Effects of purinergic stimulation, CFTR and osmotic stress on amiloride-sensitive Na+ transport in epithelia and Xenopus oocytes

Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl-secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl(-)dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in chocytes even after exposure to hypertonic or bypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.

Neuroprotectant effects of iso-osmolar D-mannitol to prevent Pacific ciguatoxin-1 induced alterations in neuronal excitability: A comparison with other osmotic agents and free radical scavengers

The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 MM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-clamp recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 MM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 MM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Na-v channels, as well as the increased rate of recovery from inactivation of TTX-resistant Nav channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant I-Na amplitude by P-CTX-1. Additional experiments using hyper- and isoosmolar D-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox (R) and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na-v channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Nav channel subtype, possibly to alter or reduce toxin association. (c) 2005 Elsevier Ltd. All rights reserved.

Sensitive and specific detection of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease of sugarcane, with a polymerase chain reaction-based assay

A sensitive, specific polymerase chain reaction-based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli. This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis, nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.

Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay

Mycophenolic acid is an immunosuppressant administered as a bioavailable ester, mycophenolate mofetil. The pharmacokinetics of mycophenolic acid have been reported to be variable. Accurate measurement of concentrations of this drug could be important to adjust doses. The aim of this study was to compare the enzyme-multiplied immunoassay technique (EMIT [Dade Behring; San Jose, CA, U.S.A.]) for mycophenolic acid with a high-performance liquid chromatographic (HPLC) assay using samples collected from renal transplant recipients. The HPLC assay used solid phase extraction and a C18 stationary phase with ultraviolet (UV) detection (254 nm). The immunoassay required no manual sample preparation. Plasma samples (n = 102) from seven patients, collected at various times after a dose, were analyzed using both methods. Both assays fulfilled quality-control criteria. Higher concentrations were consistently measured in patient samples when using EMIT. The mean (+/- standard deviation [SD]) bias (EMIT-HPLC) was 1.88 +/- 0.86 mg/L. The differences in concentrations were higher in the middle of a dosage interval, suggesting that a metabolite might have been responsible for overestimation. Measurement of glucuronide concentrations by HPLC demonstrated only a weak correlation between assay differences and glucuronide concentrations. If the crossreacting substance is active, EMIT could provide a superior measure of immunosuppression; if inactive, further work is needed to improve antibody specificity. In conclusion, it was found that EMIT overestimates the concentration of mycophenolic acid in plasma samples from renal transplant recipients compared with HPLC analysis.

Osmotic properties of the sealed tubular system of toad and rat skeletal muscle

A method was developed that allows conversion of changes in maximum Ca2+-dependent fluorescence of a fixed amount of fluo-3 into volume changes of the fluo-3-containing solution. This method was then applied to investigate by confocal microscopy the osmotic properties of the sealed tubular (t-) system of toad and rat mechanically skinned fibers in which a certain amount Of fluo-3 was trapped. When the osmolality of the myoplasmic environment was altered by simple dilution or addition of sucrose within the range 190-638 mosmol kg(-1), the sealed t-system of toad fibers behaved almost like an ideal osmometer, changing its volume inverse proportionally to osmolality However, increasing the osmolality above 638 to 2,550 mosmol kg(-1) caused hardly any change in t-system volume. In myoplasmic solutions made hypotonic to 128 mosmol kg(-1), a loss of Ca2+ from the sealed t-system of toad fibers Occurred, presumably through either stretch-activated cationic channels or store-operated Ca2+ channels. In contrast to the behavior of the t-system in toad fibers, the volume of the sealed t-system of rat fibers changed little (by

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues, The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-mul amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C-18 (150X4.6 mm, 5 mum) column at 35 degreesC. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored. (C) 2001 Elsevier Science B.V. All rights reserved.

The effects of interleukin-10 depletion in vivo on the immune response to Porphyromonas gingivalis in a murine model

