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Biomedical subjects

K J Wilson

Publications and source records attributed to K J Wilson.

At least 55 records · Page 3Linked to original sources
PubMed1986

Salmonella cytotoxin: a component of the bacterial outer membrane.

Salmonella cytotoxin present in cell-free sonic lysates causes rounding and detachment of Chinese hamster ovary (CHO) cells. Although the precise role of this toxin in the pathogenesis of salmonellosis is unclear, cytotoxin production by Salmonella could account for tissue damage or possibly, facilitate invasion. A variety of other bacteria (e.g. Shigella, Escherichia, Legionella) have been shown to form soluble cytotoxins, many of which may be involved in pathogenesis. The data in this report indicate that the Salmonella cytotoxin in cell-free sonic lysates is firmly associated with cell membrane fragments that can be pelleted by ultracentrifugation (270,000 g for 2.4 h). Furthermore, lysozyme treatment of filter-sterilized sonic extracts of Salmonella species followed by isopycnic sucrose gradient ultracentrifugation allowed separation of the outer and inner membrane components. The outer membrane (OM) peak contained the cytotoxic activity when assayed for detachment of CHO cells. The importance of these data resides in the observation that the Salmonella cytotoxin is an outer membrane component. Its mere location places it in a position of direct contact with host cells and suggests a possible role in cell damage and/or invasion. Furthermore, ultracentrifugation provides a method by which much of the Salmonella cytotoxin in sonic extracts can be removed allowing expression of the Salmonella enterotoxin, whose CHO cell elongation effect is usually obscured by the presence of the cytotoxin causing cell rounding and detachment.

Animals↗
PubMed1985

Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1).

Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.

Amino Acid Sequence↗
PubMed1984

Characterization of translational inhibitors from Phytolacca americana. Amino-terminal sequence determination and antibody-inhibitor conjugates.

Two translational inhibitors (pokeweed antiviral protein and pokeweed antiviral protein II) isolated from the leaves of the pokeweed plant, Phytolacca americana, were characterized as to their behavior during reverse-phase HPLC and their amino-terminal sequences. Alignment of the sequences demonstrated that a substantial degree of homology was present (10 of 29 identical residues). Pokeweed antiviral protein was shown by reverse-phase chromatography to be composed of at least two components, pokeweed antiviral proteina and pokeweed antiviral proteinb, which comigrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis, shared identical N-terminal amino-acid sequences through residue 31, and had similar specific activities in a cell-free translation inhibition assay. Pokeweed antiviral protein II was covalently coupled to a monoclonal antibody that recognizes the transferrin receptor (anti-transferrin receptor). The disulfide-linked conjugate inhibited protein synthesis in the human breast tumor cell line MCF-7, whereas anti-transferrin receptor, pokeweed antiviral protein II, or an immunotoxin composed of an irrelevant antiserum and pokeweed antiviral protein II, were nontoxic. The inhibitory dose 50% of anti-transferrin receptor-pokeweed antiviral protein II for MCF-7 cells was 0.7 nM, whereas the corresponding ricin A chain conjugate (anti-transferrin receptor-ricin A chain) was more potent with a inhibitory dose 50% of 0.1 nM. Pokeweed antiviral protein II can be added to the growing list of translation inhibitors that are effective as components of immunotoxins in vitro. Additional studies will be needed to determine whether pokeweed antiviral protein II immunotoxins provide advantageous properties for in vivo applications.

Amino Acid Sequence↗
PubMed1984

Contemporary methodology for protein structure determination.

The techniques used for the characterization of protein and peptide structure have undergone great changes that have improved the speed, reliability, and applicability of the process. High-performance liquid chromatography and gel electrophoresis have made the purification of proteins and peptides a routine procedure, even when the compound of interest is a minor component of a complex biological mixture. The chemistry and instrumentation used in amino acid analysis and amino acid sequencing now permit the analysis of as little as 5 to 50 picomoles of samples. This represents an increase in sensitivity of more than a thousandfold over the last 10 years and has made possible the structural analysis of a wide variety of scarce but important compounds.

