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Biomedical subjects

K R Williams

Publications and source records attributed to K R Williams.

At least 127 records · Page 7Linked to original sources
PubMed1986

High pressure liquid chromatography purification of UP1 and UP2, two related single-stranded nucleic acid-binding proteins from calf thymus.

Two single-stranded nucleic acid-binding proteins, UP1 and UP2, that were originally reported by Herrick and Alberts (Herrick, G., and Alberts, B. (1976) J. Biol. Chem. 251, 2124-2132) have been purified to apparent homogeneity from calf thymus by high performance liquid chromatography. The amino acid sequence of UP1 (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) reveals that UP1 contains 195 amino acids, including one dimethylarginine residue near its COOH terminus. Further analysis of this sequence now demonstrates that UP1 contains a 91-residue internal repeat such that when residues 3-93 (the "A" region) are aligned with residues 94-194 (the "B" region), 32% of the amino acids in these two regions are identical and an additional 39% of those changes that are seen could be accomplished by single base changes. The high degree of internal homology between residues 51-61 and 143-152 and in particular the high density of aromatic and positively charged amino acids in these two regions suggest that residues 51-61 and 143-152 may constitute two independent DNA-binding sites. Solid-phase sequencing of three tryptic peptides that together account for 9% of the 39,500-dalton UP2 protein demonstrate that there is a high degree of sequence homology between UP1 and UP2. Of the 34 residues that have been sequenced in UP2, 44% are identical in both UP1 and UP2. The blocked NH2 terminus, amino acid composition, particularly with regard to its high glycine content and the presence of dimethylarginine, and molecular weight of UP2 suggest this protein is related to proteins that have previously been found associated with heterogeneous RNA. Taken together, these data indicate that both UP1 and UP2 belong to a new family of single-stranded nucleic acid-binding proteins that may be closely related to heterogeneous ribonucleoproteins.

Amino Acid Sequence↗
PubMed1986

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single-stranded DNA binding protein (ssDBP) UP I were shown to be cross-reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti-ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re-evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Acidic lipids enhance cathepsin D cleavage of the myelin basic protein.

Some acidic lipids including sulfatide and phosphatidylinositol were found to increase greatly the rate of cathepsin D cleavage of the myelin basic protein. Since a similar effect was seen when the substrate was changed to cytochrome C, but not when the enzyme was changed to pepsin, these acidic lipids seem to be acting on cathepsin D rather than on myelin basic protein itself. Even so, chemical modification studies suggest that this phenomenon is only seen when the myelin basic protein is in its native conformation. Succinylation of MBP increases its rate of cleavage by cathepsin D by at least tenfold and, in addition, with this modified and presumably denatured MBP as substrate, activation of cathepsin D is no longer seen with acidic lipids. These findings suggest that the native conformation of MBP is both an important determinant of its rate of cleavage by cathepsin D and is also essential for observing activation of this reaction by acidic lipids. The acidic lipids seem to alter the "extended active site" of cathepsin D in such a way as to enable this enzyme better to utilize the native myelin basic protein as a substrate. Cathepsin D has previously been implicated as the protease responsible for the release into cerebrospinal fluid in multiple sclerosis patients of an encephalitogenic fragment derived from myelin basic protein. It is possible that the elevated levels of cathepsin D and sulfatide that have previously been found associated with multiple sclerosis plaques in vivo act in concert to bring about the rapid cleavage and subsequent loss of the myelin basic protein from these localized regions in the myelin sheath.

Animals↗
PubMed1986

Orthodontic movement of a canine into an adjoining extraction site.

The orthodontic movement of a canine tooth into an adjoining extraction site using a removable appliance is calculated using the finite element method (FEM). The calculated movements are compared, using a reflex plotter, with actual clinical measurements made on several patients. In both cases, wide variations in tooth displacement and hence rotation are found. The most important variable indicated by FEM appears to be the angle at which the load acts on the tooth. The position and magnitude of load have only a minor influence.

Cuspid↗
PubMed1986

An optical and electron microscopy study of materials implanted in the rat middle ear. I Carbon.

Glassy carbon implants in the middle ear of the rat model appear well tolerated over periods of 6 months. The implants are rapidly covered with a fibrous capsule resulting in some bonding to the surrounding tissue. At a later stage (12 wk), further development of this bond takes place through growth of bone onto the implant surface from adjoining bone lamellae. No adverse tissue reactions were found in the histological sections produced.

Animals↗
PubMed1986

An optical and electron microscopy study of materials implanted in the rat middle ear. II. A bioactive glass ceramic.

