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1.
Br J Pharmacol ; 172(16): 4146-57, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26014146

ABSTRACT

BACKGROUND AND PURPOSE: 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4 ) is an essential cofactor for nitric oxide biosynthesis. Substantial clinical evidence indicates that intravenous BH4 restores vascular function in patients. Unfortunately, oral BH4 has limited efficacy. Therefore, orally bioavailable pharmacological activators of endogenous BH4 biosynthesis hold significant therapeutic potential. GTP-cyclohydrolase 1 (GCH1), the rate limiting enzyme in BH4 synthesis, forms a protein complex with GCH1 feedback regulatory protein (GFRP). This complex is subject to allosteric feed-forward activation by L-phenylalanine (L-phe). We investigated the effects of L-phe on the biophysical interactions of GCH1 and GFRP and its potential to alter BH4 levels in vivo. EXPERIMENTAL APPROACH: Detailed characterization of GCH1-GFRP protein-protein interactions were performed using surface plasmon resonance (SPR) with or without L-phe. Effects on systemic and vascular BH4 biosynthesis in vivo were investigated following L-phe treatment (100 mg·kg(-1) , p.o.). KEY RESULTS: GCH1 and GFRP proteins interacted in the absence of known ligands or substrate but the presence of L-phe doubled maximal binding and enhanced binding affinity eightfold. Furthermore, the complex displayed very slow association and dissociation rates. In vivo, L-phe challenge induced a sustained elevation of aortic BH4 , an effect absent in GCH1(fl/fl)-Tie2Cre mice. CONCLUSIONS AND IMPLICATIONS: Biophysical data indicate that GCH1 and GFRP are constitutively bound. In vivo, data demonstrated that L-phe elevated vascular BH4 in an endothelial GCH1 dependent manner. Pharmacological agents which mimic the allosteric effects of L-phe on the GCH1-GFRP complex have the potential to elevate endothelial BH4 biosynthesis for numerous cardiovascular disorders.


Subject(s)
Biopterins/analogs & derivatives , GTP Cyclohydrolase/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Phenylalanine/pharmacology , Animals , Biopterins/blood , Biopterins/metabolism , Cell Line , GTP Cyclohydrolase/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Mice, Inbred C57BL , Mice, Transgenic , Nitric Oxide/metabolism , RNA, Messenger/metabolism , Superoxides/metabolism
2.
Drug Test Anal ; 2(6): 271-7, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20564606

ABSTRACT

Etamiphylline camsylate (Millophylline V) was administered intravenously to two horses at a dose of 2.8 mg/kg. Urine and blood samples were taken up to 32 h post administration. Unhydrolyzed plasma and urine was extracted using solid phase extraction (SPE). The identity of the parent drug and metabolites was confirmed using a linear ion trap mass spectrometer and accurate mass analysis on an orbitrap mass spectrometer. Desethyletamiphylline (molecular weight 251) was the main metabolite observed in the urine and plasma samples and resulted from the N-deethylation of etamiphylline. The second metabolite detected in urine and plasma resulted from the demethylation of etamiphylline (molecular weight 265). The third minor metabolite detected in urine was proposed to have resulted from a simultaneous N-deethylation and demethylation of etamiphylline (molecular weight 238).


Subject(s)
Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Theophylline/analogs & derivatives , Animals , Horses , Injections, Intravenous , Solid Phase Extraction , Theophylline/pharmacokinetics
3.
Rapid Commun Mass Spectrom ; 23(19): 3173-82, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19718777

