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Biomedical subjects

A S Yang

Publications and source records attributed to A S Yang.

32 records · Page 2Linked to original sources
PubMed1995

HhaI and HpaII DNA methyltransferases bind DNA mismatches, methylate uracil and block DNA repair.

The hydrolytic deamination of 5-methylcytosine (5-mC) to thymine (T) is believed to be responsible for the high mutability of the CpG dinucleotide in DNA. We have shown a possible alternate mechanism for mutagenesis at CpG in which HpaII DNA-(cytosine-5) methyltransferase (M.HpaII) can enzymatically deaminate cytosine (C) to uracil (U) in DNA [Shen, J.-C., Rideout, W.M., III and Jones, P.A., Cell, 71, 1073-1080, (1992)]. Both the hydrolytic deamination of 5-mC and enzymatic deamination of C create premutagenic DNA mismatches (G:U and G:T) with the guanine (G) originally paired to the normal C. Surprisingly, we found that DNA-(cytosine-5) methyltransferases have higher affinities for these DNA mismatches than for their normal G:C targets and are capable of transferring a methyl group to the 5-position of U, creating T at low efficiencies. This binding by methyltransferase to mismatches at the recognition site prevented repair of G:U mismatches by uracil DNA glycosylase in vitro.

Base Sequence↗
PubMed1994

Structural origins of pH and ionic strength effects on protein stability. Acid denaturation of sperm whale apomyoglobin.

A recently developed approach to calculate the pH dependence of protein stability from three-dimensional structure information is applied to the analysis of acid denaturation of sperm whale apomyoglobin. The finite difference Poisson-Boltzmann method is used to calculate pKa values and these are used to obtain titration curves for the folded protein as well as for compact intermediates. The total electrostatic free energy change involved in apomyoglobin unfolding is then evaluated. Calculations are carried out of the unfolding free energy of the native (N) and the compact intermediate (I) of apomyoglobin relative to the unfolded state (U) over a range of pH at various ionic strengths. The contributions from key ionizable groups to the unfolding process are discussed. For the acid-induced partial unfolding of apomyoglobin near pH 5, the transition from N to I is found to be driven by three histidines that are exposed when the B, C, D and E helices unfold. Similarly, the unfolding of the compact intermediate I consisting of the A, G and H helices is driven primarily by a few carboxylic acids with low pKa values in the compact state. This picture is in contrast to the view which attributes acid denaturation to electrostatic repulsion resulting from the build up of positive charge. In fact, charge-charge interactions in myoglobin are found to be attractive at all pH values where the protein unfolds. pH-dependent changes in these interactions contribute to acid denaturation but other electrostatic effects, such as hydrogen bonding and solvation, are important as well. The effect of increasing ionic strength on unfolding is attributed to the decrease of attractive charge-charge interactions which destabilize the N state relative to I, but stabilize the I state relative to U by reducing the pKa shifts of a few critical carboxylic acids. The I state is found to be more stable than U at neutral pH thus accounting for its presence as an intermediate on the protein folding pathway. Our results have implications for the origins of compact intermediates or "molten globule" states.

Animals↗
PubMed1994

Two molecular pathways to transitional cell carcinoma of the bladder.

Noninvasive transitional cell carcinomas of the bladder can have two distinct morphologies suggesting they contain different genetic alterations. Papillary transitional cell carcinomas (T(a) tumors) are often multifocal and only occasionally progress, whereas flat tumors (carcinomas in situ, CIS), frequently progress to invasive disease. We examined 216 bladder tumors of various stages and histopathologies for two genetic alterations previously described to be of importance in bladder tumorigenesis. Loss of heterozygosity of chromosome 9 was observed in 24 of 70 (34%) T(a) tumors but was present in only 3 of 24 (12%) CIS and dysplasia lesions (P = 0.04). In contrast, only 1 of 36 (3%) T(a) tumors contained a p53 gene mutation compared to 15 of 23 (65%) CIS and dysplasias (P < 0.001), a frequency comparable to that observed in muscle invasive tumors (25 of 49; 51%). The presence of p53 mutations in CIS and dysplasia could explain their propensities to progress since these mutations are known to destabilize the genome. Analysis of several tumor pairs involving a CIS and an invasive cancer provided evidence that the chromosome 9 alteration may in some cases be involved in the progression of CIS to more invasive tumors, in addition to its role in the initiation of T(a) tumors. However, the CIS and secondary tumor were found to contain different genetic alterations in some patients suggesting divergent progression pathways. Bladder carcinogenesis may therefore proceed through two distinct genetic alteration pathways responsible for generating superficial tumors with differing morphologies and pathologies.

