Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.     

Evaluation of biologic gastrin activity of compound CI-988 in the isolated, vascularly perfused rat stomach

BACKGROUND: The peptoid CI-988 has previously been shown to have high affinity for the cholecystokinin (CCK)-B/gastrin receptor and has been reported to be a powerful CCK antagonist in many systems, although it has agonist activity on histidine decarboxylase in the rat. METHODS: In the present study the effect of CI-988 on acid secretion and histamine release in the totally isolated, vascularly perfused rat stomach was assessed. RESULTS: CI-988 was found to be a gastrin agonist with regard to the stimulation of both histamine release and acid secretion. CONCLUSION: Thus, in this stomach model CI-988 behaved as a CCKB/gastrin agonist. The present study underlines the importance of testing the biologic activity of ligands in models with sufficient sensitivity.     

Direct myocardial effects of OPC-18790 in human heart failure: beneficial effects on contractile and diastolic function demonstrated by intracoronary infusion with pressure-volume analysis

OBJECTIVES: We sought to determine the precise myocardial effects of OPC-18790 as demonstrated by intracoronary administration. BACKGROUND: Although previous studies have determined the cardiovascular effects of a novel intravenous inotrope, OPC-18790, the observed benefits on contractile and diastolic function may have been confounded by the marked changes in peripheral loading associated with this drug when given intravenously. METHODS: Eight heart failure patients received intracoronary OPC-18790 at 31.25 microg/min for 20 min, and then at 62.5 microg/min for another 20 min. Hemodynamic variables and pressure-volume indexes using the conductance catheter method were determined at baseline and then after the two doses. RESULTS: There were no significant effects on heart rate, cardiac output or loading conditions, including afterload as determined by systemic vascular resistance and arterial elastance (Ea) and preload as determined by end-diastolic volume (EDV). There were significant increases in end-systolic elastance (Ees) from 0.74+/-0.11 to 0.90+/-0.16 mm Hg/ml at 31.25 microg/min and to 137+/-0.33 mm Hg/ml at 62.5 microg/min (p < 0.05 by analysis of variance [ANOVA]). Diastolic function improved, as determined by the time constant for isovolumetric relaxation tau, which decreased significantly from baseline to 31.25 microg/min (94+/-9 to 79+/-9 ms, p < 0.05), and did not shorten further at 62.5 microg/min (78+/-8 ms, p=NS). There were significant decreases in right atrial pressure (9+/-1 to 7+/-1 mm Hg, p < 0.01 by ANOVA) and mean pulmonary artery wedge pressure (21+/-3 to 16+/-2 mm Hg, p < 0.05 by ANOVA). This fall in filling pressures was not accompanied by any change in EDV. Inspection of the diastolic portion of the pressure-volume curve confirmed a downward shift consistent with pericardial release in five of the eight patients. CONCLUSIONS: Intracoronary administration of OPC-18790 demonstrates that the direct myocardial effects of this agent include a modest increase in inotropy and improvement in diastolic function, both of which occur without increases in heart rate, indicating that this agent may be beneficial for the intravenous treatment of congestive heart failure.     

Porcine spleen deoxyribonuclease II. Covalent structure, cDNA sequence, molecular cloning, and gene expression

Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked alpha.beta heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708-10713). The alpha subunit, after disulfide cleavage, yields two chains, alpha1 and alpha2. The complete amino acid sequences of the alpha1, beta, and alpha2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between alpha1 and alpha2 and of three intrachain disulfides in alpha2 were assigned. Six carbohydrate attachment sites, two in beta and four in alpha2, were detected by sugar analyses. The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined. The amplified fragments shown to be a cDNA sequence of 1,292 bases. This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH2-end, two small connecting peptides in the middle, and a peptide at the COOH terminus. These are evidently removed to form mature DNase II. Thus, all three chains in the sequence alpha1, beta, and alpha2 are coded by the same cDNA. When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.     

Knowledge of New English vocabulary in amnesia: an examination of premorbidly acquired semantic memory

To assess if amnesics have intact remote memory for general semantic information, we examined memory for vocabulary words with known dates of entry into the language between 1955 and 1989. Amnesics of mixed etiology with acute onset performed normally on both a recall and a recognition task. Korsakoff patients, in contrast, were impaired on both tasks and demonstrated a gradient such that their knowledge of words acquired during more recent time periods was worse than that of words acquired during more remote time periods. The improvement in performance associated with recognition testing was larger for Korsakoff patients than for control subjects and correlated significantly with a composite measure of frontal dysfunction. These findings suggest a deficit in the controlled search and retrieval of semantic information in Korsakoff patients. The implications of the differential performance of Korsakoff and mixed etiology amnesics for explanations of temporally graded retrograde amnesia are discussed.     

Analytical performance of the Tandem-R free PSA immunoassay measuring free prostate-specific antigen

The analytical performance of the Tandem-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA-alpha 1-antichymotrypsin was determined to be < 1%. The minimum-detectable concentration was < 0.05 microgram/L. The within-run and between-day CVs were < or = 5% for samples with > 0.3 microgram/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.     

Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embedded sections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes.     

Mu opioid agonists potentiate amphetamine- and cocaine-induced rotational behavior in the rat

Opioids modulate brain dopaminergic function in various experimental paradigms. This study used the rotational model of behavior in rats with unilateral 6-hydroxydopamine-induced lesions of the nigrostriatal pathway to investigate this interaction. Doses of two presynaptically acting dopaminergic drugs, amphetamine and cocaine, were coadministered with several doses of the mu opioid agonist, morphine. Morphine, at 3.0 mg/kg, potentiated rotational behavior induced by each dose of the stimulants. To determine the receptor specificity of the actions of morphine, the mu opioid agonists buprenorphine, fentanyl, levorphanol, meperidine, and methadone, and dextrorphan, the non-opioid isomer of levorphanol, were administered alone and with 1.0 mg/kg amphetamine. Each of these drugs, as well as morphine, produced circling behavior on its own. All of the mu opioid agonists and dextrorphan increased amphetamine-induced turning; the coadministration of dextrorphan, levorphanol, meperidine, methadone and morphine with amphetamine produced turning greater than predicted by simple additivity. To determine whether an opioid receptor was involved in these interactions, the opioid antagonist, naltrexone, was administered before the amphetamine/mu opioid receptor agonist combination. Naltrexone blocked the potentiating effects of morphine, but not those of the other drugs. Moreover, naltrexone alone dose-dependently increased amphetamine-induced rotational behavior. These studies show that some mu opioid receptor agonists can potentiate stimulant-induced rotational behavior and that blockade of opioid receptors can also produce a potentiation. The role of mu opioid receptors in these effects remains unclear.