ABSTRACT

Insulin-like growth factor-I (IGF-I) is a known biomarker of recombinant human growth hormone (rhGH) abuse, and is also used clinically to confirm acromegaly. The protein leucine-rich alpha-2-glycoprotein (LRG) was recently identified as a putative biomarker of rhGH administration. The combination of an ACN depletion method and a 5-min ultra-high-performance liquid chromatography/tandem mass spectrometry (uHPLC/MS/MS)-based selected reaction monitoring (SRM) assay detected both IGF-I and LRG at endogenous concentrations. Four eight-point standard addition curves of IGF-I (16-2000 ng/mL) demonstrated good linearity (r(2) = 0.9991 and coefficients of variance (CVs) <13%). Serum samples from two rhGH administrations were extracted and their uHPLC/MS/MS-derived IGF-I concentrations correlated well against immunochemistry-derived values. Combining IGF-I and LRG data improved the separation of treated and placebo states compared with IGF-I alone, further strengthening the hypothesis that LRG is a biomarker of rhGH administration. Artificial neural networks (ANNs) analysis of the LRG and IGF-I data demonstrated an improved model over that developed using IGF-I alone, with a predictive accuracy of 97%, specificity of 96% and sensitivity of 100%. Receiver operator characteristic (ROC) analysis gave an AUC value of 0.98. This study demonstrates the first large scale and high throughput uHPLC/MS/MS-based quantitation of a medium abundance protein (IGF-I) in human serum. Furthermore, the data we have presented for the quantitative analysis of IGF-I suggest that, in this case, monitoring a single SRM transition to a trypsin peptide surrogate is a valid approach to protein quantitation by LC/MS/MS.


Subject(s)
Chromatography, High Pressure Liquid/methods , Drug Monitoring/methods , Glycoproteins/chemistry , Human Growth Hormone/administration & dosage , Insulin-Like Growth Factor I/chemistry , Tandem Mass Spectrometry/methods , Adult , Glycoproteins/blood , Humans , Male , Young Adult
4.
Psychopharmacology (Berl) ; 204(3): 465-76, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19198808

ABSTRACT

RATIONALE: Gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are prodrugs for gamma-hydroxybutyrate (GHB). Like GHB, GBL and 1,4-BD are drugs of abuse, but their behavioral effects may differ from GHB under some conditions. OBJECTIVES: The first study compared the behavioral effects of GBL (32-240 mg/kg) and 1,4-BD (32-240 mg/kg) with each other and to effects previously reported for GHB (32-420 mg/kg). A second study determined GHB pharmacokinetics following intragastric administration of GHB, GBL, and 1,4-BD. METHODS: Operant responding for food, observed behavioral effects, and a fine-motor task occurred at multiple time intervals after administration of drug or vehicle. In a separate pharmacokinetics study, blood samples were collected across multiple time points after administration of GHB, GBL, and 1,4-BD. RESULTS: Like GHB, GBL, and 1,4-BD impaired performance on the fine-motor task, but the onset of motor impairment differed across drugs. GBL and 1,4-BD dose dependently decreased the number of food pellets earned, but at lower doses than previously observed for GHB. Similar to GHB, both GBL and 1,4-BD produced sedation, muscle relaxation, gastrointestinal symptoms, and tremors/jerks. Administration of GBL and 1,4-BD produced higher maximum concentrations of GHB with shorter times to maximum concentrations of GHB in plasma when compared to GHB administration. CONCLUSIONS: GBL and 1,4-BD produced behavioral effects similar to those previously reported with GHB and the time course of effects were related to blood levels of GHB. Given their higher potency and faster onset of effects, the abuse liability of GBL and 1,4-BD may be greater than GHB.


Subject(s)
4-Butyrolactone/pharmacology , 4-Butyrolactone/pharmacokinetics , Behavior, Animal/drug effects , Butylene Glycols/pharmacology , Butylene Glycols/pharmacokinetics , GABA Modulators/pharmacology , GABA Modulators/pharmacokinetics , Prodrugs/pharmacology , Prodrugs/pharmacokinetics , Sodium Oxybate/pharmacology , Sodium Oxybate/pharmacokinetics , Animals , Area Under Curve , Conditioning, Operant/drug effects , Dose-Response Relationship, Drug , Food , Male , Motor Skills/drug effects , Papio , Reward , Substance-Related Disorders/psychology
5.
Int J Biol Macromol ; 33(4-5): 175-82, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14607362

ABSTRACT

Burkholderia cepacia (formerly Pseudomonas cepacia) grows in media containing acetamide or propionamide as C and N sources. Chromosomal DNA from a hospital isolate of B. cepacia served as a template in PCRs using primers designed for the amplification of the P. aeruginosa amiE gene that encodes an aliphatic amidase. Partial sequencing of the PCR products gave a translated sequence 100% identical with the amino acid sequence of P. aeruginosa amidase. A search of Burkholderia genomes detected a putative amidase in B. cepacia J2315 with high identity to the P. aeruginosa amidase and predicted that other Burkholderia species also possessed CN_hydrolases that use the same catalytic triad (Glu-Lys-Cys) as amidase. Superimposition of theoretical three-dimensional models suggested that differences in the amino acid sequences between amidases from B. cepacia (hospital isolate) and B. cepacia J2315 do not affect their three-dimensional structure.


