1H NMR of compounds with low water solubility in the presence of erythrocytes: effects of emulsion phase separation.

When lipophilic compounds like diethyl phthalate (DEP) were added to water, two sets of resonances appeared in the 1H NMR spectrum, whereas when added in concentrations above approximately 3.5 mM to erythrocytes in a high haematocrit suspension, only one set of resonances was observed at the low-frequency position. The appearance of one set of resonances at lower frequency was found to be common to a series of lipophilic compounds in erythrocytes. The appearance of the NMR spectra is ascribed to the existence of an emulsion, meaning two different phases of a compound: a "droplet" (resonances to lower frequency) and aqueous dissolved phase (resonances to higher frequency). The absence of the resonances from the dissolved phase in erythrocyte solution is ascribed to exchange broadening. The absolute chemical shift of the compound in its "droplet" phase was also measured using a cylindrical/spherical microcell. This arrangement mimicked the geometry of the dissolved versus the phase-separated species and thus obviated the effect of a difference in magnetic susceptibility between the "droplet" solute and its aqueous solution. Factors influencing the formation of emulsion phases such as erythrocytes, haemoglobin and smaller proteins were investigated; they are found to be effective in the order given.

Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls.

Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6. 3%) (P < 0.05). Positive IS900 PCR results occurred more often in U. S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U. S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181 study subjects, both IS900 PCR and ELISA were performed. Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive. Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients. Gamma interferon (IFN-gamma) release by peripheral blood leukocytes after M. avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S. (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05). Interleukin-5 responses were not different among CD, UC, or control groups. Collectively, the PCR, ELISA, and IFN-gamma tests for M. avium subsp. paratuberculosis together with the unexpected observation that BCG vaccination influenced M. avium subsp. paratuberculosis detection, lead us to conclude that M. avium subsp. paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients. Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.

Random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing in the analysis of a hospital outbreak of Salmonella enteritidis.

Isolates of Salmonella Enteritidis from 81 patients from Herlev Hospital or from Copenhagen County were analysed by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE) and phage-typing. Fourteen polymorphic markers from five decamer primers unambiguously placed all isolates into six RAPD groups: 65 isolates of phagetype 6 (PFGE type I) were resolved into three RAPD groups constituting 86, 12, and 2%, respectively. A fourth RAPD group of 10 isolates was coincident with phage type 8 (PFGE type II) and two isolates, one phage-type 1, the other phage-type 4 (both PFGE type I) formed the fifth group. The sixth group of four isolates was not phage typeable and was PFGE type III. Forty outbreak-related isolates of phage-type 6 were resolved into three strains. No diversity of phage-type 6 was found among isolates unrelated to the outbreak. It is concluded that RAPD is useful as a tool in investigations of microbial outbreaks in its own right, or to supplement phage-typing and PFGE of Salmonella Enteritidis.

Cefuroxime resistance in Klebsiella pneumoniae. Susceptibility to cefotaxime and ceftazidime despite production of ESBLs.

The production of beta-lactamases, the outer membrane protein (OMP) patterns, some clinical impacts and the prevalence of resistance among cefuroxime-resistant Danish clinical isolates of Klebsiella pneumoniae were investigated. Fifteen resistant and five susceptible strains were collected from 14 patients during 1991-1994. Isolates from five patients produced extended-spectrum beta-lactamases (ESBLs). Cefuroxime resistance was accompanied by a 10-fold elevation of ciprofloxacin minimal inhibitory concentration (MIC), and for some isolates by an alteration of the OMP pattern. The relationship between alterations of the OMP patterns and cross-resistance to ciprofloxacin and the other antibiotics tested was not universal. Ten of the cefuroxime-resistant strains had elevated MICs of cefotaxime or ceftazidime, but the MICs were still below the breakpoint for susceptibility. The MICs of imipenem were not affected. Nosocomial infection or long-term colonization with resistant strains may be of importance since five patients were not treated with cefuroxime prior to isolation of the resistant strain, and all patients had either serious diseases or stayed at the hospital for a long period of time. The prevalence of cefuroxime and ciprofloxacin resistance among clinical isolates from Copenhagen county during 1990-1995 was 8.3% and 7.5%, respectively, but higher for urinary tract specimens. A greater consumption of cefuroxime as compared to cefotaxime and ceftazidime in this study, as seen generally in Denmark, indicated that ESBLs produced by the investigated strains of K. pneumoniae may be selected with cefuroxime.

Cefuroxime resistance in Escherichia coli. Resistance mechanisms and prevalence.