Amino Acid Sequence↗
PubMed1984

Utility of the gas-phase sequencer for both liquid- and solid-phase degradation of proteins and peptides at low picomole levels.

The utility of the commercially available gas-phase sequencer for complete analysis of peptide samples was investigated. Using the program supplied with the instrument, significant extractive loss of samples in Polybrene was observed, even at input levels up to 500 pmol. In order to reduce this loss, the sequencer program was modified by increasing the phenylisothiocyanate (PITC)-coupling steps from two to three and lengthening the duration of ethyl acetate (S2) delivery while reducing the delivery rate. These changes gave improved results with peptides, e.g., all eight residues of angiotensin II were identified at the 25-pmol level. In addition, background contamination was decreased and repetitive yields were increased. The instrument was also found to function well with samples coupled to solid supports; however, some of the methodologies that work adequately for covalent attachment of peptides to solid supports at the level 1-10 nmol were found to give unacceptable coupling/sequenceable yields at or below the 100-pmol level. The coupling methods tried were (1) reaction of homoserine lactone with aminopropyl (AP)-glass, (2) reaction of alpha- and epsilon-NH2 groups with p-phenylenediisothiocyanate (DITC)-glass, and (3) reaction of alpha-COOH groups with aminoaryl (AA)-glass via EDAC (1-ethyl-3,3'-dimethylaminopropyl-carbodiimide). Of these, the first method gave combined yields of 42-94% while the latter two were only 9-35% efficient. The covalently bound samples provided sequence information even at the resulting low levels, e.g., 9/13 residues of dynorphin including Lys-13 at 11 pmol. In general, sequencer runs on solid-phase samples gave "cleaner" analyses and slightly higher repetitive yields (1-2%). Sequence information has also been obtained on peptides made by solid-phase synthesis prior to cleavage from the polystyrene support. With improved coupling efficiencies, solid-phase techniques would provide an alternative to immobilization of peptides in Polybrene films for low picomole level gas-phase sequencing.

Amino Acid Sequence↗
PubMed1984

Concentrations of vancomycin in bone and serum of normal rabbits and those with osteomyelitis.

The concentrations of vancomycin in the bone and serum of rabbits with Staphylococcus aureus osteomyelitis were assessed after each rabbit was given a single dose of vancomycin. Simultaneous mean concentrations of vancomycin in infected rabbits 1 h after administration of the antibiotic were 36.4 +/- 4.6 micrograms/ml (serum), 5.3 +/- 0.8 microgram/g (infected bone), and 3.0 +/- 0.2 micrograms/g (noninfected bone). Concentrations of vancomycin in serum of normal controls were higher than concentrations of vancomycin in serum of osteomyelitic rabbits after 1, 2, 3, and 6 h.

Animals↗
PubMed1984

Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa.

Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation. The A. tumefaciens and E. coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants. A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid. These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules. Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules. There was no evidence of bacterial release from the infection threads. In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells. No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed. In contrast to the A. tumefaciens-induced pseudonodules , the E. coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules. Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa.

Base Sequence↗
PubMed1983

The covalent structure of mitochondrial aspartate aminotransferase from chicken. Identification of segments of the polypeptide chain invariant specifically in the mitochondrial isoenzyme.