Subject(s)
Amidohydrolases/genetics , Aminohydrolases/genetics , Burkholderia cepacia/genetics , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Amino Acid Sequence , Aminohydrolases/metabolism , Genome, Bacterial , Humans , Molecular Sequence Data , Multigene Family , Phylogeny , Protein Conformation , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Structural Homology, Protein
7.
J Bone Joint Surg Br ; 84(4): 600-6, 2002 May.
Article in English | MEDLINE | ID: mdl-12043787

ABSTRACT

Damage to articular cartilage is a common injury, for which there is no effective treatment. Our aims were to investigate the temporal sequence of the repair of articular cartilage and to define a critical-size defect. Full-thickness defects were made in adult male New Zealand white rabbits. The diameter (1 to 4 mm) of the defects was varied in order to determine the effect that the size and depth of the defect had on its healing. The defects were made in the femoral groove of the knee with one defect per knee and eight knees per group. The tissues were fixed in formalin at days 3, 7, 14, 21, 28, 42, 84 and 126 after operation and the sections stained with Toluidine Blue. These were then examined and evaluated for several parameters including the degree of metachromasia and the amount of subchondral bone which had reformed in the defect. The defects had a characteristic pattern of healing which differed at different days and for different sizes of defect. Specifically, the defects of 1 mm first peaked in terms of metachromasia at day 21, those of 2 mm at day 28, followed by defects of 3 mm and 4 mm. The healing of the subchondral bone was slowest in defects of 1 mm.


Subject(s)
Cartilage, Articular/injuries , Cartilage, Articular/physiology , Knee Joint , Regeneration , Animals , Cartilage, Articular/pathology , Male , Mathematics , Rabbits , Time Factors
8.
Hybridoma ; 20(4): 273-9, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11604114

ABSTRACT

Amidase from Pseudomonas aeruginosa was purified by anionic exchange chromatography and used to immunise female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A selected IgM subclass MAb was purified from in vitro hybridoma cell line supernatant by a two-step anionic exchange chromatography. The MAb was specific for amidase from P. aeruginosa as determined by Western blotting and recognized the native and denatured forms of the enzyme.


Subject(s)
Amidohydrolases/immunology , Antibodies, Monoclonal/biosynthesis , Pseudomonas aeruginosa/enzymology , Amidohydrolases/isolation & purification , Amidohydrolases/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Blotting, Western , Chromatography, Ion Exchange , Female , Immunoglobulin M/immunology , Mice , Mice, Inbred BALB C
9.
Kidney Int ; 59(5): 1881-90, 2001 May.
Article in English | MEDLINE | ID: mdl-11318960

ABSTRACT

BACKGROUND: This study evaluated the effects of dietary sodium manipulation in dogs on the regulation of canine angiotensin receptors (cAT1 and cAT2) in the kidney and adrenal. METHODS: Isolated glomeruli and membranes from renal medulla and the adrenal gland were used in radioligand binding assays from two groups of dogs: dogs maintained on low-sodium diet for two weeks followed by a high-sodium diet for two weeks (H), and dogs were maintained on the reverse schedule (L). RESULTS: Analysis of the binding data showed that dietary sodium manipulation had no significant effects on cAT1 and cAT2 receptor binding affinities in glomeruli, renal medulla, and adrenal tissues. In contrast, dietary sodium loading induced a marked increase in cAT1 receptor expression in both the glomeruli and adrenal compared with receptor expression in salt-restricted animals [H/L ratio: glomeruli (1.5), renal medulla (1.1), adrenal (1.6)] that inversely correlated with the activity of the plasma renin angiotensin system. Conversely, adrenal cAT2 receptor expression was regulated in an inverse manner in the H and L animal groups [H/L ratio: 0.7]. CONCLUSIONS: This study demonstrates that renal glomerular and adrenal AT1 receptors in the dog are coordinately down-regulated by dietary sodium restriction compared with sodium loading, which is distinctly different from the reciprocal regulation observed for rat AT1 receptors in these tissues. Collectively, these data suggest that postreceptor events in dogs are determinants of the aldosterone response observed during sodium restriction. These findings have important implications for the regulation of the renin-angiotensin system in humans, and suggest that coordinate regulation of AT1 receptors in the adrenal and glomeruli represent a negative feedback mechanism that when functioning normally prevents fluctuations of arterial blood pressure and development of arterial hypertension in response to changes in dietary sodium.


