Detection of modified amino acids in lantibiotic peptide mutacin II by chemical derivation and electrospray ionization-mass spectroscopic analysis.
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Amino Acids↗
Biomedical subjects
M Kirk
Publications and source records attributed to M Kirk.
Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry.
Over the past decade, a number of nuclear and cytoplasmic proteins have been identified that are modified by single N-acetylglucosamine residues attached to the hydroxyl side chain of serines or threonines (O-GlcNAc). O-GlcNAc is a dynamic modification and therefore may act in a regulatory capacity analogous to phosphorylation. To undertake site-directed mutagenesis studies of O-GlcNAc's function, it is necessary to identify the sites of glycosylation on various proteins. The current method of site mapping, which involves galactosyltransferase labeling, generation of glycopeptides by proteolysis, purification by several rounds of HPLC, and gas-phase and manual Edman sequencing, is very tedious and requires about 10 pmol of pure, labeled glycopeptide. In this report, synthetic glycopeptides were generated and used to demonstrate that O-GlcNAc-modified peptides can be rapidly identified in complex mixtures by HPLC-coupled electrospray mass spectrometry due to the partial loss of the O-linked glycan (204 amu) at a modest orifice potential. Furthermore, the exact site of glycosylation was directly identified in the low picomole range by collision-induced dissociation (CID) of the glycopeptide after removal of the O-GlcNAc by alkaline beta-elimination. The conversion of glycosylserine to 2-aminopropenoic acid (2-ap) by beta-elimination both decreased the mass of the glycopeptide by 222 amu and resulted in a CID fragment ion representing the loss of 69 amu (2-ap) instead of 87 amu (Ser) at the position of the glycosylserine. Finally, we tested this method on an identical synthetic, alpha-linked O-GalNAc-modified peptide. Like O-GlcNAc, the O-GalNAc moiety was selectively removed at a modest orifice potential; however, the beta-elimination conditions that efficiently removed the O-GlcNAc only liberated about 20% of the O-GalNAc. We conclude that the selectivity and the sensitivity of this method will make it a powerful tool for determining the sites of O-GlcNAc modification on proteins of low abundance such as transcription factors and oncogenes.
Acetylglucosamine↗
Urine toxicology screens in drivers suspected of driving while impaired from drugs.
OBJECTIVE: Police departments, in conjunction with the National Highway Traffic Safety Administration, have developed a standardized evaluation aimed at identifying drivers impaired by drugs other than ethanol. These evaluations are performed by specially trained police officers known as Drug Recognition Experts. METHODS: We retrospectively reviewed the evaluations of 242 drivers detained for driving while impaired in the City and County of Denver from January 1, 1988 to June 30, 1990. RESULTS: All drivers had urine toxicology screens performed, which were positive for a mean 1.2 +/- 0.9 SD (range zero to four) for drugs having the potential for causing driving impairment. The 193/242 urine screens (79.8%) testing positive showed the following drugs: cannabis 162 (66.9%), stimulants (including cocaine metabolites) 80 (33.1%), depressants (benzodiazepines and barbiturates) 24 (9.9%), narcotics 12 (5.0%), inhalants (toluene) 1 (0.4%), hallucinogens (LSD) 1 (0.4%), and other 3 (1.2%). Drug Recognition Experts, based on their initial evaluation, were able to predict correctly some or all of the drugs found on the urine screens in 178/242 (73.6%) of cases. Overall agreement between the Drug Recognition Experts opinions and urine screen results had a kappa value (p < 0.05) of 0.41. CONCLUSIONS: There was a high rate (79.8%) of positive urine toxicology screens in drivers suspected of nonethanol drug impairment. In most cases, Drug Recognition Experts were able to reliably predict the results of these screens.
Adolescent↗
Nitric oxide inhibition of lipoxygenase-dependent liposome and low-density lipoprotein oxidation: termination of radical chain propagation reactions and formation of nitrogen-containing oxidized lipid derivatives.
