Generating positive attention from the press.
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Mass Media↗
Biomedical subjects
K J Wilson
Publications and source records attributed to K J Wilson.
Cloning and nucleotide sequence of the Vibrio proteolyticus aminopeptidase gene.
The gene encoding the Vibrio proteolyticus aminopeptidase was cloned and sequenced and its amino acid sequence was deduced. The gene encodes a 54 kDa protein, larger than the previously reported size of 30 kDa for the purified aminopeptidase. Sequence alignments revealed a 43-45% homology with two other Vibrio sp. extracellular proteinases.
Amino Acid Sequence↗
The time dependent UV resonance Raman spectra, conformation, and biological activity of acetylcholine analogues upon binding to acetylcholine binding proteins.
In order to obtain quantitative data on the relation between the conformation of acetylcholine and its interaction with biologically significant proteins, a series of acetylcholine analogues with absorption bands in the region 200-300 nm have been synthesized or obtained commercially. Each of these compounds were assayed to measure its activity as an ion channel activator of the nicotinic acetylcholine receptor protein (AChR). In addition, the suitability of some of these compounds as substrates for hydrolysis by acetylcholine esterase (AChE) was determined. One of these analogues, dimethylthionocarbamylcholine (DMTC-Ch), has the ester carbonyl oxygen replaced by a thionyl sulfur. DMTC-Ch has been found to be quite active as an ion channel activator when bound to AChR and was found to react with the enzyme AChE as a suicide substrate. It forms a thionoester of the serine at the AChE active site by an ester exchange reaction that releases the choline as the first product. However, the second or acid product is not released even at pH 7.5 over a period of days. This acetylcholine analog has an absorption band at about 240 nm and exhibits very strong ultraviolet resonance Raman (UVRR) spectra using 239 nm excitation from a frequency modified Nd:YAG laser. This technique allows observation of both conformational changes of the ligand molecule that result in frequency changes as well as changes in the excited state electronic structure that results in changes in the relative intensity of the Raman bands. The time dependence of the UVRR spectrum of the ligand upon binding to both AChE and AChR has been studied from 0.1 msec to minutes. Some time dependence in the conformation of DMTC-Ch upon binding to AChE has been found for very short (0.1-0.5 msec) times. However, no change in the conformation of this neurotransmitter analog is found in the available time range upon binding to AChR. From these data it is concluded that a previous suggestion that acetylcholine has a conformational change upon binding to AChR may be incorrect since the solution behavior of the carbamyl cholines and acetylcholine are similar. Even if acetylcholine does change conformation upont binding to AChR, it is unlikely that such a conformational change plays a significant role in channel activation. We present strong evidence that acetylcholine and its analogues can be active in a variety of conformations.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholine↗
Effect of ivermectin on the volume of blood ingested by two species of ticks (Acari: Ixodidae) feeding on cattle.
Females of the lone star tick, Amblyomma americanum (L.), and the American dog tick, Dermacentor variabilis (Say), were fed on ivermectin (Ivomec)-treated and untreated bovines to determine the effect of the acaricide on volume of blood ingested and to compare the weight differences between the treatment and control groups at various time intervals after attachment. Adult females from each genus were collected from Bos tarus hosts and subjected to hematin assays on three collection dates to estimate the volume of blood ingested. Before feeding, lone star ticks contained an average of 2.0 microliters of blood and had an average weight of 5.2 mg. Unengorged American dog tick females had an average blood volume of 3.3 microliters and a mean weight of 5.8 mg. Ticks of both species reacted to ivermectin by expressing lower mean weights, and they consumed smaller quantities of blood. Lone star tick females were significantly affected in terms of amount of blood consumed and body weight changes when compared with control ticks. After feeding on treated cattle, lone star tick females contained smaller quantities of blood than pretreatment females, but there were no significant differences observed until day 12 between the control and the treated groups. American dog tick females on treated hosts had measurable quantities of blood that were significantly different among the experimental groups.
Animals↗
Micro-level protein and peptide separations.
The current major limitation in protein micro-structural and -functional studies is our inability to purify and manipulate microgram or smaller quantities of material. Both high performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE) have been widely used in attempts to address such needs. This review summarizes the current status of preparative and analytical applications for both methods, and suggests which directions the micro-separations field will take within the next few years.
Microchemistry↗
Structural Similarities between Spinach Chloroplast and Cytosolic Class I Fructose 1,6-Bisphosphate Aldolases : Immunochemical and Amino-Terminal Amino Acid Sequence Analysis.
