Biomedical subjects

J Moss

Publications and source records attributed to J Moss.

At least 19 recordsLinked to original sources

PubMed1999

Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation.

Mono-ADP-ribosylation, a post-translational modification in which the ADP-ribose moiety of NAD is transferred to an acceptor protein, is catalyzed by a family of amino acid-specific ADP-ribosyltransferases. ADP-ribosyltransferase 5 (ART5), a murine transferase originally isolated from Yac-1 lymphoma cells, differed in properties from previously identified eukaryotic transferases in that it exhibited significant NAD glycohydrolase (NADase) activity. To investigate the mechanism of regulation of transferase and NADase activities, ART5 was synthesized as a FLAG fusion protein in Escherichia coli. Agmatine was used as the ADP-ribose acceptor to quantify transferase activity. ART5 was found to be primarily an NADase at 10 microM NAD, whereas at higher NAD concentrations (1 mM), after some delay, transferase activity increased, whereas NADase activity fell. This change in catalytic activity was correlated with auto-ADP-ribosylation and occurred in a time- and NAD concentration-dependent manner. Based on the change in mobility of auto-ADP-ribosylated ART5 by SDS-polyacrylamide gel electrophoresis, the modification appeared to be stoichiometric and resulted in the addition of at least two ADP-ribose moieties. Auto-ADP-ribosylated ART5 isolated after incubation with NAD was primarily a transferase. These findings suggest that auto-ADP-ribosylation of ART5 was stoichiometric, resulted in at least two modifications and converted ART5 from an NADase to a transferase, and could be one mechanism for regulating enzyme activity.

ADP Ribose Transferases↗

PubMed1999

Induction of human beta-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island.

Helicobacter pylori is an etiological agent of gastritis, peptic ulcer, and gastric cancer. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which belongs to one of the most important host defense systems against bacterial infection in several epithelial tissues. We studied the effect of H. pylori on the expression of hBD-2 mRNA in MKN45 gastric mucosal cells. H. pylori, but not culture filtrate, increased the hBD-2 mRNA level in MKN45 cells; the inductive effect of H. pylori was not detected with Intestine 407 cells. Among H. pylori strains, strain OHPC0002, which lacks a cag Pathogenicity Island (PAI), did not induce hBD-2 mRNA in MKN45 cells. These results suggested that H. pylori cag PAI is critical for the induction of hBD-2 mRNA in MKN45 cells. Exposure of MKN45 cells to Salmonella typhimurium, S. enteritidis, S. typhi, and S. dublin, but not Escherichia coli ML35, also resulted in induction of hBD-2 mRNA.

Blood Bactericidal Activity↗

PubMed1999

Thiols mediate superoxide-dependent NADH modification of glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is covalently modified by NAD in the presence of nitric oxide (NO) and dithiothreitol. Replacement of NAD with NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased modification 25-fold. We now demonstrate that in contrast to NO-mediated attachment of NAD, covalent attachment of NADH to GAPDH proceeds in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species; inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that linkage to GAPDH occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at Cys-149, the active-site thiol. This thiol linkage was stable to HgCl2. Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO, and is mediated by superoxide.

Animals↗

PubMed1999

Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells.

Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.

ADP Ribose Transferases↗

PubMed1999

Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors.

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol. Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP. p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made. Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity. The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically. Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6. Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at </=50 microM. The inactive BFA analogue B36 did not inhibit the Sec7 domain or p200. Thus, the Sec7 domain of p200, like that of Sec7 itself (Sata, M., Donaldson, J. G., Moss, J., and Vaughan, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4204-4208), plays a role in BFA inhibition as well as in GEP activity, although the latter is markedly modified by other structural elements.

ADP-Ribosylation Factor 1↗

PubMed1999

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.

Adenosine Diphosphate↗

PubMed1999

Identification of critical, conserved vicinal aspartate residues in mammalian and bacterial ADP-ribosylarginine hydrolases.

NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyze opposing arms of a putative ADP-ribosylation cycle. ADP-ribosylarginine hydrolases from mammalian tissues and Rhodospirillum rubrum exhibit three regions of similarity in deduced amino acid sequence. We postulated that amino acids in these consensus regions could be critical for hydrolase function. To test this hypothesis, hydrolase, cloned from rat brain, was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by glutathione-Sepharose affinity chromatography. Conserved amino acids in each of these regions were altered by site-directed mutagenesis. Replacement of Asp-60 or Asp-61 with Ala, Gln, or Asn, but not Glu, significantly reduced enzyme activity. The double Asp-60 --> Glu/Asp-61 --> Glu mutant was inactive, as were Asp-60 --> Gln/Asp-61 --> Gln or Asp-60 --> Asn/Asp-61 --> Asn. The catalytically inactive single and double mutants appeared to retain conformation, since they bound ADP-ribose, a substrate analogue and an inhibitor of enzyme activity, with affinity similar to that of the wild-type hydrolase and with the expected stoichiometry of one. Replacing His-65, Arg-139, Asp-285, which are also located in the conserved regions, with alanine did not change specific activity. These data clearly show that the conserved vicinal aspartates 60 and 61 in rat ADP-ribosylarginine hydrolase are critical for catalytic activity, but not for high affinity binding of the substrate analogue, ADP-ribose.

Adenosine Diphosphate Ribose↗

PubMed1999

Cutting edge: C1q protects against the development of glomerulonephritis independently of C3 activation.

C1q-deficient (C1qa-/-) mice develop antinuclear Abs and glomerulonephritis (GN) characterized by multiple apoptotic bodies. To explore the contribution of C3 activation to the induction of spontaneous GN, C1qa-/- mice were crossed with factor B- and C2-deficient (H2-Bf/C2-/-) mice. GN was present in 64% of the 45 C1qa/H2-Bf/C2-/- mice compared with 8% of the 65 H2-Bf/C2-/- mice and none of the 24 wild-type controls. IgG was detected in the glomeruli of diseased C1qa/H2-Bf/C2-/- kidneys. However, glomerular staining for C3 was absent. Increased numbers of glomerular apoptotic bodies were detected in undiseased C1qa/H2-Bf/C2-/- kidneys. These findings support the hypothesis that C1q may play a role in the clearance of apoptotic cells without the necessity for C3 activation and demonstrate that the activation of C3 is not essential for the development of GN in this spontaneous model of lupus-like disease.

Animals↗

PubMed1999

Purification and cloning of a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors.

Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: approximately 200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and approximately 50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a approximately 209-kDa protein 99.5% identical in deduced amino acid sequence (1, 849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is approximately 74% identical (1, 785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a approximately 202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5'-3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for approximately 50-kDa BFA-insensitive ARF GEPs are known, these new clones for the approximately 200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA-sensitive ARF function in Golgi transport.

ADP-Ribosylation Factor 1↗

PubMed1999

Structural elements of ADP-ribosylation factor 1 required for functional interaction with cytohesin-1.

ADP-ribosylation factor 1 (ARF1) is a 20-kDa guanine nucleotide-binding protein involved in vesicular trafficking. Conversion of inactive ARF-GDP to active ARF-GTP is catalyzed by guanine nucleotide exchange proteins such as cytohesin-1. Cytohesin-1 and its Sec7 domain (C-1Sec7) exhibit guanine nucleotide exchange protein activity with ARF1 but not ARF-like protein 1 (ARL1), which is 57% identical in amino acid sequence. With chimeric proteins composed of ARF1 (F) and ARL1 (L) sequences we identified three structural elements responsible for this specificity. Cytohesin-1 increased [35S]guanosine 5'-(gamma-thio)triphosphate binding to L28/F (first 28 residues of L, remainder F) and to a much lesser extent F139/L, and mut13F139/L (F139/L with random sequence in the first 13 positions) but not Delta13ARF1 that lacks the first 13 amino acids; therefore, a nonspecific ARF N terminus was required for cytohesin-1 action. The N terminus was not, however, required for that of C-1Sec7. Both C-1Sec7 and cytohesin-1 effectively released guanosine 5'-(gamma-thio)triphosphate from ARF1, but only C-1Sec7 displaced the nonhydrolyzable GTP analog bound to mut13F139/L, again indicating that structure in addition to the Sec7 domain is involved in cytohesin-1 interaction. Some element(s) of the C-terminal region is also involved, because replacement of the last 42 amino acids with ARL sequence in F139L decreased markedly the interaction with cytohesin-1. Participation of both termini is consistent with the crystallographic structure of ARF in which the two terminal alpha-helices are in close proximity. ARF1 residues 28-50 are also important in the interaction with cytohesin-1; replacement of Lys-38 with Gln, the corresponding residue in ARL1, abolished the ability to serve as substrate for cytohesin-1 or C-1Sec7. These studies have defined multiple structural elements in ARF1, including switch 1 and the N and C termini, that participate in functional interactions with cytohesin-1 (or its catalytic domain C-1Sec7), which were not apparent from crystallographic analysis.