Subject(s)
Adrenal Glands/drug effects , Adrenal Glands/metabolism , Kidney Glomerulus/drug effects , Kidney Glomerulus/metabolism , Receptors, Angiotensin/drug effects , Receptors, Angiotensin/metabolism , Sodium, Dietary/administration & dosage , Aldosterone/blood , Angiotensin II/blood , Animals , Blood Pressure/drug effects , Blood Pressure/physiology , Diet, Sodium-Restricted , Dogs , Feedback , Hydrocortisone/blood , Kidney Medulla/drug effects , Kidney Medulla/metabolism , Kinetics , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Renin/blood , Renin-Angiotensin System/drug effects , Renin-Angiotensin System/physiology
11.
Mol Biotechnol ; 16(1): 5-16, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11098465

ABSTRACT

A mutant strain, KLAM59, of Pseudomonas aeruginosa has been isolated that synthesizes a catalytically inactive amidase. The mutation in the amidase gene has been identified (Glu59Val) by direct sequencing of PCR-amplified mutant gene and confirmed by sequencing the cloned PCR-amplified gene. The wild-type and altered amidase genes were cloned into an expression vector and both enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide followed by gel filtration chromatography. The mutant enzyme was catalytically inactive, and it was detected in column fractions by monoclonal antibodies previously raised against the wild-type enzyme using an ELISA sandwich method. The recombinant wild-type and mutant enzymes were purified with a final recovery of enzyme in the range of 70-80%. The wild-type and mutant enzymes behaved differently on the affinity column as shown by their elution profiles. The molecular weights of the purified wild-type and mutant amidases were found to be 210,000 and 78,000 Dalton, respectively, by gel filtration chromatography. On the other hand, the mutant enzyme ran as a single protein band on SDS-PAGE and native PAGE with a M(r) of 38,000 and 78,000 Dalton, respectively. These data suggest that the substitution Glu59Val was responsible for the dimeric structure of the mutant enzyme as opposed to the hexameric form of the wild-type enzyme. Therefore, the Glu59 seems to be a critical residue in the maintenance of the native quaternary structure of amidase.


Subject(s)
Amidohydrolases/chemistry , Amidohydrolases/metabolism , Glutamic Acid , Pseudomonas aeruginosa/enzymology , Valine , Amidohydrolases/isolation & purification , Amino Acid Substitution , Catalysis , Chromatography, Affinity/methods , Chromatography, Gel/methods , Cloning, Molecular/methods , DNA Primers , Electrophoresis, Polyacrylamide Gel/methods , Escherichia coli/genetics , Gene Amplification , Kinetics , Molecular Weight , Polymerase Chain Reaction/methods , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
12.
J Chromatogr B Biomed Sci Appl ; 748(1): 77-87, 2000 Oct 01.
Article in English | MEDLINE | ID: mdl-11092588

ABSTRACT

A pentafluorophenylpropyl (PFPP) bonded silica column has been used for the high-performance liquid chromatography-electrospray ionization-mass spectrometry-mass spectrometry assay (HPLC-ESI-MS-MS) of cocaine (COC) and its metabolite, ecgonine methyl ester (EME) in human urine. COC and EME were used as model basic solutes to demonstrate that a PFPP phase yields excellent results for the assay and validation of drugs in biological fluids. The assay was linear over three orders of magnitude (1.0-1000 ng/ml) and precision and accuracy of the assay was 4 and 15%, respectively. The limit of detection (LOD) for COC and EME was 1.6 and 2.8 pg on column, respectively. In addition, only a simple 1:10 dilution of the urine was necessary for the sample preparation procedure thus saving time on a laborious extraction step. The major advantage of the PFPP phase was the enhancement of the ESI-MS signal by providing good retention and good peak shape of COC and EME with a mobile phase of 90% acetonitrile. The MS signal for COC was a factor of 12 times greater on the PFPP phase than on the C18 phase.