The primary structure of mitochondrial aspartate aminotransferase from chicken is reported. The enzyme is a dimer of identical subunits. Each subunit contains 401 amino acid residues; the calculated subunit molecular weight of the apoform is 44,866. The degree of sequence identity with the homologous cytosolic isoenzyme from chicken is 46%. A comparison of the primary structures of the mitochondrial and the cytosolic isoenzyme from pig and chicken shows that 40% of all residues are invariant. The degree of interspecies sequence identity both of the mitochondrial and the cytosolic isoenzyme from chicken and pig (86% and 83%, respectively) markedly exceeds that of the intraspecies identity between mitochondrial and cytosolic aspartate aminotransferase in chicken (46%) or in pig (48%). Based on these values, the duplication of the aspartate aminotransferase ancestral gene is estimated to have occurred approximately 1000 million years ago, i.e. at the time of the emergence of eukaryotic cells. By sequence comparison it is possible to identify amino acid residues and segments of the polypeptide chain that have been conserved specifically in the mitochondrial isoenzyme during phylogenetic evolution. These segments comprise about a third of the total polypeptide chain and appear to cluster in a certain surface region. The cluster carries an excess of positively charged residues which exceeds the overall charge difference between the cytosolic (pI approximately 6) and the mitochondrial isoenzyme (pI approximately 9).

Amino Acid Sequence↗
PubMed1983

Ca2+-binding proteins: a comparative study of their behavior during high-performance liquid chromatography using gradient elution on reverse-phase supports.

Reverse-phase high-performance liquid chromatography has been shown to be applicable to the isolation of Ca2+-binding proteins, specifically parvalbumins, from tissue extracts or from preparations first purified by "conventional" chromatography. Through an investigation of the behavior of a series of Ca2+-binding proteins as a function of buffer composition, pH, and organic eluant it has been possible to define mild conditions allowing for chromatography of the proteins in their native states. The elution positions of parvalbumins were not observed to correlate with the "overall" protein hydrophobicity, calculated using hydrophobicity values for the individual amino acids, thus indicating that factors such as hydrophobic/hydrophilic surface areas are important in determining the degree of association with the support. The usefulness of reverse-phase chromatography as an analytical tool for determining protein homogeneity is illustrated. Samples which had been isolated via "conventional" chromatography methods, and thought to be homogeneous, were observed to contain multiple species of the same protein.

Animals↗
PubMed1983

Characterization of isoprotein patterns in tissue extracts and isolated samples of metallothioneins by reverse-phase high-pressure liquid chromatography.

Reverse-phase high-pressure ('performance') liquid chromatography was used to characterize the isometallothioneins in preparations isolated from tissues of a variety of animals by 'conventional' chromatographic methods. The resolution was such that isoproteins differing by a single serine leads to leucine difference in 61 residues could be easily separated. Yields from the reverse-phase support were typically 60-70% for the isoproteins. Comparisons of isometallothionein patterns after Cd2+-induction in rabbits indicated that total metallothionein concentrations were about 4-fold higher in liver than kidney extracts from the same animal. In the extracts a minimum of four and six isometallothionein peaks were detected in kidney and liver respectively. Under acidic conditions, where the metals are removed from the protein, the chromatographic properties, i.e. hydrophobicities, of the isoproteins from kidney were identical with those of four of those found in liver. Although the same peaks appeared in tissue extracts from individual animals, concentration differences were apparent. Remarkably, no differences were observed between the isoprotein patterns of liver or kidney as a consequence of either Cd2+- or Zn2+-induction. Chromatography of the metal-containing forms at neutral pH in Tris buffer indicated that the relative ratios of the complexed metal ions in the isoproteins were found to be effectively identical, not only before and after chromatography, but also within the separated forms from a single tissue source.

Amino Acids↗
PubMed1982

Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin.

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.

Animals↗
PubMed1982

An around-the-house device for the clinical determination of respiratory driving pressure: a note on making simple even simpler.

A noninvasive method for determining a client's ability to generate respiratory driving pressure is described. This method is an off-spring, of simpler form, from a method previously offered in this journal. The key to its simplification is the use of a pressure-indication device that can be constructed from a small collection of items found around the house. The usefulness of this off-spring method is discussed with regard to evaluation and management concerns and its advantages over its parent method are considered.

Equipment Design↗
PubMed1982

Primary structure of parvalbumin from rat skeletal muscle.