ADP-Ribosylation Factor 1↗

PubMed1999

Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member.

A phospholipase D (PLD) superfamily was recently identified that contains proteins of highly diverse functions with the conserved motif HXKX4DX6G(G/S). The superfamily includes a bacterial nuclease, human and plant PLD enzymes, cardiolipin synthases, phosphatidylserine synthases, and the murine toxin from Yersinia pestis (Ymt). Ymt is particularly effective as a prototype for family members containing two conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to have PLD-like activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis(para-nitrophenol) phosphate as a substrate. Using these substrates, the amino acids essential for Ymt function were determined. Specifically, substitution of histidine or lysine in the conserved motifs reduced the turnover rate of bis(para-nitrophenol) phosphate by a factor of 10(4) and phospholipid turnover to an undetectable level. The role of the conserved residues in catalysis was further defined by the isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the catalytic reaction. Based on these data, a unifying two-step catalytic mechanism is proposed for this diverse family of enzymes.

Animals↗

PubMed1999

Structural basis for the inhibitory effect of brefeldin A on guanine nucleotide-exchange proteins for ADP-ribosylation factors.

Protein secretion through the endoplasmic reticulum and Golgi vesicular trafficking system is initiated by the binding of ADP-ribosylation factors (ARFs) to donor membranes, leading to recruitment of coatomer, bud formation, and eventual vesicle release. ARFs are approximately 20-kDa GTPases that are active with bound GTP and inactive with GDP bound. Conversion of ARF-GDP to ARF-GTP is regulated by guanine nucleotide-exchange proteins. All known ARF guanine nucleotide-exchange proteins contain a Sec7 domain of approximately 200 amino acids that includes the active site and fall into two classes that differ in molecular size and susceptibility to inhibition by the fungal metabolite brefeldin A (BFA). To determine the structural basis of BFA sensitivity, chimeric molecules were constructed by using sequences from the Sec7 domains of BFA-sensitive yeast Sec7 protein (ySec7d) and the insensitive human cytohesin-1 (C-1Sec7). Based on BFA inhibition of the activities of these molecules with recombinant yeast ARF2 as substrate, the Asp965-Met975 sequence in ySec7d was shown to be responsible for BFA sensitivity. A C-1Sec7 mutant in which Ser199, Asn204, and Pro209 were replaced with the corresponding ySec7d amino acids, Asp965, Gln970, and Met975, exhibited BFA sensitivity similar to that of recombinant ySec7d (rySec7d). Single replacement in C-1Sec7 of Ser199 or Pro209 resulted in partial inhibition by BFA, whereas replacement of Gln970 in ySec7d with Asn (as found in C-1Sec7) had no effect. As predicted, the double C-1Sec7 mutant with S199D and P209M was BFA-sensitive, demonstrating that Asp965 and Met975 in ySec7d are major molecular determinants of BFA sensitivity.

ADP-Ribosylation Factors↗

PubMed1999

Structural basis for the differential toxicity of cholera toxin and Escherichia coli heat-labile enterotoxin. Construction of hybrid toxins identifies the A2-domain as the determinant of differential toxicity.

Cholera toxin (Ctx) and E. coli heat-labile enterotoxin (Etx) are structurally and functionally similar AB5 toxins with over 80% sequence identity. When their action in polarized human epithelial (T84) cells was monitored by measuring toxin-induced Cl- ion secretion, Ctx was found to be the more potent of the two toxins. Here, we examine the structural basis for this difference in toxicity by engineering a set of mutant and hybrid toxins and testing their activity in T84 cells. This revealed that the differential toxicity of Ctx and Etx was (i) not due to differences in the A-subunit's C-terminal KDEL targeting motif (which is RDEL in Etx), as a KDEL to RDEL substitution had no effect on cholera toxin activity; (ii) not attributable to the enzymatically active A1-fragment, as hybrid toxins in which the A1-fragment in Ctx was substituted for that of Etx (and vice versa) did not alter relative toxicity; and (iii) not due to the B-subunit, as the replacement of the B-subunit in Ctx for that of Etx caused no alteration in toxicity, thus excluding the possibility that the broader receptor specificity of EtxB is responsible for reduced activity. Remarkably, the difference in toxicity could be mapped to a 10-amino acid segment of the A2-fragment that penetrates the central pore of the B-subunit pentamer. A comparison of the in vitro stability of two hybrid toxins, differing only in this 10-amino acid segment, revealed that the Ctx A2-segment conferred a greater stability to the interaction between the A- and B-subunits than the corresponding segment from Etx A2. This suggests that the reason for the relative potency of Ctx compared with Etx stems from the increased ability of the A2-fragment of Ctx to maintain holotoxin stability during uptake and transport into intestinal epithelia.