Subject(s)
Chromatography, High Pressure Liquid/methods , Cocaine/urine , Spectrometry, Mass, Electrospray Ionization/methods , Calibration , Cocaine/analogs & derivatives , Humans , Reproducibility of Results , Sensitivity and Specificity
13.
J Pharm Biomed Anal ; 23(2-3): 597-605, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10933554

ABSTRACT

Cyanopropyl (CN) and pentafluorophenylpropyl (PFPP) modified silica columns give good retention and good peak shape for the high performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS) analysis of several classes of basic drugs. These phases enhance the ESI-MS signal by providing good retention of basic drugs with a mobile phase containing 90% acetonitrile. With C18 columns, in order to achieve good retention of basic drugs, only a mobile phase containing less than 40% acetonitrile can be used. Higher concentrations of acetonitrile produce a larger MS signal in the ESI process; the MS signal was a factor of 9 and 12 times greater on the CN and PFPP phases when compared with the C18 phase for the analysis of codeine. The C18 phase required only 4.0-6.0% acetonitrile to obtain the same retention time for codeine. The CN and PFPP stationary phases can be used for the analysis of a range of basic drugs, including many compounds which are poorly retained on the popular C18 and C8 stationary phases. Applications of CN and PFPP columns in the HPLC/ESI/MS of basic drugs include the analysis of antimalarials, such as quinine, bronchodilators, such as salbutamol and tulobuterol, cardioactive drugs, such as procainamide and beta-blockers, tricyclic antidepressants (TCAs), such as protriptyline and trimipramine and alkaloids, such as morphine and codeine. The CN and PFPP phases are also useful for the analysis of bufuralol and its metabolite, hydroxy-bufuralol. All the above analyses were performed using the same mobile phase, 90% acetonitrile; thus the HPLC method development process was expedited. The CN and PFPP phases also gave reproducible retention times and peak shape after more than 8 h of analyses.


Subject(s)
Chromatography, High Pressure Liquid/methods , Cyanides/chemistry , Fluorine/chemistry , Mass Spectrometry/methods , Pharmaceutical Preparations/analysis , Silicon Dioxide/chemistry , Chromatography, High Pressure Liquid/instrumentation , Silica Gel
14.
J Chromatogr A ; 873(2): 257-67, 2000 Mar 24.
Article in English | MEDLINE | ID: mdl-10757303

ABSTRACT

The analysis and use of fullerenes in capillary electrophoresis (CE) was investigated. Sodium dodecyl sulfate (SDS) was used to solubilize fullerenes C60, C70, and a mixture of C60 and C70 in water. The behavior of the solutions of the C60- and C70-SDS complexes was examined by CE with on-line UV-Vis diode array detection. This study included the use of a C60-SDS complex as a new method of micellar electrokinetic chromatography (MEKC) for the separation of polycyclic aromatic hydrocarbons (PAHs) using CE with uniwavelength detection. Since SDS micelles act as a pseudostationary phase in which the PAH compounds partition with their hydrophobic interior, the addition of C60 within the micelles enhanced separation of the PAHs. The preliminary results using C60-MEKC with SDS were compared to those obtained with MEKC with SDS. The capillary electrophoretic separations were performed in 10 mM borate-phosphate buffer with 100 mM SDS at pH 9.5.


Subject(s)
Carbon/analysis , Electrophoresis, Capillary/methods , Fullerenes , Carbon/chemistry , Spectrophotometry, Ultraviolet
15.
Anal Chem ; 72(7): 1666-71, 2000 Apr 01.
Article in English | MEDLINE | ID: mdl-10763267

ABSTRACT

Cultures of the phytoplankton diatom, Pseudonitzschia multiseries, have been harvested under controlled growth conditions ranging from late logarithmic to late stationary phase (17-58 days). The amount of domoic acid (DA) present in the growth media and in the homogenized cells has been determined by HPLC. Defined samples of media, homogenized cells, whole cells, and whole cells in media have been laser excited at 251 nm for the purpose of selectively exciting intense UV resonance Raman spectra from DA in the samples. Neither media nor cell component spectra from algae seriously interfere with DA spectra. The spectral cross sections for the dominant 1652-cm-1 mode of DA have been determined for 242-, 251-, and 257-nm excitation. Maximum sensitivities are achieved with 251-nm excitation because cross sections for DA are a maximum, and interference from other algal components becomes very small. DA concentrations that have been determined with 251-nm excitation by resonance Raman methods correlate closely with values determined independently with HPLC, especially at higher DA concentrations. The UV resonance Raman analysis of DA in phytoplankton algae is shown to be very sensitive and quantitative as well as rapid and nonintrusive.