Immunochemical studies using polyclonal antisera prepared individually against highly purified cytosolic and chloroplast spinach leaf (Spinacia oleracea) fructose bisphosphate aldolases showed significant cross reaction between both forms of spinach aldolase and their heterologous antisera. The individual cross reactions were estimated to be approximately 50% in both cases under conditions of antibody saturation using a highly sensitive enzyme-linked immunosorbent assay. In contrast, the class I procaryotic aldolase from Mycobacterium smegmatis and the class II aldolase from yeast (Saccharomyces cerevisiae) did not cross-react with either type of antiserum. The 29 residue long amino-terminal amino acid sequences of the procaryotic M. smegmatis and the spinach chloroplast aldolases were determined. Comparisons of these sequences with those of other aldolases showed that the amino-terminal primary structure of the chloroplast aldolase is much more similar to the amino-terminal structures of class I cytosolic eucaryotic aldolases than it is to the corresponding region of the M. smegmatis enzyme, especially in that region which forms the first "beta sheet" in the secondary structure of the eucaryotic aldolases. Moreover, results of a systematic comparison of the amino acid compositions of a number of diverse eucaryotic and procaryotic fructose bisphosphate aldolases further suggest that the chloroplast aldolase belongs to the eucaryotic rather than the procaryotic "family" of class I aldolases.
Journal Article↗
Microanalysis of SDS-PAGE electroblotted proteins.
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Amino Acid Sequence↗
Comparative evaluation of the diffusion of tobramycin and cefotaxime out of antibiotic-impregnated polymethylmethacrylate beads.
Both tobramycin and cefotaxime diffuse from antibiotic-impregnated polymethylmethacrylate (PMMA) beads in quantities sufficient to inhibit the growth of bacteria on agar lawns or in broth cultures over a 28-day period. Extraction of antibiotic from tobramycin or cefotaxime-impregnated PMMA beads revealed that substantial amounts of both antibiotics remained within the beads despite 28 days of diffusion. Diffusion of antibiotic from the PMMA beads during the initial 3-5 days is much greater than occurs for the remainder of the 4-week period. The results of the study suggest that perhaps tobramycin of cefotaxime-impregnated PMMA beads would produce local levels of antibiotic high enough to sterilize a given dead space for a period of 28 days.
Anti-Bacterial Agents↗
Applications of tandem microbore liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/electroblotting in microsequence analysis.
Protein isolation by microbore HPLC is compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/electroblotting methods for several major proteins from rabbit muscle. Although single-mode HPLC or SDS-PAGE/electroblotting provides excellent speed and sensitivity for submicrogram-level protein purification, neither one alone has adequate resolution for separating such a complex protein mixture. Tandem procedures, utilizing two different modes of HPLC in separate steps or a combination of single HPLC separation and SDS-PAGE/electroblotting, offer the necessary versatility. One of the major concerns in this investigation was to evaluate electroblotting techniques for microsequencing. The Aebersold et al. procedure (R.H. Aebersold, D.B. Teplow, L.E. Hood, and S.B.H. Kent (1986) J. Biol. Chem. 261, 4229-4238) was substantially modified and improved; the details of this work will be published elsewhere. These changes significantly improve repetitive yields at the low microgram level without producing high backgrounds. At lower levels the recovery of sequenceable protein currently limits our ability to obtain useful results. Starting with 250-750 micrograms of rabbit muscle crude extract, several proteins (15-70 kDa) were isolated by tandem microbore LC and PAGE/electroblotting for amino-terminal sequence analysis. It appears that the combination of electroblotting and microbore LC represents a powerful approach for microsample preparation.
Amino Acid Sequence↗
Effect of buffer pH and peptide composition on the selectivity of peptide separations by capillary zone electrophoresis.
A series of 10 synthetic peptides containing varying degrees of charge and hydrophobicity was used to study the effects of peptide composition and buffer pH on the selectivity of separations by capillary zone electrophoresis (CZE). A simple model is used to explain the effect of buffer pH on the separation. It was found that pH is an important parameter affecting the selectivity of CZE separations. Furthermore, it is shown that the selectivity of the separation is such that peptides differing in neutral amino acid composition can be resolved, and that even differences in a peptide's amino acid sequence can be detected. A protease digest of beta-lactoglobulin A is shown as a practical example of a separation of a complex peptide mixture.
Amino Acids↗
Environmental ultrasound in laboratories and animal houses: a possible cause for concern in the welfare and use of laboratory animals.
Many laboratory animals are known to be sensitive to sounds (ultrasounds) beyond the nominal upper limit (20 kHz) of the human hearing range. Sources of sound in laboratories and animal houses were examined to determine the extent of ambient ultrasound. Of 39 sources monitored, 24 were found to emit ultrasonic sounds. Many of these (e.g. cage washers and hoses) also produced sound in the audible range. Running taps, squeaky chairs and rotating glass stoppers created particularly high sound pressure levels and contained frequencies to over 100 kHz. The oscilloscopes and visual display units investigated provided particular cause for concern as they emitted sounds that were entirely ultrasonic and therefore were apparently silent. Ambient ultrasound therefore appears to be common in laboratories and animal houses. It is suggested that its effect on laboratory animals should be investigated and guidelines on acceptable levels be formulated.
Animal Welfare↗
Identification of a long form of cystatin from human saliva by rapid microbore HPLC mapping.
