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Biomedical subjects

K L Knight

Publications and source records attributed to K L Knight.

At least 127 records · Page 7Linked to original sources
PubMed1985

Affinity labeling of a tyrosine residue in the ATP binding site of the recA protein from Escherichia coli with 5'-p-fluorosulfonylbenzoyladenosine.

We have covalently modified the recA protein from Escherichia coli with the adenine nucleotide analog 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA). The rate at which the protein is modified shows a sigmoidal dependence on the concentration of 5'-FSBA suggesting that binding of the analog is characterized by positive cooperativity. Covalent modification of the protein results in irreversible inactivation of its single-stranded DNA-dependent ATPase activity such that 100% inactivation is achieved when 25% of the enzyme monomers have been modified. Attachment of 5'-FSBA is specific for the ATP-binding site of recA protein as judged by the following criteria: (i) attachment of the affinity label to the protein appears to saturate at 1 mol of 5'-FSBA/mol of protein; (ii) binding of 5'-FSBA to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine-5'-O-(thiotriphosphate), ADP, UTP, and GTP, but not by adenosine; (iii) attachment of 5'-FSBA to the protein occurs at a single site as determined by high pressure liquid chromatography peptide separation. Following trypsin digestion of recA protein that had been covalently modified with [3H]5'-FSBA we isolated a single labeled peptide (T31) containing the exclusive site of 5'-FSBA attachment. A secondary proteolytic digestion was performed on both 5'-FSBA modified T31 and unmodified T31 using Staphylococcus aureus V8 protease, and by comparison of the amino acid compositions of the resulting peptides we identified Tyr-264 as the exclusive site of 5'-FSBA attachment in recA protein.

Adenosine↗
PubMed1985

Tyrosine 264 in the recA protein from Escherichia coli is the site of modification by the photoaffinity label 8-azidoadenosine 5'-triphosphate.

The photoaffinity label 8-azidoadenosine 5'-triphosphate (N3-ATP) was used to covalently modify the recA protein from Escherichia coli within its ATP-binding site. We have previously demonstrated that N3-ATP modification of recA protein is specific for the ATP-binding site and have isolated a unique tryptic peptide (T31), spanning residues 257-280, that contains the exclusive site of attachment of this ATP analog (Knight, K. L., and McEntee, K. (1985) J. Biol. Chem. 260, 867-872). We performed a secondary proteolytic digestion of the [alpha-32P]N3-ATP-labeled T31 peptide using Staphylococcus aureus V8 protease and purified the resulting peptide fragments by high-pressure liquid chromatography (HPLC). Based on a comparison of the amino acid compositions of all purified fragments and sequence analysis of one labeled fragment we determined that Tyr-264 is the exclusive site of N3-ATP attachment in recA protein. Photoaffinity labeling of recA protein was also performed in the presence of single-stranded DNA. Following trypsin treatment and separation of peptides by HPLC we showed that tryptic peptide T31 contained the exclusive site of N3-ATP attachment. A secondary proteolytic digestion was performed on both [alpha-32P]N3ATP-modified T31 and unmodified T31 using alpha-chymotrypsin. Comparison of the HPLC profiles and amino acid compositions of the resulting fragments was consistent with Tyr-264 as the exclusive site of N3-ATP attachment to recA protein.

Adenosine Triphosphate↗
PubMed1985

Covalent modification of the recA protein from Escherichia coli with the photoaffinity label 8-azidoadenosine 5'-triphosphate.

We have covalently modified the recA protein from Escherichia coli with the photoaffinity ATP analog 8-azido-[alpha-32P]ATP (N3-ATP). Covalent attachment of N3-ATP to recA protein is dependent on native protein conformation and is shown to be specific for the site of ATP hydrolysis by the following criteria. (i) Binding of the probe to recA protein is inhibited by ATP and competitive inhibitors of its ATP hydrolytic activity, e.g. adenosine 5'-O-(thiotriphosphate), ADP, and UTP, but not by adenosine; (ii) N3-ATP is efficiently hydrolyzed by recA protein in the presence of single-stranded DNA; (iii) labeling of recA protein occurs at a single site as judged by two-dimensional thin-layer peptide mapping and high-performance liquid chromatography peptide separation. We have purified and identified a tryptic fragment, spanning amino acid residues 257-280, which contains the primary site of attachment of N3-ATP. This peptide is likely to be contained within the ATP hydrolytic site of recA protein.