Subject(s)
Kainic Acid/analogs & derivatives , Phytoplankton/chemistry , Spectrophotometry, Ultraviolet/methods , Spectrum Analysis, Raman/methods , Kainic Acid/analysis
16.
J Chromatogr A ; 869(1-2): 159-70, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10720235

ABSTRACT

Stationary phases were investigated for HPLC coupled with electrospray ionization mass spectrometry (ESI-MS) for the analysis of basic drugs. Tricyclic antidepressants (TCAs) and beta-blockers were used as model solutes. The functional groups, pentafluorophenyl (PFP), OH, CN or CH3 were attached to the silica via a propyl chain. The effects of these stationary phases as well as C8 and C18 phases on retention and peak shape of the basic drugs were studied. The CN and PFP phases adequately retained (tR of 2 to 6 min) the basic drugs when the mobile phase was composed of 90% acetonitrile, whereas with the C4, C8 and C18 phases, less than 40% acetonitrile had to be used to provide adequate retention of the basic drugs. Because acetonitrile provides better desolvation in ESI than an aqueous solvent, it produces an increased MS signal. As an example of the HPLC-ESI-MS analysis of the beta-blocker, pindolol, on a CN phase, the use of 90% acetonitrile in the mobile phase increased the ESI-MS signal by 790% when compared to a C18 phase which could use only 5% acetonitrile in the mobile phase for retention of the solute. In addition, the CN and PFP phases provided better peak shape than the OH phase and the hydrophobic phases (C4, C8 and C18) and ion-pairing or ion-suppressing agents were not required. The retention behavior of the TCAs and beta-blockers on each of the phases is described.


Subject(s)
Adrenergic beta-Antagonists/analysis , Antidepressive Agents, Tricyclic/analysis , Chromatography, High Pressure Liquid/methods , Mass Spectrometry
17.
Anal Biochem ; 276(2): 242-50, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10603247

ABSTRACT

Sphingolipids are an important class of lipids due to their role as biologically active molecules and as intracellular second messengers. Sphingolipid metabolites are involved in a wide variety of important biological processes including signal transduction and growth regulation. Simple, quantitative analytical methods are needed to assay these complex lipids, in order to study their biological functions. The current methods used to quantify ceramides and long-chain sphingoid bases are primarily based on derivatization with uv or fluorescent tags and with radioactive-based enzymatic assays. A method was developed to separate ceramides and sphingoid bases by normal-phase high-performance liquid chromatography and detect them directly with evaporative light-scattering detection. Ceramides and the sphingoid bases phytosphingosine, dihydrosphingosine, sphingosine, and sphingosine 1-phosphate were resolved with a rapid and quantitative assay in the nanomole range. Yeast extracts grown to various time points were assayed for ceramide and sphingoid bases using a simple, isocratic HPLC system. Both ceramide and phytosphingosine, the primary sphingoid base present in yeast cell extracts, were detected in yeast cell extracts. Phytosphingosine was resolved as a sharp peak with the addition of triethylamine and formic acid modifiers to a chloroform/ethanol mobile phase. This method demonstrates the first direct assay of both ceramides and sphingoid bases.


