrucarin A was of comparable irritancy to T2 on rat skin. Diacetoxyscirpenol and nivalenol were less potent.

Animals↗
PubMed1986

Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells.

The expression of epidermal growth factor (EGF-R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr approximately 170,000, with a migration in sodium dodecyl sulfate-polyacrylamide gels identical to the EGR-R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr approximately 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the v-erb-B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I-labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF-R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.

Brain Neoplasms↗
PubMed1986

The in vitro penetration and distribution of T-2 toxin through human skin.

The penetration and distribution of T-2 toxin in excised human abdominal skin has been determined for a dose range of 1.0-2.6 micrograms/cm2 skin using an ethanol vehicle and a saline receptor solution. In all cases the overall percentage penetration of T-2 after 48 h was low, the greatest amounts of toxin being present in the stratum corneum with less in the epidermis and relatively little in the dermis. Vehicle: skin partition coefficients support this finding. Neither penetration nor distribution were changed by a rabbit serum receptor solution. Electron micrographs showed that at 1.8 micrograms/cm2 and above the contents of the intercellular space are leached out to leave the integument as a porous membrane. The distribution of T-2 within the skin after 48 h would suggest that for doses up to 2.6 micrograms/cm2 the irritative and inflammatory effects on the skin would be of more immediate concern than would systemic toxicity.

Diffusion↗
PubMed1985

Serine kinase activity associated with Maloney murine sarcoma virus-124-encoded p37mos.

An antiserum directed against amino acid residues 37-55 [anti-mos (37-55) serum] of the predicted v-mos sequence was used to precipitate p37mos from Moloney murine sarcoma virus-124 (Mo-MuSV-124) acutely infected 3T3 cells. Proteins with sizes ranging from p37mos to 43 kDa (p43) were found to be phosphorylated when anti-mos (37-55) immune complexes containing p37mos were incubated with [gamma-32P]ATP and Mn2+. The phosphorylation of p37mos and p43 could be specifically blocked when the anti-mos (37-55) serum was incubated with 37-55 cyclic mos peptide prior to immunoprecipitation, but not if the serum was preincubated with an unrelated peptide representing amino acids of the myc protein sequence. Anti-mos (37-55) immune complexes from uninfected 3T3 cells did not produce any phosphorylated proteins the size of p37mos or p43. However, a 50-kDa protein (p50) was phosphorylated in both unblocked and mos peptide-blocked anti-mos (37-55) immune complexes from infected 3T3 cells, and in immune complexes from uninfected cells. Quercetin, an inhibitor of some protein kinases, inhibited the kinase phosphorylating p50 but not the kinase phosphorylating p37mos and p43. Preabsorption of the cell extract prior to immunoprecipitation with an excess of formalin-fixed Staphylococcus aureus, complexed with preimmune normal rabbit serum IgG, specifically removed the kinase phosphorylating p50. The amount of in vitro phosphorylated p37mos and p43 in the immune-complex kinase assay reached a maximum in extracts of 3T3 cells 2-3 days postinfection with Mo-MuSV 124 but decreased to trace levels after 5 days. Metabolically and in vitro phosphorylated p37mos generated an identical pattern of phosphopeptides upon partial V8 protease digestion. Based on peptide mapping and a kinetic analysis of the in vitro phosphorylation reaction, p37mos appears to be a precursor to the p43 phosphorylated species. Phosphoamino acid analyses revealed only phosphoserine in in vitro phosphorylated p37mos and p43mos. It was concluded that p37mos is closely associated with a serine kinase activity and that the in vitro phosphorylation of p37mos may lead to formation of a highly modified mos protein (p43) by way of superphosphorylation.

Animals↗
PubMed1985

Further characterization of the in vitro products generated by proteolytic cleavage of Gazdar murine sarcoma virus p65gag.