Lipoxygenase-induced lipid oxidation contributes to plasma lipoprotein oxidation and may be an underlying pathogenic mechanism of atherogenesis. Since inactivation of the vasorelaxant actions of nitric oxide (.NO) plays a critical role in the impaired function of atherosclerotic vessels and because .NO reacts rapidly with other radical species, we assessed the influence of .NO on lipoxygenase-catalyzed oxidation of linoleic and linolenic acid, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PC) liposomes, hypercholesterolemic rabbit beta-very-low-density lipoprotein, and human low-density lipoprotein. Soybean lipoxygenase (SLO)-induced lipid oxidation was assessed by accumulation of conjugated dienes, formation of lipid hydroperoxides, oxygen consumption, and liquid chromatography-mass spectrometry. Different rates of delivery of .NO to lipid oxidation systems were accomplished either by infusion of .NO gas equilibrated with anaerobic buffer or via .NO released from S-nitrosoglutathione. Nitric oxide alone did not induce lipid peroxidation, while exposure to SLO yielded significant oxidation of fatty acids, PC liposomes, or lipoproteins in a metal ion-independent mechanism. Low concentrations of .NO, which did not significantly inhibit the activity of the iron-containing lipoxygenase, induced potent inhibition of lipid peroxidation in a dose-dependent manner. Mass spectral analysis of oxidation products showed formation of nitrito-, nitro-, nitrosoperoxo-, and/or nitrated lipid oxidation adducts, demonstrating that .NO serves as a potent terminator of radical chain propagation reactions. The formation of Schiff's base fluorescent conjugates between SLO-oxidized linoleic or linolenic acid and bovine serum albumin (BSA) was also inhibited by .NO via reaction with lipid hydroperoxyl radicals (LOO.), thus preventing the reaction of LOO. with polypeptide amino groups. Mass spectrometry analysis showed that both lipid peroxidation products and nitrogen-containing oxidized lipid species decreased in the presence of BSA. We conclude that .NO can play a potent oxidant-protective role in the vessel wall by inhibiting lipoxygenase-dependent lipid and lipoprotein oxidation. This occurs via termination of lipid radical chain propagation reactions catalyzed by alkoxyl (LO.) and LOO. intermediates of lipid peroxidation rather than by inhibition of lipoxygenase-catalyzed initiation reactions.
Animals↗
Vgr-1/BMP-6 induces osteoblastic differentiation of pluripotential mesenchymal cells.
The transforming growth factor-beta (TGF-beta) superfamily is a group of secreted growth factors that appears to play a central role in mesenchymal differentiation, including cartilage and bone formation. The present study examines the role of one member of this family, vgr-1, also called bone morphogenetic protein-6, in mesenchymal cell differentiation. This factor may be considered as a prototype for the largest subgroup of related factors within the TGF-beta superfamily, the function of which has as yet been poorly defined. vgr-1 has been localized previously to hypertrophic cartilage and has been shown to induce endochondral bone formation in vivo. To further characterize the role of vgr-1 in bone and cartilage differentiation, we stably transfected the pluripotent mesenchymal cell line ROB-C26 with a vector to overexpress vgr-1. Overexpression of this factor did not affect cell shape or morphology, but it enhanced osteoblastic differentiation in vitro and altered cellular responsiveness to retinoic acid. Furthermore, the extracellular matrix produced by these vgr-1-overexpressing cells induced ectopic bone formation in vivo and osteoblastic differentiation in vitro, similar to the matrix produced by C26 cells treated with retinoic acid. The osteoinductive effect of the matrix from vgr-1-overexpressing cells was blocked using a neutralizing vgr-1 antibody but not with a neutralizing TGF-beta 1 antibody, indicating that vgr-1 alone was required for this osteogenic effect. In contrast, the osteoinductive effect of matrix from retinoic acid-treated cells was blocked with both vgr-1 and TGF-beta 1 antibodies, suggesting that TGF-beta 1 may act prior to vgr-1 during osteoblastic differentiation. We further demonstrated that osteoinduction by vgr-1 was dependent on presentation of vgr-1 within the matrix, because the osteoinductive effect of matrix from vgr-1-overexpressing cells could not be mimicked with the addition of soluble vgr-1 to parental C26 cells. Finally, overexpression of MyoD within the C26 cells overexpressing vgr-1 converted the cells to myoblasts, indicating that vgr-1 had induced early osteoblastic.
Alkaline Phosphatase↗
[The organization of veterinary services in African animal husbandry systems: chances for the exchange of interdisciplinary learning processes].