Adenosine Triphosphate↗
PubMed1985

Quadriceps strengthening with the DAPRE technique: case studies with neurological implications.

The Daily Adjustable Progressive Resistive Exercise (DAPRE) technique was developed clinically in an effort to provide an objective means of increasing resistance concurrently with strength increases during knee rehabilitation subsequent to injury/surgery. The key to the DAPRE technique is that on the third and fourth sets of exercise the patient performs as many repetitions as possible. The number of repetitions performed during the third and fourth sets is used to determine the amount of weight that is added to (or sometimes removed from) the working weight for the next set and session, respectively. Consequently, patients exercise nearer their optimal capacity during each weight rehabilitation session, and their strength redevelopment occurs at a much faster rate. This report describes the quariceps muscle strength gains by 21 athletes who used the DAPRE technique following knee immobilization for a minimum of 3 wk. These patients averaged an increase of 4.3 +/- 2.2 (SD) kg X d-1 for a period of 6.4 +/- 2.2 d, as measured by a six repetitions maximum test. It seems unlikely that morphological changes were responsible for these strength increases. It is postulated that strength redevelopment following immobilization involves changes in neural pathways and/or overcoming possible neural inhibitors.

Athletic Injuries↗
PubMed1985

Organization and polymorphism of rabbit immunoglobulin heavy chain genes.

Germline genes encoding C mu, C gamma, C alpha, and C epsilon heavy chains of rabbit immunoglobulins have been isolated from recombinant phage and cosmid libraries. The JH, C mu, C gamma, and C epsilon are found in a 5'-JH-C mu-C gamma-C epsilon-3' orientation on a 90kb stretch of DNA. Four C alpha genes have been cloned and presumably reside 3' to the other CH genes. Southern blot analysis of rabbit sperm DNA indicates that the rabbit genome contains a single C gamma gene, one C mu gene, and as many as 10 C alpha genes. Restriction site polymorphism is found for C mu, C gamma, and C alpha genes of rabbits of various heavy chain haplotypes. The organization of the rabbit CH genes differs from that of mouse and human CH genes in that the rabbit has multiple C alpha genes, whereas mouse and human have one or two C alpha genes, respectively. In addition, mouse and human have four C gamma genes, whereas rabbit has only one C gamma gene. The presence of a single C gamma gene indicates that at least in the rabbits examined, no germline gene encoding latent or unexpected, C gamma allotypes is present. The genetic control of the expression of latent C gamma allotypes is discussed.

Animals↗
PubMed1985

Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes.

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.

Amino Acid Sequence↗
PubMed1985

Two B-cell subpopulations identified by flow cytometry.

Ig+ spleen cells were analysed by light scatter analysis on a flow cytometer and two distinct subpopulations were identified. The large Ig+ cells (10-11 micron in diameter) were found in spleen and bone marrow, whereas the small Ig+ cells (7-8 micron in diameter) were found in all lymphoid tissues. Of the total Ig+ splenic lymphocytes, 40-60% were large Ig+ cells and had surface IgM, Ia and Fc gamma receptors. The large Ig+ cells were highly enriched for responsiveness to the B-cell mitogens, anti-Ig, Nocardia water-soluble mitogen and LPS, whereas the small Ig+ splenic cells had little or no responsiveness to these mitogens.

Animals↗
PubMed1985

Guidelines for rehabilitation of sports injuries.

Rehabilitation involves a functional progression through a systematic program of physical reconditioning involving the re-establishment of intact articulations and muscles, pain-free joints and muscles, joint flexibility, muscular strength, muscular endurance, muscular speed, integrated and coordinated movement (skill patterns), and cardiovascular endurance. Specific demands must be imposed upon the body to bring about redevelopment of each phase. A proper diagnosis prior to beginning, and constant monitoring of the patient's progress during, rehabilitation are necessary so that the demands of the therapeutic regimen can be adjusted according to the patient's progress. The DAPRE technique objectifies isotonic and loaded isometric strength development and therefore stimulates greater strength gains during rehabilitation than other techniques do.