The bioactive glass Ceravital is rapidly covered by a fibrous encapsulating layer when placed in the rat middle ear. In addition, the surface of the glass becomes gradually modified from purely a silicate to a phosphosilicate. It is suggested that this phosphosilicate layer acts as a reservoir for the gradual mineralization of the encapsulating collagenous fibrils, resulting eventually in the formation of a strong bony bond.

Animals↗
PubMed1986

Ceravital in ossiculoplasty: experimental studies and early clinical results.

The biocompatibility of the bioactive glass-ceramic Ceravital was investigated experimentally both in vivo and in vitro. In the former, ceramic discs were interposed in 30 rat middle ears for periods ranging from 6 weeks to 12 months. In the latter, Ceravital otological prosthesis were placed in human fibroblast culture. Reactions to the biomaterial were assessed by means of scanning electron microscopy (SEM) with chemical analysis facilities (EDAX, WDAX) and light microscopy. The interface reactions were similar in both in vivo and in vitro studies and confirmed the findings of other authors regarding the implants' biocompatibility and bioactivity. The technique of ion etching, however, enabled clear demonstration of this bioactivity on the implant surface, exposed merely to the middle-ear secretions, thus questioning the necessity of placing bone pâté on the implant head at surgery, in order to encourage bioactive bonding with the overlying drum. A clinical trial of Ceravital in 128 patients with an average follow-up period of 2 years is reported. Forty per cent of these patients underwent a type II tympanoplasty, 60 per cent a type III. Though relatively short-term, the results to date have been encouraging: 88 per cent of the implants have been well-tolerated; 70 per cent of the cases have yielded a satisfactory hearing result; and only 3 per cent of the implanted prostheses have been extruded.

Animals↗
PubMed1986

Coding sequence of the precursor of the beta subunit of rat propionyl-CoA carboxylase.

A cDNA encoding the cytoplasmic precursor of the beta subunit of the mitochondrial enzyme propionyl-CoA carboxylase (EC 6.4.1.3) was cloned and sequenced. The DNA sequence of 2070 nucleotides is almost identical in size to the major hybridizing mRNA from rat liver (2000 +/- 50 nucleotides), suggesting that the cloned DNA represents nearly all of the mRNA sequence. A polypeptide expressed in vitro from an mRNA transcript of this cDNA is indistinguishable in size from the beta subunit precursor (58,500 Da). An open reading frame of 1623 nucleotides, flanked by stop codons, encodes a polypeptide of 541 amino acids; the predicted amino acid sequence was confirmed as that of the beta subunit of propionyl-CoA carboxylase by matching it to the amino acid sequences of five peptides derived from pure mature rat enzyme. Although the exact length of the cleavable, NH2-terminal leader peptide has not been determined because the NH2-terminal residue of the mature subunit is blocked, the leader is most likely 40-42 amino acids in length and is highly positively charged. Computer-aided analysis of secondary structure suggests that the leader peptide consists of two alpha-helical segments, with the two most NH2-terminal arginine residues occupying opposite sites of the first helix; this helix has no apparent hydrophobic moment.

Amino Acid Sequence↗
PubMed1986

Gene 32 protein, the single-stranded DNA binding protein from bacteriophage T4, is a zinc metalloprotein.

Gene 32 protein (g32P) isolated from bacteriophage T4-infected Escherichia coli and from an overproduction vector derived from the plasmid pKC30 contains 1 mol of tightly incorporated Zn(II) per mol of protein. A linear incorporation of three molar equivalents of p-hydroxymercuriphenylsulfonate (PMPS) results in a linear release of 1.1 mol of Zn(II) from the protein. Reversal of formation of the g32P-PMPS complex with thiol in the presence of EDTA results in a zinc-free apo-g32P. Cd(II) and Co(II) can be exchanged with the intrinsic Zn(II) ion. The Cd(II) protein shows a charge-transfer band at approximately 250 nm. The Co(II) protein shows a set of absorption bands typical of a tetrahedral Co(II) complex (epsilon max = 660 M-1 X cm-1 at 645 nm), and two intense charge-transfer bands are present at 355 nm (epsilon = 2,250 M-1 X cm-1) and 320 nm (epsilon = 3,175 M-1 X cm-1). These observations are consistent with three cysteines as ligands to the Zn(II) ion in g32P. Zn(II) g32P undergoes precise limited proteolysis by trypsin to produce the small fragments A and B and the core, g32P-(A + B). Under identical conditions, apo-g32P is hydrolyzed rapidly beyond the g32P-(A + B) stage to produce many proteolyzed fragments. Fluorescence quenching experiments show that at low protein concentration apo-g32P has markedly altered binding affinity for poly(dT) relative to native g32P. Three of the four cysteines of g32P are found in a tyrosine-rich sequence corresponding to residues 72-116 and implicated in DNA binding by 1H NMR investigations. Zn(II) appears to provide a conformational element contributing to DNA binding by coordinating the cysteine and possibly histidine side chains in the sequence -Cys-X3-His-X5-Cys-X2-Cys-, residues 77-90, located in the DNA binding domain of g32P.

