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Biomedical subjects

K R Williams

Publications and source records attributed to K R Williams.

At least 109 records · Page 6Linked to original sources
PubMed1988

Tympanosclerosis, grommets and shear stresses.

This paper deals with grommet induced tympanosclerosis. In the first part, patients from a controlled trial of the treatment of bilateral glue ear using unilateral grommet insertion are reviewed 15 years later. Tympanosclerosis due to grommet insertion is summated using computer graphics. Two areas of maximal tympanosclerosis are found, anteriorly and posteriorly midway between the annulus and the umbo. These maximal areas join inferiorly in the classical horse shoe shape. In an attempt to identify reasons for this distribution, the structure of the lamina propria is reviewed, and the changes that occur in tympanosclerosis considered. With this in mind, the tympanic membrane is modelled using finite element methodology to determine the areas of maximal shear stresses. These are found to be in the same position as tympanosclerosis. Such stresses would be increased by the weight of a grommet in situ. These stresses will damage the fibrils interconnecting between the fibrous layers of the lamina propria and this may lead to tympanosclerosis. This raises the question of whether a lighter grommet would decrease the incidence of grommet-induced tympanosclerosis.

Ear, Middle↗
PubMed1987

Photoaffinity labeling of the thymidine triphosphate binding domain in Escherichia coli DNA polymerase I: identification of histidine-881 as the site of cross-linking.

Using the technique of ultraviolet-mediated cross-linking of substrate deoxynucleoside triphosphates (dNTPs) to their acceptor site [Abraham, K. I., & Modak, M. J. (1984) Biochemistry 23, 1176-1182], we have labeled the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) with [alpha-32P]dTTP. Covalent cross-linking of [alpha-32P]dTTP to the Klenow fragment is shown to be at the substrate binding site by the following criteria: (a) the cross-linking reaction requires dTTP in its metal chelate form; (b) dTTP is readily competed out by other dNTPs as well as by substrate binding site directed reagents; (c) labeling with dTTP occurs at a single site as judged by peptide mapping. Under optimal conditions, a modification of approximately 20% of the enzyme was achieved. Following tryptic digestion of the [alpha-32P]dTTP-labeled Klenow fragment, reverse-phase high-performance liquid chromatography demonstrated that 80% of the radioactivity was contained within a single peptide. The amino acid composition and sequence of this peptide identified it as the peptide spanning amino acid residues 876-890 in the primary sequence of E. coli Pol I. Chymotrypsin and Staphylococcus aureus V8 protease digestion of the labeled tryptic peptide in each case yielded a single smaller fragment that was radioactive. Amino acid analysis and sequencing of these smaller peptides further narrowed the dTTP cross-linking site to within the region spanning residues 876-883. We concluded that histidine-881 is the primary attachment site for dTTP in E. coli DNA Pol I, since during amino acid sequencing analysis of all three radioactive peptides loss of the histidine residue at the expected cycle is observed.

Amino Acid Sequence↗
PubMed1987

The function of zinc in gene 32 protein from T4.

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II) bound in a tetrahedral complex to -S- ligands, proposed on spectral evidence to include Cys-77, Cys-87, and Cys-90 [Giedroc, D. P., Keating, K. M., Williams, K. R., Konigsberg, W. H., & Coleman, J. E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452]. The Zn(II) can be completely removed by treatment with the mercurial reagent p-(hydroxymercuri)benzenesulfonate and ethylenediaminetetraacetic acid. The resultant apo-g32P is rapidly digested by trypsin in contrast to the zinc protein which undergoes specific limited proteolysis to yield a resistant DNA-binding core. Rebinding of Zn(II) to the apoprotein restores the same limited susceptibility to proteolysis displayed by the native Zn(II) protein. In the presence of 150 mM NaCl, Zn(II) g32P reduces the melting temperature Tm of poly[d(A-T)] by 47 degrees C, while apo-g32P is unable to melt poly[d(A-T)] at this salt concentration, as the protein thermally unfolds before melting can take place. At 25 mM NaCl, however, apo-g32P lowers the Tm of poly[d(A-T)] by 36 degrees C, but the melting curve is broad compared to the steep cooperative melting induced by Zn(II) g32P. Association constants Ka calculated from the poly[d(A-T)] melting curves for Zn(II) and apo-g32P differ by 3 orders of magnitude, 4.8 X 10(10) M-1 and 4.3 X 10(7) M-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoproteins↗
PubMed1987

Ferrate oxidation of Escherichia coli DNA polymerase-I. Identification of a methionine residue that is essential for DNA binding.