Athletic Injuries↗
PubMed1984

Identification of the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli: recA441 and recA629.

We have identified the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli. The recA441 mutant, which shows constitutive expression of the recA-mediated SOS response at 42 degrees C, contains two amino acid substitutions, glutamic acid to lysine at residue 38 and isoleucine to valine at residue 298. The recA629 mutant is an unusual pseudorevertant of recA441 that is no longer capable of spontaneous expression of SOS functions at 42 degrees C. Purified recA629 protein is cold-labile for several of the wild-type enzymatic activities and is shown here to contain three amino acid substitutions, the two found in the recA441 protein at residues 38 and 298, as well as an aspartic acid-to-glycine change at residue 32. The mutation at residue 32 was verified by restriction digestion of the 5' region of the recA629 structural gene.

Alleles↗
PubMed1984

The reaction of ozone with glyceraldehyde-3-phosphate dehydrogenase.

Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GPDH) by ozone can be correlated with oxidation of the active-site -SH residue. Oxidation of peripheral -SH groups, and tryptophan, methionine, and histidine residues occurs concomitantly, but loss of activity depends solely on active-site oxidation. Inactivation is only slightly reversible by dithiothreitol. Kinetic studies show that inhibition of GPDH by ozone mimics noncompetitive inhibition and is characterized as irreversible enzyme inactivation. Analysis of products resulting from ozone oxidation of glutathione suggests that cysteic acid is the product of protein-SH oxidation. Despite oxidation of the active-site -SH , no significant decrease in the Racker band absorbance occurs. This is explained by the appearance of a new chromophore in this region of the absorbance spectrum. Increased absorbance at 322 nm following ozone treatment indicates that tryptophan is converted quantitatively to N-formylkynurenine. When the active-site -SH is reversibly blocked by tetrathionate, enzyme activity is completely recoverable following reaction of the derivatized enzyme with a 1.3X excess of ozone over enzyme monomer. Activity is fully recovered despite the oxidation of peripheral -SH, tryptophan, and histidine residues. Circular dichroism spectra of ozone-treated enzyme show that reaction of GPDH with up to a threefold excess of ozone over enzyme monomer results in no significant disruption of protein secondary structure. Spectra in the near-uv show distinct changes that reflect tryptophan oxidation.

Amino Acids↗
PubMed1984

Genes encoding alpha-heavy chains of rabbit IgA: characterization of cDNA encoding IgA-g subclass alpha-chains.

cDNA molecules encoding rabbit IgA alpha-heavy chains have been synthesized and six of these have been characterized. The complete nucleotide sequence of one cDNA, p 19 (942bp), showed that it encoded all but the N-terminal 57 amino acid residues of the constant region of alpha-chains. The cDNA molecules were subcloned into the expression vector pUC8 and E. coli were transformed. Radioimmunoassay of the molecules synthesized by these clones showed that all six cDNA molecules encoded alpha-chains of the IgA-g subclass. Comparison of the amino acids encoded by the alpha-cDNA with the amino acid sequence of mouse and human alpha-chains showed that although all of the intradomain disulfide bonds appear to be conserved, some positions, probably involved in interchain disulfide bonds, are not conserved. We propose that secretory component is covalently bound to cysteine 299 and/or cysteine 301 of the CH2 domain of mouse and human alpha-chains. The results from Southern blot analysis of genomic DNA with 32P-cDNA suggests that the rabbit genome has multiple C alpha genes.

Animals↗
PubMed1984

The role of rabbit Ia molecules in immune functions as determined with the use of an anti-Ia monoclonal antibody.