The structure of the veterinary services to date hinders--together with massive intervention in the land tenure systems and the animal keepers' rights in arid and semi-arid areas--the future organization of productive animal-keeping systems adapted to the ecological conditions. The consensus of the findings of research in the social as well as in the natural sciences, is that state (agrarian) policies and international development cooperation have led to centralism and authoritarian administrations which formalize formerly informal, locally specific rules for securing the health of the animals and the maintenance of grazing and water resources. As a result, the animal-keeping groups have lost their rights, social conflicts arose and the environment suffered. Thus, decentralization, subsidarity and--above all--comprehensive participation on the part of the animal keepers are regarded as the guidelines for the future organization of state and/or private services for maintaining the health of the animals. Thereby, the planning at the various administrative levels is faced by great challenges in view of the progressing differentiation in the animal-keeping systems (e.g. peri-urban systems; urban absentee herd owners; marginalized, subsistence-oriented households).
Africa↗
Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives.
Superoxide (O2-.), nitric oxide (.NO), and their reaction product peroxynitrite (ONOO-) have all been shown to independently exert toxic target molecule reactions. Because these reactive species are often generated in excess during diverse inflammatory and other pathologic circumstances, we assessed the influence of .NO on membrane lipid peroxidation induced by O2-., H2O2, and .OH derived from xanthine oxidase (XO) and by ONOO-. Experimental conditions in lipid oxidation systems were adjusted to yield different rates of delivery of .NO, relative to rates of O2-. and H2O2 generation, by infusion of either .NO or via .NO released from S-nitroso-N-acetylpenicillamine or S-nitrosoglutathione. Peroxidation of phosphatidylcholine liposomes was assessed by formation of thiobarbituric acid-reactive products and by liquid chromatography-mass spectrometry. Liposomes exposed to XO-derived reactive species in the presence of .NO exhibited both stimulation and inhibition of lipid peroxidation, depending on the ratio of the rates of reactive oxygen species production and .NO introduction into reaction systems. Nitric oxide alone did not induce lipid peroxidation. Linolenic acid emulsions peroxidized by XO-derived reactive species showed similar dose-dependent regulation of lipid peroxidation by .NO. Mass spectral analysis of oxidation products showed formation of nitrito-, nitro-, nitrosoperoxo-, and/or nitrated lipid oxidation adducts, demonstrating that .NO serves as a potent terminator of radical chain propagation reactions. Electron spin resonance (ESR) analysis of incubation mixtures provided no evidence for formation of paramagnetic iron-lipid-nitric oxide complexes in reaction systems. Peroxynitrite-dependent lipid peroxidation, which predominantly occurs by metal-independent mechanisms, was also inhibited by .NO. Peroxynitrite-mediated benzoate hydroxylation was partially inhibited by .NO, inferring reaction between .NO and ONOOH. It is concluded that .NO can both stimulate O2-./H2O2/.OH-induced lipid oxidation and mediate oxidant-protective reactions in membranes at higher rates of .NO production, with the prooxidant versus antioxidant outcome critically dependent on relative concentrations of individual reactive species. Prooxidant reactions of .NO will occur after O2-. reaction with .NO to yield potent secondary oxidants such as ONOO- and the antioxidant effects of .NO a consequence of direct reaction with alkoxyl and peroxyl radical intermediates during lipid peroxidation, thus terminating lipid radical chain propagation reactions.
Hydroxyl Radical↗
The implementation of a quality management system in a department of diagnostic radiology.
The quality management systems of the Departments of Diagnostic Radiology of St George's Hospital, London, and Bristol Royal Infirmary (BRI) have been certified with the British Standard 5750. These were the first clinical departments in the UK to be certified to this internationally recognized quality assurance (QA) standard. Quality is a much used term; in this context it is defined as 'fitness for purpose', and quality assurance is the mechanism by which quality is achieved. The paper describes the features of the quality system which have been implemented at St George's Hospital, and how a management concept for manufacturing industries has been adapted successfully to a National Health Service Department. The paper also demonstrates how the quality system has led to the setting and achieving of departmental objectives.
England↗
The effects of changes in plasma nonesterified fatty acid levels on oxidative metabolism during moderate exercise in patients with non-insulin-dependent diabetes mellitus.