The primary structure of parvalbumin from rat skeletal muscle has been determined principally by automated sequencing of tryptic peptides using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate as the Edman reagent on a solid-phase sequencer. Remaining positions and most peptide overlaps were identified by analysis of peptides arising from CNBr, chymotryptic and Staphylococcus aureus protease cleavages and through digestions with carboxypeptidases A, B and Y. Reverse-phase high-performance liquid chromatography on C-18 supports was employed for all peptide separations. Structural homology between rat and rabbit parvalbumins helped to confirm the alignments of the tryptic peptides T4-T3, T2-T6 and to define the position of the Lys triplet (36-38). A comparison of the two mammalian proteins revealed 14 amino acid differences, which are all located on the surface of the molecule. A prediction of the secondary structure has been made and found to be very similar for the rat and rabbit proteins with the exception of the sequence region 72-78, located between the Ca2+, Mg2+-binding CD and EF domains.

Amino Acid Sequence↗
PubMed1981

The behaviour of peptides on reverse-phase supports during high-pressure liquid chromatography.

High-pressure ('performance') liquid chromatography has been used to investigate the reverse-phase chromatographic behaviour of peptides, ranging in length from 2 to 65 amino acid residues, which have originated from primary-sequence determinations or solution/solid-phase syntheses. By using a pyridine/formate-pyridine/acetate/propan-1-ol buffer system, as previously described [Hughes, Winterhalter & Wilson (1979) FEBS Lett. 108, 81-86], the influence of various experimental parameters were examined. (a) Peptide retention was observed to be temperature-independent between 25 and 55 degrees C. (b) The dependence of chromatographic retention on pH decreases with increasing peptide hydrophobicity. (c) Chromatographic results from C8- and C18-chain-length, as well as from 5 micrometers- and 10 micrometers-particle-size, supports were comparable. (d) The hydrophobic strength of the organic solvent in the mobile phase was observed to decrease: propan-1-ol approximately equal to propan-2-ol greater than acetonitrile much greater than methanol. (e) When gradient rates (% of buffer B/unit time) were systematically decreased, peptide retention decreased in a hyperbolic manner. Comparisons of the peptides chromatographed with respect to their measured retention properties and calculated hydrophobicities were performed by computer analysis. Deviation of peptide chromatographic behaviour was observed to be essentially independent of hydrophobicity, chain length and charge. On the basis of the measured retention properties of the chromatographed peptides, hydrophobic constants for the various amino acid side chains were determined and compared with similar constants available from the literature.

Amino Acids↗
PubMed1981

Comparison of buffers and detection systems for high-pressure liquid chromatography of peptide mixtures.

The chromatographic properties of peptides ranging in length from 2 to 65 residues have been compared on reverse-phase packings in three buffer systems at low pH. Of the buffers examined, two are widely used in connection with u.v. detection [(i) phosphate/acetonitrile or (ii) phosphate/propan-2-ol] and the third for fluorescence detection [(iii) pyridine/formate-pyridine/acetate/propan-1-ol]. The addition of a chaotropic salt, NaClO4, to the phosphate buffers, as first described by Meek (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1632-1636, is shown to significantly improve the chromatographic behaviour of more hydrophobic peptides. The two most commonly used detection systems, u.v. and fluorescence, are compared in terms of ease of use and sensitivity.

1-Propanol↗
PubMed1981

Chemical modification of peptides by hydrazine.

High pressure ('performance') liquid chromatography on reverse-phase supports has been used to characterize the products arising from the hydrazine treatment of peptides. In addition to converting arginine residues into ornithine, the reaction was found to cleave predominately Gly-Xaa, Xaa-Gly, Asn-Xaa and Xaa-Ser peptide bonds. Peptide-bond cleavage and deguanidation was studied as a function of time of exposure to hydrazine, hydrazine concentration and temperature. The convenience of this method of chromatography for the rapid low-cost separation and isolation of peptides, as well as their reaction products, is illustrated at the level of material required for solid-phase microsequencing.

Amino Acids↗