In vitro proteolytic cleavage of the Gazdar murine sarcoma virus (Gz-MuSV) p65gag polypeptide (Gz-p65gag) was facilitated by detergent-disrupted Moloney murine leukaemia virus (Mo-MuLV). Incubation of radioactively labelled Gz-p65gag in the presence of unlabelled Mo-MuLV under optimal conditions resulted in the cleavage of Gz-p65gag to proteins of 40000 (P40) and 25000 (P25) Mr. P40 and P25 appeared to be similar in both mobility and antigenicity to Mo-MuLV intermediates, Pr40gag and Pr25gag, previously found in infected cells. Additional proteins of 30000 (Gz-p30), 15000 (Gz-p12), 12000 (Gz-p15) and 10000 (Gzp10) Mr were also generated upon cleavage of Gz-p65gag and contained antigenic determinants of Mo-MuLV structural proteins p30, pp12, p15 and p10, respectively. Both detergent-disrupted Mo-MuLV and Rauscher murine leukaemia virus produced similar cleavage profiles. Trypsin and detergent-disrupted mouse mammary tumour virus generated cleavage patterns very different from that produced by Mo-MuLV. Both visual and quantitative time studies of the reaction indicated that P40 gave rise to Gz-p30 and Gz-p10. Tryptic peptide mapping of Gz-p65gag and its cleavage products supported the results obtained from both immunoprecipitation studies with anti-gag sera and the kinetics of cleavage of Gz-p65gag. Both Mo-MuLV Pr65gag and Gz-p65gag were found to be very similar in primary sequence as judged by peptide mapping. P40 produced tryptic peptides that co-migrated with Mo-MuLV p30 peptides; P25 contained tryptic peptides that were also found in Mo-MuLV p15. Gz-p30 and Gz-p15 contained the tryptic peptides of Mo-MuLV p30 and p15, respectively, that were found in P40 and P25. The Gz-p10 fraction contained a tryptic peptide that was also found in P40, but not p30. These results provide good evidence that the protease packaged within Mo-MuLV can cleave, in vitro, the gag-related polyprotein of Gz-MuSV in a manner very similar to the processing of Mo-MuLV Pr65gag in infected cell culture systems.

Amino Acid Sequence↗
PubMed1985

A cAMP-independent serine/threonine kinase activity is associated with the mos sequences of ts110 Moloney murine sarcoma virus-encoded P85gag-mos.

Two proteins, termed P85gag-mos and P58gag, are encoded by the temperature-sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85gag-mos is believed to be important for the transforming potential of ts110 MuSV and has been found to be associated with a thermolabile kinase activity that phosphorylates both P85gag-mos and P58gag in immune complexes. Modifications of the original kinase assay conditions are reported here that have allowed a 30-fold increase in the specific activity of P85gag-mos phosphorylated in vitro. The in vitro P85gag-mos-phosphorylating activity was found to be unresponsive to 10 microM-cAMP or 10 microM-cGMP. Addition of 1 mM-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85gag-mos and P58gag; the molar phosphate incorporation per mole of P85gag-mos increased from 0.032 to 0.9, whereas the specific activity of in vitro-phosphorylated P58gag increased 18-fold, from 0.013 to 0.234. pH curves of the in vitro kinase reaction further confirmed the presence of phosphatase activity; in the absence of pyrophosphate, a sharp optimum at pH 4 to 5 was observed, whereas it shifted broadly to pH 7.0 in the presence of pyrophosphate. Under the latter conditions, several experiments were performed in order to determine if the kinase was associated with either gag or mos sequences of P85gag-mos. Antisera directed against p15, p12 and p30 sequences of the gag protein region of P85gag-mos yielded immune complexes that allowed phosphorylation in vitro of P85gag-mos. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62gag. An anti-mos serum generated against a synthetic peptide representing the predicted v-mos amino acid residues 37 to 55 recognizes P85gag-mos and allowed phosphorylation of P85gag-mos in vitro in the absence of P58gag. Peptide mapping of both phosphorylated P85gag-mos and P58gag, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85gag-mos became phosphorylated not only on gag sequences, but also at the N-terminal portion of v-mos. Phosphoamino acid analyses of P85gag-mos and P58gag phosphorylated in vitro under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically 32P-labelled P85gag-mos and P58gag were also found to contain phosphoserine and phosphothreonine. Based on these results, we conclude that a cAMP-independent, serine/threonine protein kinase activity is associated with the mos sequences of P85gag-mos.

Antigen-Antibody Complex↗