Amino Acid Sequence↗

PubMed1999

Identification and characterization of the Aeromonas sobria hemolysin glycoprotein receptor on intestine 407 cells.

Aeromonas sobria hemolysin is important in the pathogenesis of diarrhoea caused by this enteropathogenic bacterium. By immunoprecipitation analysis using hemolysin and anti-hemolysin antibody, a 66 kDa protein (p66) was identified as a receptor for A. sobria hemolysin on Intestine 407 cells. Treatment of p66 with N-glycosidase F reduced the apparent sized of p66 to 60 kDa on SDS-polyacrylamide gels. p66, released from Intestine 407 cells following incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, bound A. sobria hemolysin. Thus treatment of Intestine 407 cells with PI-PLC resulted in the remarkable decrease of the sensitivity to A. sobria hemolysin. These results are consistent with the hypothesis that p66, the binding protein for A. sobria hemolysin, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the surface of Intestine 407 cells and probably plays a role as a receptor for A. sobria hemolysin on the intestinal cells.

Aeromonas↗

PubMed1999

Effects of intravenous methylnaltrexone on opioid-induced gut motility and transit time changes in subjects receiving chronic methadone therapy: a pilot study.

In this preliminary study, we evaluated the effects of methylnaltrexone, a peripheral opioid-receptor antagonist, on chronic opioid-induced gut motility and transit changes in four subjects with chronic methadone-induced constipation. Subjects participated in this single blind, placebo controlled study for up to 8 days. We gave placebo the first day; for the remainder of the study, we gave intravenous methylnaltrexone (0.05-0.45 mg/kg) twice daily. During the study period, we recorded oral-cecal transit time and opioid withdrawal symptoms, as well as laxation response based on the frequency and consistency of the stools. Subjects 1 and 2 who were administered methylnaltrexone 0.45 mg/kg, a dose previously administered in normal volunteers, showed immediate positive laxation. Subject 2, after positive laxation response, had severe abdominal cramping, but showed no opioid systemic signs of withdrawal. The subject was discontinued due to the cramping. In Subjects 3 and 4, we reduced the methylnaltrexone dose to 0.05-0.15 mg/kg. The latter two subjects also had an immediate laxation response during and after intravenous medication without significant side effects. The stool frequency of these four subjects increased from 1-2 times per week before the study to approximately 1.5 stool per day during the treatment period. Oral-cecal transit times of Subjects 1, 3, and 4 were reduced from 150, 150 and 150 min (after placebo) to 90, 60 and 60 min (with methylnaltrexone), respectively. Our preliminary results demonstrate that low dose intravenous methylnaltrexone effectively reversed chronic methadone-induced constipation and delay in gut transit time. Thus, we anticipate that cancer patients receiving chronic opioids may also have increased sensitivity to methylnaltrexone, and that low dose methylnaltrexone may have clinical utility in managing opioid-induced constipation in chronic-pain patients.

Adult↗

PubMed1999

Antihistamine prophylaxis permits rapid vancomycin infusion.

OBJECTIVE: To determine whether pretreatment with intravenous antihistamines attenuates the symptoms of red-man syndrome associated with rapid vancomycin administration. DESIGN: Prospective, randomized, double-blinded, placebo-controlled study of patients undergoing elective arthroplasty. SETTING: Preoperative unit in a tertiary care center. PATIENTS: Forty preoperative patients (American Society of Anesthesiologists status I-III, receiving vancomycin prophylaxis for elective prosthetic joint replacement or revision. INTERVENTIONS: Elective orthopedic patients were randomly allocated to receive intravenous antihistamines (diphenhydramine, 1 mg/kg, and cimetidine, 4 mg/kg) or placebo before rapid vancomycin infusion (1 g over 10 mins). Hemodynamic measurements, symptoms of histamine release, and plasma histamine levels were obtained in each patient during vancomycin administration. Rapid vancomycin infusion was discontinued in cases of decreases in mean blood pressure of > or =20% or intolerable itching. MEASUREMENTS AND MAIN RESULTS: Clinical symptomatology of red-man syndrome and histamine levels were assessed using Fisher's exact test or Student's t-test. Comparison of baseline and peak histamine levels for both the treated (mean +/- SD, 0.2 +/- 0.2 vs. 4.7 +/- 2.4 ng/mL; p < .0001) and placebo patients (mean +/-SD, 0.2 +/- 0.1 vs. 3.5 +/- 3.4 ng/mL; p = .0002) was statistically significant. Although there was a significant increase in plasma histamine levels during vancomycin infusion, it did not differ between the treatment groups. Only two (11%) of the treated patients developed hypotension, vs. 12 (63%) of the placebo patients (p = .002). Rash was partially attenuated. Twelve (63%) of the treated patients developed rash, compared with 19 (100%) of the placebo patients (p = .008). The rapid infusion was discontinued in two (11%) of the treated patients, compared with 11 (58%) of the placebo patients (p = .005). Four treated patients had no symptoms of histamine release. CONCLUSIONS: Pretreatment with intravenous H1 and H2 antihistamines permitted rapid vancomycin administration in 89% of treated patients. Although protection was incomplete, rash did not predict a need to stop the rapid infusion of vancomycin in our patients.