Treatment of Escherichia coli DNA polymerase-I with potassium ferrate (K2FeO4), a site-specific oxidizing agent for the phosphate group-binding sites of proteins, results in the irreversible inactivation of enzyme activity as judged by the loss of polymerization as well as 3'-5' exonuclease activity. A significant protection from ferrate-mediated inactivation is observed in the presence of DNA but not by substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme also exhibits loss of template-primer binding activity, whereas its ability to bind substrate triphosphates is unaffected. In addition, comparative high pressure liquid chromatography tryptic peptide maps obtained before and after ferrate oxidation demonstrated that only five peptides of the more than 60 peptide peaks present in the tryptic digest underwent a major change in either peak position or intensity as a result of ferrate treatment. Amino acid analyses and/or sequencing identified four of these affected peaks as corresponding to peptides that span residues 324-340, 437-455, 456-464, and 512-518, respectively. However, only the last peptide, which has the sequence: Met-Trp-Pro-Asp-Leu-Gln-Lys, was significantly protected in the presence of DNA. This latter peptide was also the only peptide whose degree of oxidation correlated directly with the extent of inactivation of the enzyme. Amino acid analysis indicated that methionine 512 is the target site in this peptide for ferrate oxidation. Methionine 512, therefore, appears to be essential for the DNA-binding function of DNA polymerase-I from E. coli.

Chromatography, High Pressure Liquid↗
PubMed1987

Tissue response of several polymeric materials implanted in the rat middle ear.

Implantation of three different polymeric composite materials in the rat middle ear showed that the nylon prosthesis was encapsulated more rapidly than the two poly(methyl methacrylate) based implants. Several PMMA based implants were only partially covered at the 6 mnth stage by a tenuous capsule made up of collagen and a mixed inflammatory cell infiltrate. The nylon material was covered at the 3 mnth stage with a thin capsule and healthy fibroblasts.

Acrylic Resins↗
PubMed1987

Biomechanical studies of elite female distance runners.

The experiments showed the elite women runners to be predominantly midfoot strikers. Their ground reaction forces showed peaks of 3.3 times body weight in the vertical component, 0.8 times body weight in the braking phase, and 0.3 times body weight in the mediolateral direction. The asymmetry in their ground reaction forces was expressed mainly in the mediolateral component. Only minor differences were found between ground reaction forces in racing and training shoes. More abduction during foot placement was associated with greater rearfoot motion and with greater change in mediolateral component of velocity. Among the numerous moderate to high correlations of interest were those between vertical oscillation and peak vertical force, crossover and change in mediolateral velocity, and between movements of the swing leg and mediolateral force values. There were very few correlations between running economy and biomechanical variables, and stride length correlated poorly with stature. The elite runners were shorter in stature, lighter, had shorter legs and considerably less iliac crest fat than a typical non-athletic female population. The runners also had narrower pelves than a student population of similar age. They were reasonably homogeneous and symmetrical in anthropometric dimensions. Compared with a group of elite male runners at the same running velocity, the elite women exhibited more hip flexion, greater angular velocities in hip flexion and extension, and longer stride lengths relative to leg length during running. Their vertical oscillation (expressed relative to leg length) was less than that shown by their male counterparts. On the issue of relative pelvic width, the women had greater relative bitrochanteric and bi-iliac crest widths but similar bispinous widths compared to the elite male runners (all values expressed relative to leg length). Clearly the issue of male-female pelvic width differences at the elite level is not as simple as had previously been suggested.

Adult↗
PubMed1987

Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA.

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).

Amino Acid Sequence↗
PubMed1987

Amino acid sequence of UP1, an hnRNP-derived single-stranded nucleic acid binding protein from calf thymus.