Alleles↗
PubMed1993

On the pH dependence of protein stability.

This paper treats the free energy contribution of ionizable groups to protein stability. A method is presented for the calculation of the pH dependence of the denaturation free energy of a protein, which yields results that can be compared directly to experiment. The first step in the treatment is the determination of the average charges of all the ionizable groups in both the folded and unfolded protein. An expression due to Tanford then relates the pH dependence of the unfolding free energy to the difference in net charge between the two states. In order to determine absolute rather than relative unfolding free energies, it is necessary to calculate the total contribution of ionizable groups to protein stability at some reference pH. This is accomplished through a statistical mechanical treatment similar to the one used previously in the calculation of pKas. The treatment itself is rigorous but it suffers from uncertainties in the pKa calculations. Nevertheless, the overall shape of experimentally observed plots of denaturation free energy as a function of pH are reasonably well reproduced by the calculations. A number of general conclusions that arise from the analysis are: (1) knowledge of titration curves and/or effective pKa values of ionizable groups in proteins is sufficient to calculate the pH dependence of the denaturation free energy with respect to some reference pH value. However, in order to calculate the absolute contribution of ionizable groups to protein stability, it is necessary to also know the intrinsic pKa of each group. This is defined as the pKa of a group in a hypothetical state of the protein where all other groups are neutral. (2) Due to desolvation effects, ionizable groups destabilize proteins, although the effect is strongly dependent on pH. There are however, strongly stabilizing pairwise Coulombic interactions on the surface of proteins. (3) Plots of stability versus pH should not be interpreted in terms of a group whose pKa corresponds to the titration midpoint, but rather to a group with different pKas (that correspond approximately to the titration end points) in each state. (4) Any residual structure in the GuHCl-denatured state of proteins appears to have little effect on the pH dependence of stability. (5) pH-dependent unfolding, for example to the "molten globule" state, appears due to individual groups with anomalous pKas whose locations on the protein surface may determine the nature of the unfolded state.

Electricity↗
PubMed1993

Distinct pattern of p53 mutations in bladder cancer: relationship to tobacco usage.

A distinct mutational spectrum for the p53 tumor suppressor gene in bladder carcinomas was established in patients with known exposures to cigarette smoke. Single-strand conformational polymorphism analysis of exons 5 through 8 of the p53 gene showed inactivating mutations in 16 of 40 (40%) bladder tumors from smokers and 13 of 40 (33%) tumors from lifetime nonsmokers. Overall, 13 of the 50 (26%) total point mutations discovered in this and previous work were G:C-->C:G transversions, a relatively rare mutational type in human tumors. In six tumors, identical AGA (Arg)-->ACA (Thr) point mutations at codon 280 were observed, suggesting a mutational hotspot in these tumors. Comparison of the mutational spectra from smokers and nonsmokers revealed no obvious differences in the types or positions of inactivating mutations; however, 5 of 15 tumors containing point mutations from cigarette smokers had double mutations, four of which were tandem mutations on the same allele. No double mutations were found in tumors from nonsmoking patients. None of the mutations in smokers were G:C-->T:A transversions, which would be anticipated for exposure to the suspected cigarette smoke carcinogen 4-aminobiphenyl. The results suggest that, although cigarette smoke exposure may not significantly alter the kinds of mutations sustained in the p53 gene, it may act to increase the extent of DNA damage per mutagenic event.

Base Sequence↗
PubMed1993

On the calculation of pKas in proteins.