Microbore HPLC methodology permits rapid and sensitive mapping of human saliva proteins. Saliva is sampled and processed in less than one hour, greatly reducing the likelihood of artifactual protein degradation. As little as 50 microliters of saliva yields proteins in sufficient quantities and purity to obtain amino terminal sequences directly. By this route we have discovered a 14 kDa protein extremely homologous to Cystatin S, but amino-terminally extended by eight amino acids.
Amino Acid Sequence↗
Effects of abscisic acid on K+ channels in Vicia faba guard cell protoplasts.
Potassium channels were resolved in Vicia faba guard cell protoplasts by patch voltage-clamp. Whole-cell currents and single K+ channels had linear instantaneous current-voltage relations, reversing at the calculated Nernst potential for K+. Whole cell K+ currents activated exponentially during step depolarizations, with half-activation times of 400-450 msec at +80 mV and 90-110 msec at +150 mV. Single K+ channel conductance was 65 +/- 5 pS with a mean open time of 1.25 +/- 0.30 msec at 150 mV. Potassium channels were blocked by internal Cs+ and by external TEA+, but they were insensitive to external 4-aminopyridine. Application of 10 microM abscisic acid increased mean open time and caused long-lasting bursts of channel openings. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology.
Abscisic Acid↗
Partial primary structures of human and murine macrophage colony stimulating factor (CSF-1).
Approximately 40 amino-terminal residues and 20 internal residues of CSF-1 purified from the media of cultured human pancreatic carcinoma (MIA PaCa) cells and of cultured murine L cells have been identified. Results indicated that the two subunits in each molecule of biologically active CSF-1 are identical in their amino-terminal portions. The twelve amino-terminal residues of MIA PaCa CSF-1 were found to be identical to those of human-urinary CSF-1, suggesting that the polypeptide portions of the two human proteins may be identical. Approximately 75% of the amino acids identified in both MIA PaCa CSF-1 and murine CSF-1 were found to be common to both. No homology to other proteins was observed. This study suggests a subunit polypeptide Mr nearer to 17K than to 26K predicted from cDNA.
Amino Acid Sequence↗
Microbore flow-rates and protein chromatography.
Reversed-phase chromatography of proteins on microbore columns can achieve sensitivities that exceed those for standard-bore columns by a factor of 10-20, when operated at the same linear velocities. These gains in sensitivity are accompanied by proportional reductions in peak volume. Sensitivities on standard- (4.6 mm I.D.) and narrow-bore (2.1 mm I.D.) columns have been further improved by reducing the flow-rates to those typical for microbore (1 mm I.D.) columns. We have investigated the role of flow-rate in determining peak volumes for a constant time gradient and found that flow-rate affects peak volume to a much greater extent than column diameter. Column length was not found to have a significant effect on either peak volume or sensitivity. We have found that a four-fold reduction in flow-rate results in at least a two-fold reduction in peak volume over the flow-rate range from 25 to 400 microliters/min. Recovery of proteins in smaller volumes should prove beneficial to subsequent protein characterization methodologies.
Chromatography, High Pressure Liquid↗
Properties of Single K and Cl Channels in Asclepias tuberosa Protoplasts.
Potassium and chloride channels were characterized in Asclepias tuberosa suspension cell derived protoplasts by patch voltage-clamp. Whole-cell currents and single channels in excised patches had linear instantaneous current-voltage relations, reversing at the Nernst potentials for K(+) and Cl(-), respectively. Whole cell K(+) currents activated exponentially during step depolarizations, while voltage-dependent Cl(-) channels were activated by hyperpolarizations. Single K(+) channel conductance was 40 +/- 5 pS with a mean open time of 4.5 milliseconds at 100 millivolts. Potassium channels were blocked by Cs(+) and tetraethylammonium, but were insensitive to 4-aminopyridine. Chloride channels had a single-channel conductance of 100 +/- 17 picosiemens, mean open time of 8.8 milliseconds, and were blocked by Zn(2+) and ethacrynic acid. Whole-cell Cl(-) currents were inhibited by abscisic acid, and were unaffected by indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology and development.
Journal Article↗
Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.
Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nodulation mutant that fails to nodulate pigeonpea, elicits uninvaded nodules on siratro, and elicits normal, nitrogen-fixing nodules on groundnut. In addition, nine mutants defective in nitrogen fixation (Fix- phenotype) were isolated. Three fail to supply symbiotically fixed nitrogen to all three host plants. Surprisingly, nodules elicited by one of these mutants exhibit high levels of acetylene reduction activity, demonstrating the presence of the enzyme nitrogenase. Three more mutants have partially effective phenotypes (Fix +/-) in symbiosis with all three host plants. The remaining three mutants fail to supply fixed nitrogen to one of the host plants tested while remaining partially or fully effective on the other two hosts; two of these mutants are Fix- in pigeonpea and Fix +/- on groundnut and on siratro, whereas the other one is Fix- on groundnut but Fix+ on siratro and on pigeonpea. These latter mutants also retain significant nodule acetylene reduction activity, even in the ineffective symbioses. Such bacterial host-specific fixation (Hsf) mutants have not previously been reported.
DNA Transposable Elements↗
Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition.
We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro.
Amino Acid Sequence↗