Blood levels of intermediary metabolites were measured and indirect calorimetry was performed in 10 otherwise healthy, non-insulin-dependent diabetic (NIDDM) patients before, during, and after 30 minutes of moderate exercise on three occasions in random order at weekly intervals with (1) heparin treatment to increase preexercise plasma nonesterified fatty acid (NEFA) levels (HEPARIN); (2) acipimox, a nicotinic acid analogue, to reduce preexercise plasma NEFA levels (ACIPIMOX); and (3) no manipulation of preexercise plasma NEFA levels (NIL). With ACIPIMOX, preexercise blood levels were significantly reduced for NEFAs and glycerol (P < .01) and marginally reduced for acetoacetate and 3-hydroxybutyrate (NS) compared with preexercise levels for the other two treatments; these low levels seen with acipimox treatment increased only slightly during exercise and the postexercise period. Plasma NEFA levels increased by approximately 150% (P < .001) with HEPARIN at the same times. The levels of ketone bodies during either NIL or HEPARIN increased rapidly postexercise by approximately 90% to 110% for both acetoacetate and 3-hydroxybutyrate (both P < .01). Plasma insulin levels tended to be lowest (despite similar plasma glucose levels during the three treatments) with ACIPIMOX, while growth hormone (hGH) and, perhaps, noradrenaline levels were highest both during and after exercise. The respiratory quotient (RQ) was highest with ACIPIMOX (P < .05 for exercise and postexercise periods compared with the other two treatments), which, compared with NIL, reduced fat oxidation by 27% and 60% and increased carbohydrate oxidation by 29% and 74% during and after exercise, respectively (all P < .05). These changes in substrate oxidation due to ACIPIMOX were almost opposite to those observed with HEPARIN.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Hydroxybutyric Acid↗
Immediate and sustained reduction in serum cholesterol achieved in 4-week Heart Tune program.
Currently, there is limited information about educational models effective in assisting individuals to lower their serum cholesterol. Dietitians and nutritionists involved in cholesterol-reduction programs need to inform other health professionals about programs utilized and the success of those programs. The National Cholesterol Education Program has published guidelines for the detection, evaluation, and treatment of high blood cholesterol in adults. Educational programs should be systematically evaluated to confirm their success in cholesterol reduction. The Heart Tune program is a 4-week class developed by a dietitian to assist clients in cholesterol reduction. An evaluation of the lipoprotein levels of 49 program participants demonstrated that serum total cholesterol levels significantly decreased at the completion of the 4-week class (0.65 mmol/L) and after 1 year (0.80 mmol/L).
Adult↗
Bromocriptine-associated headache: possible life-threatening sympathomimetic interaction.
We present two cases of severe headache associated with the use of bromocriptine for lactation suppression in otherwise healthy women. In each case, the additional use of a therapeutic sympathomimetic agent resulted in extreme worsening of symptoms with development of hypertension and life-threatening complications (ventricular tachycardia and cardiac dysfunction in one case, seizures and cerebral vasospasm in the other). Sympathomimetics in combination with bromocriptine in patients with a bromocriptine-associated headache during the puerperium may be dangerous.
Adult↗
The validity and reliability of the navicular fat stripe as a screening test for detection of navicular fractures.
STUDY OBJECTIVE: The purpose of this study was to determine if the navicular fat stripe (NFS) is a valid and reliable screening tool in assessing potential navicular fractures. DESIGN: Retrospective analysis. SETTING: Minor trauma section of the Los Angeles County-University of Southern California Medical Center emergency department. TYPE OF PARTICIPANTS: Study population included patients seen for wrist injury between January 1987 and December 1988. The individuals who interpreted the radiographs included eight observers with varying degrees of radiographic experience. INTERVENTIONS: None MEASUREMENTS AND MAIN RESULTS: The positive predictive value averaged 12% or 15% depending on how we counted a straight NFS, and the negative predictive value averaged 93% or 95%, again depending on how we counted a straight NFS. A straight NFS was ten times as likely to occur among normal wrists than among those with navicular fractures. Interobserver reliability was found to be high with a Hoyt reliability coefficient of .79. CONCLUSIONS: An abnormal NFS is a poor predictor of a navicular fracture while both a straight and a normal NFS are good predictors of no navicular fracture. A normal NFS may allow less stringent treatment by the emergency physician. Extremes of radiographic experience were found to have no significant effect on NFS interpretation.
Adipose Tissue↗
Evaluation of four newer antimicrobial agents in the Avantage susceptibility test system.