Subject(s)
Ceramides/isolation & purification , Chromatography, High Pressure Liquid/methods , Sphingosine/isolation & purification , Ceramides/analysis , Ceramides/chemistry , Light , Saccharomyces cerevisiae/chemistry , Scattering, Radiation , Sphingosine/analogs & derivatives , Sphingosine/analysis
18.
Med J Aust ; 171(1): 18-21, 1999 Jul 05.
Article in English | MEDLINE | ID: mdl-10451666

ABSTRACT

OBJECTIVE: To measure the extent and outcome of HIV antibody testing at reception into Australian prisons. DESIGN: Cross-sectional survey at reception into prison. PARTICIPANTS AND SETTING: People received into Australian prisons from 1991 to 1997. MAIN OUTCOME MEASURES: Number of people tested for HIV infection and prevalence of diagnosed HIV infection. RESULTS: In 1991-1997, HIV antibody testing was carried out for 72% of prison entrants in Australia; the percentage tested declined significantly from 76% in 1991 to 67% in 1997 (P < 0.001). In New South Wales, the percentage of entrants tested at reception into prison dropped from almost 100% in 1991-1994 to 45% in 1997, whereas in the Northern Territory, South Australia and Western Australia the extent of testing increased significantly (P < 0.001). HIV prevalence was 0.2% among people received into Australian prisons in 1991-1997, and did not differ by sex. Most people with HIV infection (242/378; 64%) received into prison in 1991-1997 had been diagnosed at a previous entry; 136 people (36% of the total number of diagnoses) were newly diagnosed at reception into prison. CONCLUSIONS: A national monitoring system in place from 1991 indicates generally high rates of HIV antibody testing and a low prevalence of HIV infection among people entering Australian prisons. In each year, people not previously known to the prison health service to have HIV infection were received into prison, indicating continuing HIV infection in the population entering Australian prisons.


Subject(s)
HIV Infections/epidemiology , Prisons , Australia , Cross-Sectional Studies , Female , Hepatitis C/complications , Humans , Male , Substance Abuse, Intravenous/complications
19.
Biochem J ; 340 ( Pt 3): 711-4, 1999 Jun 15.
Article in English | MEDLINE | ID: mdl-10359655

ABSTRACT

Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.


Subject(s)
Amidohydrolases/chemistry , Cysteine/metabolism , Pseudomonas aeruginosa/enzymology , Amidohydrolases/genetics , Amidohydrolases/isolation & purification , Amidohydrolases/metabolism , Amino Acid Substitution , Binding Sites , Circular Dichroism , Cross Reactions , Crystallization , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/genetics , Dithionitrobenzoic Acid/metabolism , Escherichia coli/genetics , Immunodiffusion , Molecular Weight , Protein Conformation , Protein Folding , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sulfhydryl Compounds/metabolism
20.
Anal Chem ; 71(24): 5481-92, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10624156

ABSTRACT

The effects of various mobile-phase additives, solution pH, pKa, and analyte concentration on electrospray ionization mass spectra of a series of purine and pyrimidine nucleoside antiviral agents were studied in both positive and negative ion models. The use of 1% acetic acid resulted in good HPLC separation and the greatest sensitivity for [M + H]+ ions. In the negative ion mode, 50 mM ammonium hydroxide gave the greatest sensitivity for [M - H]- ions. The sensitivities as [M + H]+ ions were significantly larger than the sensitivities as [M - H]- ions for purine antiviral agents. Vidarabine monophosphate and pyrimidine antiviral agents, however, showed comparable or greater sensitivities as [M - H]- ions. The sensitivity as [M + H]+ showed no systematic variation with pH; however, the sensitivity as [M - H]- did increase with increasing pH. At constant pH, the ion intensity of the protonated species increased with increasing pKa. At higher analyte concentrations, dimer (M2H+) and trimer (M3H+) ions were observed. [M + Na]+ adducts were the dominant ions with 0.5 mM sodium salts for these compounds. The spectra of the more basic purine antiviral agents showed no [M + NH4]+ adduct ions, but [M + NH4]+ ions were the major peaks in the spectra of the less basic pyrimidine antiviral agents with ammonium salts. The ammonium adduct ion was formed preferentially when the proton affinity of the analyte was close to that of NH3. Abundant [M + OAc]- ions were observed for all of the antiviral agents except vidarabine monophosphate from solutions with added HOAc, NaOAc, and NH4OAc. The utility of mobile phases containing 1% HOAc or 50 mM NH4OH was demonstrated for chromatographic separations.


Subject(s)
Antiviral Agents/chemistry , Hydrogen-Ion Concentration , Mass Spectrometry/standards , Nucleosides/chemistry , Chromatography, High Pressure Liquid , Ions , Sensitivity and Specificity , Solutions/chemistry , Thermodynamics
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