Biomedical subjects
Publications and source records attributed to S H Friend.
Stable protein complexes between the large T antigens of mouse, monkey, baboon, or human polyomaviruses and the retinoblastoma protein were detected by an in vitro coimmunoprecipitation assay. All of the large T antigens tested were able to bind to both human and mouse retinoblastoma polypeptides, showing that these interactions have been conserved during evolution.
We have isolated a cDNA clone of the murine homologue of the human retinoblastoma (Rb) susceptibility gene. DNA sequence analysis reveals a high degree of conservation with the human Rb sequence, both in the coding and in the noncoding regions. The predicted amino acid sequence of the mouse Rb protein shows 91% identity to that of the human protein. Both proteins were found to contain a peptide sequence reminiscent of a leucine-repeat motif ("leucine-zipper") that is also found in the myc, fos, and jun oncogenes. Synthetic peptide antiserum directed against a portion of the mouse Rb protein detects three proteins of 104-110 kDa in cells that were transiently transfected with a mouse Rb gene expression construct. In the mouse embryo the expression of Rb mRNA was ubiquitous, with maximal expression being observed around 13 days of gestation. In the embryo, the highest level of expression was observed in liver and brain. In contrast, the Rb gene was found to be expressed at a very low level in adult mouse liver with high levels being found in lung, thymus, and spleen. A shorter Rb transcript was detected in mouse testes.
One of the cellular targets implicated in the process of transformation by the adenovirus E1A proteins is a 105K cellular protein. Previously, this protein had been shown to form stable protein/protein complexes with the E1A polypeptides but its identity was unknown. Here, we demonstrate that it is the product of the retinoblastoma gene. The interaction between E1A and the retinoblastoma gene product is the first demonstration of a physical link between an oncogene and an anti-oncogene.
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Retinoblastoma is a childhood tumor that can arise because of mutant alleles acquired as somatic or germinal mutations. The mutant allele can be carried in the germ line. The mutations creating these alleles act by inactivating copies of a recessive oncogene located within band q14 of chromosome 13 and termed the RB1 locus. We have reported isolation of a cDNA fragment that recognizes chromosomal sequences possessing many of the attributes of the retinoblastoma gene associated with the RB1 locus. We now report that this segment is additionally the target of somatic mutations in mesenchymal tumors among patients having no apparent predisposition to retinoblastoma and no previous evidence of retinoblastoma. These tumors provide additional evidence that the cloned sequences are representative of a gene that is a frequent target of inactivation during tumorigenesis. Sequence analysis of this cDNA provides little insight into its normal functional role.
In many ways, parents are the true consumers of their children's health care. How parents understand the cause, presentation, and treatment of their child's illness strongly affects doctor-patient communication, compliance in treatment, and follow-up. To better understand and enhance compliance, we have studied mothers' concepts of illness using Piaget's stages of cognitive development. Fifty-nine mothers who brought their children to the emergency department (ED) of a large urban children's hospital were asked, "How does a child get a cold," and "What makes a cold go away?" We directly transcribed answers and compiled information on maternal education, employment status, method of payment for health care, and number of children in the family. Forty-four percent of mothers had a prelogical conceptualization of illness, and only 17% had a formal-logical conceptualization. Only employment in the health care fields correlated with maternal conceptual levels regarding childhood illness. Physicians need to take care to communicate with the parent at her level of conceptualization to ensure understanding.
The acid-denaturation behavior of eleven cetacean myoglobins has been studied at two ionic strengths, 0.01 and 0.10 M, at 25.0 degrees C. The myoglobulins studied fall into four phylogenetic suborders, representing the sperm whales, dolphins, baleen whales, and beaked whales. The differences in response to acid denaturation among these closely related myoglobins are small but statistically significant. In three cases, free-energy differences between myoglobins can be ascribed to one amino acid difference and in three others to two differences. The differences in response were analyzed in terms of the changes in noncovalent interactions occurring in the native structure. The effects of changes in electrostatic interactions over the whole charge array were calculated for each myoglobin species by using the modified Tanford-Kirkwood theory. The predicted changes in stability correlated well with the experimental observations in most cases. When differences in hydrogen-bonding capability were considered at a first approximation, substantial effects were predicted. When these effects were taken in conjunction with the electrostatic interactions, the correlation with experiment was improved. Additionally, restrictions in motional freedom and packing constraints appeared to be significant in the single-site analysis. The detectable differences in stability due to single amino acid substitutions along with the small differences in stability between the cetacean suborders indicate that compensatory interactions provide the mechanism for the conservation of stability among the myoglobins studied.
The modified Tanford-Kirkwood electrostatic theory has been employed to evaluate pK values for all charge sites in the bovine pancreatic trypsin inhibitor (BPTI). 13C NMR titration data were obtained for all titrating groups except arginine residues in BPTI at nearly constant ionic strength in 0.1 M NaCl, at 41 degrees C. The chemical shifts of 46 resonances were found to be sensitive to pH. The pK values of these titrating resonances compared well with those computed by the modified Tanford-Kirkwood electrostatic theory. A conformational change involving the NH2- and COOH-terminal and nearby residues is shown to be partly electrostatically driven by the formation of a salt bridge between the alpha-amino and alpha-carboxyl groups at mid-pH values. The computed total electrostatic free energy of the molecule is found to be stabilizing at neutral pH despite the substantial net positive charge borne by the protein under such conditions.