We have produced a mouse anti-rabbit Ia monoclonal antibody (MAb) that detects an isotypic determinant on all rabbit Ia molecules. This MAb precipitates three polypeptide chains with molecular weights of 28,000, 31,000 and 35,000, corresponding to the Ia beta, Ii and alpha chains, respectively. The anti-Ia MAb inhibits the mixed lymphocyte culture by 80%. In secondary in vitro immune response cultures, the anti-Ia MAb inhibits the proliferative response to bovine insulin and poly (Glu50Tyr50). In studies on mitogenesis it was found that the anti-Ia MAb inhibited the response to LPS but not to concanavalin A or phytohaemagglutinin. The effect of the anti-Ia MAb on other mitogens was found to vary from rabbit to rabbit.

Animals↗
PubMed1984

Characterization of functionally distinct subpopulations of rabbit T lymphocytes.

Two subpopulations of rabbit spleen and mesenteric lymph node T cells were identified by a monoclonal antibody, 8AC8. These subpopulations were separated on the flow cytometer and were analysed for their response to T cell mitogens and antigens, their responsiveness in mixed lymphocyte cultures, and their ability to function as cytotoxic effector cells. The 8AC8+ T cell subpopulation contained cells highly responsive to T cell mitogens, to antigen, and to allogeneic or autologous stimuli in a mixed lymphocyte reaction. In contrast, the 8AC8- T cell subpopulation was non-responsive to T cell mitogens, and responded, poorly to antigen, and to allogeneic and autologous stimuli in an MLR. Both the 8AC8+ and 8AC8- subpopulations exhibited xenogenic cytotoxic effector function. Thus, the 8AC8 MAb identified a subpopulation of mature differentiated rabbit T cells; these 8AC8+ cells share many of the characteristics of the human OKT4 helper/inducer T cell subpopulation.

Animals↗
PubMed1984

Molecular genetic analysis of genes encoding the heavy chains of rabbit IgG.

We analyzed the genes which encode the heavy chain constant region of rabbit IgG molecules. Five DNA clones derived from the chromosomal region which spans the C gamma coding sequences were isolated from a recombinant phage library of rabbit liver DNA. Four of these clones can be grouped together by overlaps; together they represent 37 kb of genomic DNA, and contain one C gamma gene and one tentatively identified C epsilon gene. A second C gamma gene was identified which did not overlap with the group of four clones because of restriction site differences found in the flanking regions 5' to the C gamma genes. Nucleotide sequences of the two C gamma genes were identical. Comparisons of the restriction maps of the two cloned C gamma genomic regions suggest that they may represent allelic regions of chromosomes of two different heavy chain haplotypes with polymorphisms in the regions flanking the C gamma genes.

Amino Acid Sequence↗
PubMed1983

Serologic cross-reactions among rabbit secretory IgA molecules: evidence for multiple subclasses of secretory IgA-f molecules.

Serologic cross-reactions among allotypes of rabbit secretory IgA (sIgA) of the f-subclass were examined by quantitative radiobinding assays with various allo-anti-alpha chain reagents. Numerous cross-reactions were observed which demonstrated the complexity of the C alpha f allotypes. One of the reagents used in these studies reacted not only with all sIgA molecules of the immunogen C alpha f allotype but also with all sIgA molecules of the other C alpha f allotypes. Aliquots of the antiserum were each adsorbed with IgA molecules of these C alpha f allotypes and then used in radiobinding studies with sIgA-f molecules of the various C alpha f allotypes. The adsorbed reagents reacted with some but not all sIgA-f molecules, thus indicating that the C alpha f allotypes each comprise more than one serologically distinguishable subset. Results from these radiobinding experiments were used to develop a model in which each of the five IgA-f allotypes comprises at least two serologically distinguishable subsets. Each of these subsets expresses a unique pattern of C alpha determinants. These C alpha determinants appear to be protein in nature rather than carbohydrate as periodate oxidation of the sIgA-f glycoproteins does not affect the reactions of the molecules with the cross-reactive alloreagents. IgA molecules of the serologically distinguishable subsets presumably differ in amino acid sequence in the constant region and, thus, would be products of distinct C alpha genes. Therefore, it is probable that the cross-reactions of these molecules, which were previously thought to comprise a single subclass, sIgA-f, may reflect the presence of more than one subclass of sIgA-f molecules, i.e., a third subclass of rabbit sIgA.

Animals↗