Antimicrobial elution disks containing amoxicillin-clavulanic acid (Augmentin), cefotetan, ciprofloxacin, or norfloxacin were tested in the Avantage automated susceptibility test system. Performance was compared against an agar diffusion procedure in a three-site collaborative study. Results of 1,500 comparison with amoxicillin-clavulanic acid showed a full accord (agreement of both systems) of 93.6% and an essential accord (agreement excluding minor discrepancies) of 97.6%. Results for cefotetan showed a full accord of 95.1% and an essential accord of 98.3% by the two methods. Results for both ciprofloxacin and norfloxacin were in full accord for more than 98% of tests with gram-negative bacilli and staphylococci, but tests with enterococci gave 38 and 26.1% minor discrepancies (the result of one method was resistant or susceptible and the result of the other method was intermediate), respectively. The results indicated that the Avantage test system is accurate and reliable and provides appropriate determination of bacterial susceptibility with the four antibiotics tested.
Amoxicillin↗
Early stage ovarian cancer: a randomized clinical trial comparing whole abdominal radiotherapy, melphalan, and intraperitoneal chromic phosphate: a National Cancer Institute of Canada Clinical Trials Group report.
Two hundred fifty-seven eligible patients with stage I, IIA "high risk" ovarian carcinoma and IIB, IIIO (disease confined to pelvis), were randomized to either total abdominal radiotherapy (arm A) 2,250 rad in 20 fractions (107 patients), melphalan (arm B) 8 mg/m2/d X 4 every 4 weeks X 18 courses (106 patients), or intraperitoneal chromic phosphate (arm C) 10 to 20 mCi (44 patients). All patients were initially treated with pelvic radiotherapy; arm A, 2,250 rad in ten fractions; and arms B and C, 4,500 rad in 20 fractions. Entry to arm C was discontinued early because of toxicity. In a multifactor analysis using proportional hazards models, no significant difference in survival was observed although there was a marginally significant difference in disease-free survival (P = .015) with arm B being superior to arm A. Stage (P less than .0001), grade (P less than .0001), and histology (P less than .008) were predictors of survival in the multifactor analysis. Performance status, age, and residual disease were significant predictors in the single factor analysis but were not predictive when correction was made for the effects of stage, grade, and histology. Five-year survival rates are 62% for arm A, 61% for arm B, and 66% for arm C. Median duration of follow-up is 8 years. Long-term complications of radiotherapy were seen in 19 patients on arm A, 11 on arm B, and 11 on arm C. Four patients who had received melphalan developed either a myelodysplastic syndrome or acute leukemia. Violations in covering the whole abdominal target volume were correlated with survival.
Chromium↗
Sensorineural hearing loss and mumps.
Out of 360 children with hearing loss seen during 1 year in the Haringey Audiology Unit, 21 had unilateral, sensorineural hearing loss. Sixteen had previously been checked to have no loss. It is strongly suspected that the hearing loss resulted from mumps, and therefore the disability in such children could be prevented by introducing mumps vaccination into the immunisation programme.
Child↗
Cardiovascular clues in the diagnosis of poisoning.
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Bradycardia↗
Genetic tests for autosomal non-disjunction and chromosome loss in mice.
Two new genetic methods for detecting autosomal non-disjunction and chromosome loss in mice are described. Both methods involve the use of marker genes and Robertsonian translocations, the latter present only in tester parents, to detect events in chromosomally normal mice. With the Rb method, the tester parent carries one or more Robertsonian translocations heterozygously; with the MBH method the tester parent carries two Robertsonian translocations showing monobrachial homology. The high rates of meiotic non-disjunction in the tester mice provide gametes with specific extra or missing chromosomes which, at fertilization, can allow the survival of a proportion of the zygotes lacking or carrying an extra specific chromosome from tested chromosomally normal parents. The Rb method has been assessed for X-ray-induced chromosome 1 loss and non-disjunction in mature oocytes and also for such chromosome 1 loss from the maternal pronuclei of 1-cell zygotes. The MBH method has been assessed for X-ray-induced chromosome 1 loss in male postmeiotic cells and for non-disjunction in spermatocytes. Both methods proved effective in detecting chromosome 1 loss. A single case of the much rarer non-disjunctional event was also found. As applied, both methods compared favourably with the numerical sex chromosome anomaly (NSA) method and have considerable potential for further development.
Animals↗