The UP1 single-stranded nucleic acid binding protein from calf thymus (Herrick, G. & Alberts, B.M. (1976) J. Biol. Chem. 251, 2124-2132) has recently been shown to be a proteolytic fragment derived from the A1 heterogeneous nuclear ribonucleoprotein (hnRNP) (Pandolfo et al. (1985) Nucleic Acids Res. 13, 6577-6590). The NH2-terminus of the 22,162 dalton UP1 protein appears to be blocked, which suggests that UP1 represents the NH2-terminal two thirds of this 32,000 dalton hnRNP protein. The complete amino acid sequence for UP1 was derived from automated sequencing of peptides that were purified by HPLC from digests with trypsin, chymotrypsin, Staphylococcus aureus protease, endoproteinase Lys-C, and cyanogen bromide. Trichloroacetic acid precipitation followed by enzymatic digestion in 2 M urea proved to be the best approach for generating UP1 peptides. By carboxymethylating after, rather than before, digestion it was possible to avoid problems associated with the insolubility of the carboxymethylated UP1. All of the resulting peptides in amounts varying from 2 to 15 nmol were coupled to aminopolystyrene prior to solid-phase sequencing. Using these methods, no difficulties were encountered in assigning glutamic acid residues or in completely sequencing peptides that contained up to 25-30 residues. The relative ease with which the UP1 protein was sequenced, requiring only about a year to complete, and the comparatively modest amount of protein required, less than 5 mg, attests to the usefulness of water soluble carbodiimide coupling and solid-phase sequencing for determining the primary structures of proteins. In addition to serving as a basis for determining structural relationships among various mammalian single-stranded nucleic acid binding proteins, the amino acid sequence of UP1 reveals that the A1 hnRNP protein contains a region of internal sequence homology that apparently corresponds to two independent nucleic acid binding sites.

Amino Acid Sequence↗
PubMed1987

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family.

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.

Animals↗
PubMed1987

Relationship between distance running mechanics, running economy, and performance.

The relationships between biocmechanical aspects of distance running, running economy (VO2 submax), and performance were investigated. A variety of biomechanical measures for 31 subjects running at 3.6 m/s was obtained, including three-dimensional angular and translational kinematics, ground reaction forces and center of pressure patterns, mechanical power, and anthropometric measures. Physiological measures obtained included maximal and submaximal O2 consumption, muscle fiber composition, and measures of the ability to store and return elastic energy during knee bends. A subset of 16 runners was also evaluated in relation to performance in a 10-km run. Biomechanical variables were identified which showed significant differences or consistent trends between groups separated on the basis of VO2 submax, establishing the importance of biomechanical influences on running economy. It appears that no single variable or small subset of variables can explain differences in economy between individuals but rather that economy is related to a weighted sum of the influences of many variables.

Aerobiosis↗
PubMed1987

Wife assault and perceptions of sanctions.

Using survey data based on telephone interviews of 1,626 coupled males, this study explores the relationship between perceptions of four possible sanctions for wife assault and involvement in this form of physical aggression. The exploratory analysis addresses the following questions: To what extent do men perceive negative reactions to wife assault as certain and severe? Do nonassaultive men perceive the reactions of others as more certain and severe than men who engage in physically aggressive behavior? Among men who assault their partners, are the perceived certainty and severity of sanctions greater for one-time offenders than for repeat offenders? The relevance of the findings on the deterrent and experimental effects of sanctions are discussed, and suggestions are offered for future studies that apply deterence theory to the phenomenon of assaults taking place between adult partners.

Adult↗
PubMed1986

Escherichia coli exonuclease VII. Cloning and sequencing of the gene encoding the large subunit (xseA).

We have determined the sequence of the gene encoding the large subunit of Escherichia coli exonuclease VII (xseA) and the amino acid sequence of the protein it encodes. The coding region of the xseA gene is 1368 base pairs. The protein encoded by the gene contains 456 amino acids and has a calculated molecular weight of 51,823. The promoter for xseA is close to that for guaB, and these two genes are transcribed in opposite directions: xseA clockwise and guaB counterclockwise on the standard E. coli genetic map. The cloned xseA gene can complement an xseA deletion mutant strain. In an xseA+ genetic background production of large quantities of the xseA gene product appeared to decrease the amount of exonuclease VII activity in cell extracts. In fact, no exonuclease VII activity at all could be detected following induction of strains in which the xseA gene was under lambda pL regulation. These observations suggest that the proper ratio of the large and small exonuclease VII subunits must be maintained in order to produce active enzyme.

Amino Acid Sequence↗
PubMed1986

Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins.