This paper describes a general method to calculate the pKas of ionizable groups in proteins. Electrostatic calculations are carried out using the finite difference Poisson-Boltzmann (FDPB) method. A formal treatment of the calculation of pKas within the framework of the FDPB method is presented. The major change with respect to previous work is the specific incorporation of the complete charge distribution of both the neutral and charged forms of each ionizable group into the formalism. This is extremely important for the treatment of salt bridges. A hybrid statistical mechanical/Tanford-Roxby method, which is found to be significantly faster than previous treatments, is also introduced. This simplifies the problem of summing over the large number of possible ionization states for a complex polyion. Applications to BPTI and serine proteases suggest that the calculations can be quite reliable. However, the necessity of including bound waters in the treatment of the Asp-70... His-31 salt bridge in T4 lysozyme and experience with other proteins suggest that additional factors ultimately need to be considered in a comprehensive treatment of pKas in proteins.

Aprotinin↗
PubMed1992

Analysis of the heat capacity dependence of protein folding.

This paper presents an analysis of plots of enthalpy versus heat capacity change at 25 degrees C for the unfolding of proteins and for the dissolution of gaseous, liquid and solid solutes, first reported by Murphy, Privalov & Gill. The negative slope in the enthalpy plot for proteins is interpreted as arising from a large penalty associated with burying polar groups in the protein interior. The small enthalpy changes that accompany protein unfolding at 25 degrees C are also discussed. It is argued that the combined effects of hydrogen bond formation and close packing predict a large positive enthalpy of unfolding. Electrostatic calculations indicate that the penalty associated with burying polar groups is large enough to effectively cancel these terms, leading to the small net enthalpy changes that are observed. The free energy changes associated with protein folding are also discussed. The free energy cost of burying polar groups largely compensates for the stabilizing contribution of the hydrophobic effect and would appear to account for the fact that proteins are marginally stable, independent of their size and of their relative hydrophobicities.

Hydrogen Bonding↗
PubMed1992

Absence of p53 gene mutations in primary nasopharyngeal carcinomas.

Alterations in the p53 tumor suppressor gene and Epstein-Barr virus status were investigated in 15 nasopharyngeal carcinoma (NPC) biopsies, 4 xenografts, and 2 cell lines from the Cantonese region of southern China. One other established NPC cell line obtained from a northern Chinese patient was also studied. Restriction fragment length polymorphism analysis revealed a loss of heterozygosity for chromosome 17p, where the p53 gene resides, in only one of 15 NPC biopsies. Polymerase chain reaction-single-stranded conformational polymorphism analysis and direct sequencing failed to detect sequence alterations in exons 5 through 8 of the p53 gene in the 15 tumors and in the 4 NPC xenografts, all of which tested positive for Epstein-Barr virus. In contrast, the 3 NPC cell lines were all negative for Epstein-Barr virus and contained G----C transversions in the p53 gene, with cell lines CNE-1 and CNE-2 harboring identical AGA (arginine) to ACA (threonine) changes at codon 280. These results suggest that p53 inactivation is not a necessary component of nasopharyngeal carcinogenesis in Cantonese but may be important in the establishment of cell lines derived from these tumors.

Base Sequence↗
PubMed1992

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.

Amino Acid Sequence↗
PubMed1991

Influence of the freezing process upon fluoride binding to hemeproteins.

Fluoride association with ferric myoglobins and hemoglobins in aqueous buffers above freezing has been well studied. We chose this reaction to investigate the feasibility of observing titration intermediates and estimating dissociation constants at the freezing temperature by electron paramagnetic resonance spectroscopy at cryogenic temperatures. Dependence of apparent dissociation constant upon protein concentration was observed, a factor of four decrease in protein accompanied by about a fourfold increase in the apparent tightness of binding in the range of protein concentration studied. Binding was also found to depend upon cooling rate and concentration of additives (serum albumin, sucrose, glycerol). These effects appear to be associated with freezing-induced concentration of ligand, a process described in the literature. Bands of high concentration of electrolyte accompany solute rejection during ice growth, sweeping by slowing moving macromolecules. Thus, just before being trapped in the solid, the protein can experience a greater concentration of salt than in the original liquid. A mathematical model of this process, based upon simplifying assumptions about nucleation and ice-crystal growth rates in super-cooled solution, shows how the average concentration of mobile solute species can depend upon the concentration of all species present. Semiquantitative computer simulations of the actual, more complex, freezing are also presented and lead to estimates of ice particle size which are then compared with estimates from the former model.

Animals↗
PubMed1989

Electrical potentials in trypsin isozymes.