The pH dependence of the summed electrostatic stabilization for deoxy- and liganded hemoglobin was computed for several ionic strength values. The computed contribution to the stabilization of deoxyhemoglobin by binding of 2,3-diphosphoglycerate in the beta cleft compared well with experimental binding behavior for human hemoglobin A0 and hemoglobin F. The contribution of diphosphoglycerate binding to the alkaline Bohr effect was computed correctly for both hemoglobins A0 and F. The computed effects of simultaneous binding of diphosphoglycerate and formation of Val-1 beta carbamino adducts suggested a competition between these effectors. A direct competition was formulated between these two effectors, with extension to include a simple anion such as chloride or bicarbonate binding in competition with diphosphoglycerate but not with Val-1 beta carbamino formation. This model was found to hold at pH 7.3-7.4 over a range of concentrations of the effectors involved and to predict the pH dependence of Val-1 beta carbamino formation over the pH range 7.0-8.0. The pH dependence of the computed differential stability of liganded vs. unliganded hemoglobin A compared well with observation.
The summed electrostatic free energy contributions to deoxyhemoglobin A0 tetramer formation were computed at a series of pH and ionic strength values as the difference between the computed values for the tetramer and for the sum of the four individual chains. The electrostatic stabilization of each monomer is similar and close to that for myoglobin. At ionic strength 0.10 M the electrostatic contribution to the stability of the tetramer is approximately 35 kcal/mol at pH 6.0 and 18 kcal/mol at pH 9.6. The specific contribution to the stabilization of the tetramer, (sigma delta G"i,el)tet, is obtained by difference and shows a broad plateau above 7 kcal/mol over the range from pH 6.0 to 8.0, which is nearly obliterated by pH 9.6. By examination of the contributions of individual sites under the above summation, it is found that sites in the alpha chains are responsible for virtually the entire stabilizing effects in tetramer formation. The major differences on tetramer formation are sensed at eight sites. The stabilization provided by four of these sites results simply from changes in solvent exposure of sites in the given monomers as the tetramer is assembled. They are offset in part by changes at three sites that sense the greatest destabilization and that are responsible for the near cancellation of effects among the beta-chain sites. The general implications for the stabilization of molecular assemblies are considered.
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The compact, largely helical structure of sperm whale and harbor seal myoglobins undergoes an abrupt one-step transition between pH 4.5 and 3.5 as monitored by changes in either the heme Soret band absorbance or circular dichroism probes of secondary structure, for which a modified Tanford-Kirkwood theory provides identification of certain dominant electrostatic interactions responsible for the loss of stability. A similar treatment permits identification of the electrostatic interactions primarily responsible for a process in which the anchoring of the A helix to other parts of the molecule is weakened. This process is detected with both myoglobins, in a pH range approximately 1 unit higher than the onset of the overall unfolding process, through changes in the circular dichroic spectra near 295 nm which correspond to the L1 O-O band of the only two tryptophan residues in these proteins, residues 7 and 14. In each case protonation of certain sites in neighboring parts of the molecule can be identified as producing destabilizing interactions with components of the A helix, particularly with lysine 6.
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The pattern of electrostatic interactions between pairs of charge sites in sperm whale ferrimyoglobin was examined as a function of pH in terms of proton site occupancy, static solvent accessibility, and distance of separation. By grouping all examples of the most stabilizing interactions and all examples of the most destabilizing interactions, we can easily show that at pH 7.50 the former is much stronger; that is, the negative contributions to electrostatic free energy far outweigh the positive contributions. Much of the electrostatic energy of stabilization in native myoglobin is provided by specific charge-pair partners that are very highly conserved among 53 mammalian myoglobin species and is invariant substantially from pH 8.5 to 3.5. Destablizing interactions that become most significant, but not actually dominant, near the acid unfolding pH range can be recognized in emerging clusters of uncompensated positive charges. Binding of azide ion by the heme iron effectively reduces the most prominent destabilizing set of such interactions. In general, thoe charged residues that experience the largest summed stabilizing interactions with other groups are the most conserved between species. The histidine residues, however, show their best correlation of conservation with low values of static accessibility. Although histidine residue 64 has an effective pK corresponding to the midpoint of the unfolding transition near pH 4.2 at an ionic strength of 0.10 M and so might be called a "trigger group", its interactions contribute only a modest fraction of the overall pH-dependent free energy change. An examination of the primary stabilizing interactions represented by the charge-pair partners indicates a probably major role of electrostatic interactions in the nucleation and docking stages of the condensation of the polypeptide chain into the compact native structure.
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The U.S. National Cancer Institute (NCI) conducts a drug discovery program in which approximately 10,000 compounds are screened every year in vitro against a panel of 60 human cancer cell lines from different organs of origin. Since 1990, approximately 63,000 compounds have been tested, and their patterns of activity profiled. Recently, we analyzed the antitumor activity patterns of 112 ellipticine analogues using a hierarchical clustering algorithm. Dramatic coherence between molecular structures and activity patterns was observed qualitatively from the cluster tree. In the present study, we further investigate the quantitative structure-activity relationships (QSAR) of these compounds, in particular with respect to the influence of p53-status and the CNS cell selectivity of the activity patterns. Independent variables (i.e., chemical structural descriptors of the ellipticine analogues) were calculated from the Cerius2 molecular modeling package. Important structural descriptors, including partial atomic charges on the ellipticine ring-forming atoms, were identified by the recently developed genetic function approximation (GFA) method. For our data set, the GFA method gave better correlation and cross-validation results (R2 and CVR2 were usually approximately 0.3 higher) than did classical stepwise linear regression. A procedure for improving the performance of GFA is proposed, and the relative advantages and disadvantages of using GFA for QSAR studies are discussed.