Adult↗

PubMed1999

Successful in vitro cultivation of Borrelia duttonii and its comparison with Borrelia recurrentis.

Borrelia duttonii, the cause of East African tick-borne relapsing fever, has until now been refractory to growth in laboratory media. This spirochaete has only be propagated in mice or by tissue culture, restricting both yield and purity of cells available for research. The successful isolation of five clinical isolates of B. duttonii from patients in Central Tanzania and their comparison with Borrelia recurrentis is reported. Electron microscopy revealed spirochaetal cells with pointed ends, a mean wavelength of 1.8 microns with an amplitude of 0.8 micron, similar to the findings for B. recurrentis. Cells contained 10 periplasmic flagella inserted at each end of the spirochaete, again comparable with the counts of 8-10 flagella found in B. recurrentis. PFGE revealed a chromosome of approximately 1 Mb, a large plasmid of approximately 200 kb, and a small plasmid of 11 kb in all strains of B. duttonii and in B. recurrentis. B. duttonii possessed a further 7-9 plasmids with sizes ranging from 20 to 90 kb. In two isolates of B. duttonii, the profiles were identical. In contrast, all 18 isolates of B. recurrentis fell into one of five plasmid patterns with 3-4 plasmids ranging from 25 to 61.5 kb in addition to those of 11 and 200 kb described above. Analysis of the SDS-PAGE profiles of B. duttonii strains revealed a high-molecular-mass band of 33.4-34.2 kDa in four strains (variable large protein, VLP) and a low-molecular-mass band of 22.3 kDa in the remaining strain (variable small protein, VSP). This resembles the protein profiles found in B. recurrentis. The G + C ratio of B. duttonii was 27.6 mol%. Nucleotide sequence of the rrs gene (16S rRNA) from four B. duttonii isolates revealed 100% identity among these strains and 99.7% homology with three strains deposited by others in GenBank. The rrs gene of eight representative clinical isolates of B. recurrentis confirmed their close similarity with B. duttonii.

Bacteriological Techniques↗

PubMed1999

Oral health of children in South Australia by socio-demographic characteristics and choice of provider.

UNLABELLED: Australia has a highly developed school dental program for providing dental care to schoolchildren which operates alongside well-established private practice delivery of dental care. While free school-based dental care has high utilisation where offered, little is known about the oral health outcomes of groups of children across different providers of dental care, or patterns of service use. This paper investigates the effects of socio-demographic characteristics and choice of dental provider on oral health amongst a random sample of secondary school students residing in Adelaide, South Australia. OBJECTIVES: To describe the oral health of children by different dental provider groups, to describe the association of socio-demographic characteristics with oral health outcomes; and to describe the differences in oral health that remained after controlling for socio-demographic confounding, by dental provider group. METHODS: The study included metropolitan Adelaide schoolchildren from Year 9 and 10 school grades (n = 373). RESULTS: Children who received care from the School Dental Service (SDS) and from both the SDS and private dentists (Mixed) had significantly lower caries experience (DMFS) than children who received care from private dentists (Private) or who had not received any care for at least 2 years (No Recent Care). There were differences in the socio-demographic characteristics between children who used different dental providers, and these socio-demographic characteristics were significantly associated with levels of decayed, missing and filled surfaces and surfaces which had been fissure sealed. After controlling for socio-demographic characteristics using multiple regression, significant differences were seen in mean caries experience (DMFS) and mean numbers of decayed surfaces and filled surfaces. CONCLUSION: Although from less advantaged backgrounds, children seen through the SDS had more favourable oral health outcomes than children seen by a private dentist.

Adolescent↗