Protein A1 (Mr approximately 32,000), a major glycine-rich protein of heterogeneous nuclear ribonucleoproteins (hnRNP), was purified to near homogeneity under nondenaturing conditions from HeLa cells. Limited proteolysis of the native protein yields a trypsin-resistant N-terminal nucleic acid-binding domain about 195 amino acids long which has a primary structure nearly identical to that of the 195-amino acid-long single-stranded DNA (ssDNA)-binding protein UP1 (Mr 22,162) from calf thymus (Williams, K.R., Stone, K. L., LoPresti, M.B., Merrill, B. M., and Planck, S.R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5666-5670). 45 of the 61 glycine residues of A1 are present in the trypsin-sensitive C-terminal domain of the protein which contains no sequences homologous to UP1. Protein A2, another major glycine-rich core hnRNP protein from HeLa, has a domain structure analogous to A1 and appears to be related to ssDNA-binding proteins UP1-B from calf liver and HDP-1 from mouse myeloma in a way similar to the A1/UP1 relationship. In contrast to ssDNA-binding proteins, A1 binds preferentially to RNA over ssDNA and exhibits no helix-destabilizing activity.

Amino Acid Sequence↗
PubMed1986

1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis.

Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface.

Amino Acid Sequence↗
PubMed1986

High-performance liquid chromatographic peptide mapping and amino acid analysis in the sub-nanomole range.

By ensuring adequate gradient mixing and eliminating all major artifact peaks we have been able to obtain reproducible high-performance liquid chromatographic tryptic peptide maps on less than 50 pmol of protein. Likewise, a 10-20 fold improvement in the sensitivity of amino acid analysis has been achieved by analyzing the phenylthiocarbamyl derivatives of the free amino acids rather than the free amino acids themselves. This approach enables accurate amino acid compositions to be obtained on less than 50 ng of protein, providing that a simple correction is made for the background level of serine and glycine. We have been able to reduce the background level of these two amino acids to ca. 10 pmol each per sample by incinerating the hydrolysis tubes at 500 degrees C prior to introduction of the sample and by using gas-phase as opposed to liquid-phase hydrolysis. Background corrections are not necessary when over 500 ng of protein are hydrolyzed. With this amount of protein, amino acid compositions, based on phenylthiocarbamyl amino acid analyses were, on average, found to be accurate to within +/- 10%.

Amino Acids↗
PubMed1986

DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein. Primary structure and homology with protein phosphatase inhibitor-1.

The complete amino acid sequence of bovine brain DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein, which is a potent and specific inhibitor of the catalytic subunit of protein phosphatase-1, has been determined. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage by endoproteinase Lys-C, endoproteinase Arg-C, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease, and to chemical cleavage by cyanogen bromide. The overlapping sets of peptides were purified by high performance liquid chromatography and subjected to amino acid sequencing by automated Edman degradation to deduce the complete sequence. The protein consists of a single NH2-terminal blocked polypeptide chain of 202 residues, with a calculated molecular mass of 22,591 daltons, excluding the unidentified NH2-terminal blocking group. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or hydrodynamic measurements. The threonine residue that is phosphorylated by cyclic AMP-dependent protein kinase (Hemmings, H. C., Jr., Williams, K. R., Konigsberg, W. H., and Greengard, P. (1984) J. Biol. Chem. 259, 14486-14490), and that must be phosphorylated for the expression of inhibitory activity, is located at position 34. The molecule contains only 1 cysteine residue and 1 tryptophan residue, at positions 72 and 161, respectively. DARPP-32 is very hydrophilic, and contains a stretch of 16 consecutive acidic residues from position 119 to 134. The predicted secondary structure suggests the presence of 47% alpha-helix, 7% beta-sheet, and 46% random coil, with 11 beta-turns. Comparison of the complete amino acid sequence of bovine DARPP-32 with that of rabbit skeletal muscle protein phosphatase inhibitor-1 revealed a significant amount of sequence identity in the NH2-terminal regions of these two proteins. The active region of inhibitor-1 has been localized to an NH2-terminal fragment (Aitken, A., and Cohen, P. (1982) FEBS Lett. 147, 54-58), the part of the molecule that is most similar to DARPP-32. These data suggest that these two protein phosphatase inhibitors may share a common structural basis for their inhibitory activity and may be related by a common ancestral gene.

Amino Acid Sequence↗