In order to characterize cefuroxime resistance in Escherichia coli 22 clinical isolates were investigated for susceptibility to different beta-lactam antibiotics and ciprofloxacin. The production of beta-lactamases, the pattern of the major outer membrane proteins (OMPs), and the plasmid profiles were determined for these isolates. Ten of the isolates were resistant to ceftazidime, two to cefotaxime, and none was resistant to imipenem or ciprofloxacin. The dominating resistance mechanism was hyperproduction of the chromosomally encoded beta-lactamase to some extent accompanied by alterations of the OMP's. Two isolates with low ampicillin MIOs seemed solely to have alteration of the OMPs. None of the isolates produced plasmid-mediated extended-spectrum beta-lactamases. In addition, the prevalence of cefuroxime resistance was investigated. The prevalence as attained in 8704 clinical isolates of E. coli collected from Copenhagen County during a 5-year period (1990-1994) was 4.4%, but there was considerable variation among specimens from different sites of the body. Isolates from blood were much less resistant (2.5%) than isolates from the respiratory tract (9.7%).

1H NMR spin-echo spectroscopy of human erythrocytes. Transformation of exogenous compounds.

1H NMR spin-echo spectroscopy has been used to study the metabolism of exogenous compounds in human erythrocytes. The non-invasive nature of the technique and the continuous monitoring makes it ideal to investigate both the conversion of substrates and the occurrence and the nature of the products. Hydrolysis rates may be determined and the hydrolysis of alkyl, vinyl, and aryl acetates are shown to be due to A-esterase activity. The conversion products are normally observed, but in the case of dimethyl phthalate and methyl acetate only part of the methanol is found. An interesting case is the hydrolysis of vinyl acetate and the consecutive conversion of the vinyl alcohol to acetaldehyde (hydrate). Coupling of glutathione to a variety of vinyl derivatives is also observed. Oxidation of acetaldehyde can also be followed. Styrene and 5-chloro-2-methylaniline are slowly converted in what is believed to be oxidation reactions. Changes in 1H chemical shifts are observed for a number of compounds upon uptake into human erythrocytes, a shift that is suggested to be due to binding to haemoglobin.

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 induced calcium transients in human red cells.

A transient increase of cellular calcium was induced by addition of the divalent cation ionophore A23187 to human red cells in the absence or presence of drugs. The peak height of the calcium transient was increased about five times at pH 6.9 and up to eighteen times at pH 7.4 by trifluoperazine (0.30 mM), and two to three times at pH 6.9 by compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). The time-dependent changes of cellular calcium were analysed by the aid of a pump-leak model based partly on the calcium dependent parameters obtained from calcium ATPase experiments, partly on the A23187 induced calcium fluxes determined in experiments with ATP depleted cells. The transient increase of cellular calcium induced within few minutes after the addition of ionophore A23187 could be explained satisfactorily by the model both in the absence and presence of the four drugs, whereas the final level of cellular calcium in the drug experiments was more difficult to predict from the pump-leak model. Comparison of experimental and model calcium transients suggested that trifluoperazine and TMB-8 affected both pump and leak, whereas compound 48/80, probably due to low membrane-permeability, mainly affected the leak and verapamil affected the pump only.

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 mediated calcium uptake in ATP depleted human red cells.

The A23187 induced calcium uptake in ATP depleted cells was determined at pH 6.9 in the presence of trifluoperazine (TFP, 0.30 mM), compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). Apart from verapamil the drugs all increased the maximum rate of ionophore-mediated calcium flux by 50-60 per cent. After the ionophore addition some time elapsed before the calcium flux attained the maximum value, and this time dependence could be interpreted as a slow uptake of A23187 into the membrane: five seconds after the addition of A23187 half of the added ionophore was able to transport calcium through the membrane. The effect of pH on the ionophore-mediated calcium uptake was determined in the absence and presence of TFP. At pH 7.4 the maximum rate of calcium flux in the absence of TFP was two to three times higher than that at pH 6.9 and TFP increased the uptake rate by 98 per cent.

Hysteretic activation of the Ca2+ pump revealed by calcium transients in human red cells.

The enzymatic basis for the Ca2+ pump in human red cells is an ATPase with hysteretic properties. The Ca2+-ATPase shifts slowly between a ground state deficient in calmodulin and an active state saturated with calmodulin, and rate constants for the reversible shifts of state were recently determined at different Ca2+ concentrations (Scharff, O. and Foder, B. (1982) Biochim. Biophys. Acta 691, 133-143). In order to study whether the Ca2+ pump in intact red cells also exhibits hysteretic properties we have analysed transient increases of intracellular calcium concentrations (Cai), induced by the divalent cation ionophore A23187. The time-dependent changes of Cai were measured by use of radioactive calcium (45Ca2+) and analysed with the aid of a mathematical model, based partly on the Ca2+-dependent parameters obtained from Ca2+-ATPase experiments, partly on the A23187-induced Ca2+ fluxes determined in experiments with intact red cells. According to the model a delay in the activation of the Ca2+ pump is a prerequisite for the occurrence of A23187-induced calcium transients in the red cells, and we conclude that the Ca2+ pump in human red cells responds hysteretically. It is suggested that Ca2+ pumps in other types of cell also have hysteretic properties.