Cow and rat trypsin differ in net charge by 12.5 units yet have the same enzymatic mechanism. The role of electrical potentials in the catalytic mechanism of these trypsin isozymes is investigated by using the finite difference Poisson-Boltzmann method. The calculations reveal that the active sites are effectively shielded from surface charge, thus making it possible for the two enzymes to have essentially identical potentials in their catalytically important regions. The potentials in both active sites are dominated by local interactions arising both from partial charges and from the negative charge on Asp-102. The latter is found to stabilize the transition state by about 4 kcal/mol, a value that is consistent with the extent of reduced catalytic activity in the variant Asn-102 trypsin, in which the negative charge is absent. The calculations predict that Asp-102 is ionized and that His-57 is neutral in the resting state of the enzyme. In contrast to their negligible effect on catalytic activity, the cumulative effect of surface charges is found to raise the pK of the N-terminal alpha-amino group of Ile-16 in the rat enzyme by about 1.5 units relative to that of cow trypsin. This charged amino acid forms an ionic bond with Asp-194, which stabilizes the active conformation of the enzyme. An increase in pK of Ile-16 thus provides a possible explanation for the retention of activity of rat trypsin at high pH. The results of this study could not have been obtained from an electrostatic model based on Coulombic potentials.

Amino Acids↗
PubMed1987

Determination of relative spin concentration in some high-spin ferric proteins using E/D-distribution in electron paramagnetic resonance simulations.

Lineshape simulations are presented for the multiple, overlapping X-band electron paramagnetic resonance (EPR) spectra in two non-heme, high-spin iron proteins: phenylalanine hydroxylase (PAH) and diferric transferrin. The aim of the calculations is to determine the fraction of iron contributing to each of the sites visible by EPR. The simulations are limited to the experimentally accessible transitions occurring at g-values greater than 1.7. In both PAH and transferrin, at least one of the iron sites is characterized by the ratio of zero-field splitting parameters, E/D, near 1/3 and a broad, asymmetric lineshape. A distribution in E/D-values is used in the simulations to account for this breadth and asymmetry. To test the E/D-distribution model, experimental X-band spectra of diferric transferrin at several salt concentrations are fit by simulation. In this test, first the low-field features arising from transitions between the lowest Kramers doublet levels are simulated using E/D-distributions for two sites. Second, parameters that provide a good fit for the lowest doublet transitions are shown also to fit the resonance near an effective g-value of 4.3 from the middle Kramers doublet transition. When applied to spectra of PAH in the resting state, the E/D-distribution approach accounts for the intensity of one of the two major species of iron. The other species is characterized by E/D = 0.032, and the spectrum of this portion of the resting enzyme may be simulated using a frequency-swept Gaussian lineshape. Spectra for the enzyme in an inhibitor-saturated state are also simulated. The simulations are consistent with previous biochemical studies that indicate that only the E/D = 0.032 form of iron participates in catalysis.

Binding Sites↗
PubMedDate not supplied

Modularity, evolvability, and adaptive radiations: a comparison of the hemi- and holometabolous insects.

Despite recent attention given to the concept of modularity and its potential contribution to the evolvability of organisms, there has been little mention of how such a contribution may affect rates of diversification or how this would be assessed. A first key prediction is that lineages with relatively greater degrees of modularity in given traits should exhibit higher rates of diversification. Four general conditions for testing this prediction of the modular evolvability hypothesis are outlined here. The potential role of modularity as a deterministic factor in adaptive radiations is best examined by looking at historic patterns of diversification rather than just levels of extant diversity, the focus of most analyses of key innovations. Recent developmental evidence supports the notion that phenotypes of juvenile and adult stages of insects with "complete" metamorphosis (Holometabola) are distinct developmental and evolvable modules compared to the highly correlated life stages of insects with "incomplete" metamorphosis (Hemimetabola). Family-level rates of diversification for these two groups were calculated from the fossil record. The Holometabola was found to have a significantly and characteristically higher rate of diversification compared to the less modular Hemimetabola, consistent with the idea that intrinsic differences in modularity can influence the long-term evolvability of organisms. The modular evolvability hypothesis also makes a second key prediction: that characters in more modular clades will exhibit greater levels of variation due to their independence. This provides an independent, phenotypically based test of the hypothesis. We discuss here how this second prediction may be tested in the case of the Hemi- and Holometabola.

Adaptation, Physiological↗