Amino Acid Sequence↗
PubMed1986

Cloning of T4 gene 32 and expression of the wild-type protein under lambda promoter PL regulation in Escherichia coli.

Bacteriophage T4 gene 32 encodes a single-stranded DNA binding protein required for T4 DNA replication, recombination, and repair. Previous attempts at cloning gene 32 have failed due to a presumably deleterious effect on host cell viability. In addition, overexpression of gene 32 would be expected to be limited by the autoregulatory ability of the gene 32 product g32P. A repetitive A + T-rich sequence flanking the ribosome binding site of gene 32 has been implicated in this translational regulation. To circumvent these problems, the wild-type gene for g32P has been reconstructed in M13 using restriction fragments from T4 g32am453 and synthetic oligodeoxynucleotides so that it no longer includes its native promoter and putative autoregulatory region. The g32am453 codon TAG was changed back to TGG as in wild-type gene 32 using site-directed oligodeoxynucleotide mutagenesis. In vectors containing the lambda leftward promoter PL, gene 32 is overexpressed with the resulting transcripts being depressed at g32P concentrations that repress the wild-type gene 32 transcripts.

Bacteriophage lambda↗
PubMed1986

Experimental and clinical aspects of carbon as a middle ear prosthesis.

The biocompatibility of vitreous carbon and carbon fibre reinforced carbon (CFRC) as middle ear implants was investigated. In separate animal model systems, vitreous carbon was implanted in the middle ear of 20 DA rats for up to 6 months, whilst 3 varieties of CFRC (basic or crude, ultrasonically cleaned, glazed) were implanted in 98 guinea-pig middle ears for up to 4 months. Tissue reactions to implantation were assessed by means of stereoscopic and standard light microscopy, and scanning electron microscopy with chemical analysis facilities (EDAX, WDAX). Vitreous carbon and glazed CFRC were considered suitable for human implantation and a trial of the latter was started in 23 patients (14 partial, 9 total ossicular implants). At 9 months, only 52% of the implants were still in situ; 40% had been extruded and 8% had elicited inflammatory responses which would undoubtedly result in extrusion. Carbon particle dissemination with carbon fibre destruction and exposure as a result of peroperative prosthesis drilling were considered the likely causes for the failure of this implant.

Animals↗
PubMed1986

A model for polygenic inheritance of abdominal tergal scale pattern in Aedes aegypti.

There is much variation in the amount of white scaling on the abdominal tergites of Aedes aegypti. The genetic basis for this white scale pattern was investigated in two laboratory strains established by selection from the CARN Strain of Ae. aegypti. These experimental strains were crossed in all possible directions in single pair matings. Based on analysis of their progeny it is proposed that genes at three separate independently assorting loci control abdominal tergal scale pattern. Correlation of observed data and expected data was high. Since the abdominal tergal scale pattern in Ae. aegypti appears to be controlled by one major polygenic system with modifiers, it is proposed that it is better to consider differences in ethology, physiology, and reproductive behavior as the bases for separation of the species into intraspecific groups.

Aedes↗
PubMed1985

A monoclonal antibody that recognizes the functional domain of Escherichia coli single-stranded DNA binding protein that includes the ssb-113 mutation.

We have isolated a monoclonal antibody against Escherichia coli single-stranded DNA binding protein (SSB) that recognizes the functional domain specified by the ssb-113 temperature-sensitive mutation, a domain which is distinct from the DNA-binding site. Although the ssb-113 and ssb-1 mutations result in many similar phenotypic defects, they differ significantly in others, indicating that they affect different functional domains of the protein. Whereas the SSB-1 mutant protein is clearly defective in tetramer formation and is also unable to bind single-stranded DNA at nonpermissive temperatures, no similar in vitro defects have yet been found in the SSB-113 mutant protein. In fact, the only reported in vitro effect of the ssb-113 mutation on the protein is a slight increase in its helix destabilizing ability. Competition radioimmunoassays using a monoclonal antibody demonstrated that SSB-113 mutant protein, containing a single amino acid substitution at position 176 (the penultimate residue), did not compete with SSB while SSB-1 protein (with a single change at position 55) did compete with SSB. This analysis was refined by studies with a proteolysis fragment and with peptides derived from both SSB and SSB-113. The results indicate that the antibody recognizes a determinant near the COOH-terminal end of the protein and that the SSB-113 mutation lies within or very close to this determinant.

Antibodies, Monoclonal↗