Background: The immune response to Porphyromonas gingivalis in the mouse abscess model is known to be dependent upon CD4 T-cell activation and the regulatory role of cytokines. The role of interleukin-10 (IL-10) in this mouse model was examined in vivo. Methods: One-week-old, female BALB/c mice were divided into 4 groups. Groups 1 and 2 were given intraperitoneal (ip) injections of phosphate buffered saline (PBS) weekly for 5 weeks. Group 3 was given an ip injection of rat immunoglobulin. Group 4 was injected with rat anti-IL-10 antibodies. At week 6, group 1 was sham-immunized with PBS, and groups 2, 3, and 4 were injected with P gingivalis lipopolysaccharide (Pg-LPS) weekly for 2 weeks. One week after the final immunization, delayed-type hypersensitivity (DTH) was assessed by footpad swelling to Pg-LPS. The level of serum antibodies to Pg-LPS and IFN-gamma (IFN-gamma) was determined by enzyme-linked immunosorbent assay. Dorsal abscess formation induced by the injection of viable P gingivalis was examined daily for 30 days. Results: The footpad swelling of the anti-IL-10-treated group (group 4) was significantly higher than that of groups 1 to 3. Similarly, the serum IFN-gamma level in group 4 was much higher than that of the other experimental groups. There was no significant difference in serum IgG antibodies to Pg-LPS in any of the experimental groups. However, the level of IgM antibodies in group 4 mice was significantly lower than that in groups 2 and 3. In addition, serum IgG1 was suppressed in group 4 mice, while IgG2a antibodies were raised. However, there was no difference observed between the levels of IgG2b and IgG3 antibodies in any group of mice. The lesions in sham-immunized mice (group 1) persisted for 30 days, and those in group 2 and 3 were undetected by day 18 and 20, respectively. In sharp contrast, lesions in group 4 had healed completely by day 13. Conclusions: This study has shown that IL-10 depletion in vivo in P gingivalis LPS-induced immune response in mice led to an elevated DTH response, an increase in serum IFN-gamma levels, and raised levels of IgG and IgG2a antibodies. Treatment with anti-IL-10 antibodies resulted in suppressed IgG I and IgM responses and a more rapid healing of abscesses than in non-IL-10-depleted mice. These results suggest that IL-10 depletion in Pg-LPS-induced immune response in mice may lead to a Th1-like immune response and provide strong protection against a subsequent challenge with live P gingivalis in an abscess model.

A real-time PCR assay for the detection of Neisseria gonorrhoeae by LightCycler

The detection of Neisseria gonorrhoeae by the polymerase chain reaction (PCR) is now recognized as a sensitive and specific method of diagnosing infection by the organism. In this Study 152 urine specimens were examined for N. gonorrhoeae by a real-time PCR method using the LightCycler platform and results were compared to an in-house PCR assay using an ELISA-based detection method. N. gonorrhoeae DNA was detected in 29 (19%) specimens by LightCycler PCR (LC-PCR) and in 31 (20%) specimens by the in house PCR method. The LightCycler assay proved to be specific and 94% sensitive when compared to the in house PCR method. These features combined with the rapid turn-around time for results makes the LC-PCR particularly suitable for the detection of N. gonorrhoeae in a routine clinical laboratory. (C) 2002 Elsevier Science Inc. All rights reserved.

The role of CD4(+) cells in vivo on the induction of the immune response to Porphyromonas gingivalis in mice

Background: It has previously been suggested that CD4(+) T cells play a pivotal role in regulating the immune response to periodontal pathogens. The aim of the present study therefore was to determine delayed type hypersensitivity (DTH), spleen cell proliferation, serum and splenic anti-Porphyromonas gingivalis antibody levels, and lesion sizes following challenge with viable P. gingiualis in CD4-depleted BALB/c mice immunized with P. gingiualis outer membrane proteins (OMP). Methods: Four groups of BALB/c mice were used. Groups 1 and 2 were injected intraperitoneally (ip) with saline for 3 consecutive days and then weekly throughout the experiment. Groups 3 and 4 were injected ip with rat immunoglobulin and a monoclonal rat anti-mouse CD4 antibody, respectively. Two days later, group 1 mice were injected ip with saline only, while all the other groups were immunized ip with P. gingiualis OMP weekly for 3 weeks. One week later following the last immunization of OMP, 3 separate experiments were conducted to determine: 1) the DTH response to P. gingiualis OMP by measuring footpad swelling; 2) the levels of antibodies to P. gingiualis in serum samples and spleen cell cultures using an enzyme-linked immunosorbent assay, as well as spleen cell proliferation after stimulation with OMP; and 3) the lesion sizes after a subcutaneous challenge with viable P. gingiualis cells. Results: In CD4(+) T-cell-depleted mice (group 4), the DTH response and antigen-stimulated cell proliferation were significantly suppressed when compared to groups 2 and 3. Similarly, the levels of serum and splenic IgM, IgG, and all IgG subclass antibodies to P. gingiualis OMP were depressed. Delayed healing of P. gingivalis-induced lesions was also observed in the CD4(+) T-cell-depleted group. Conclusions: This study has shown that depletion of CD4(+) T cells prior to immunization with P. gingiualis OMP led to the suppression of both the humoral and cell-mediated immune response to this microorganism and that this was associated with delayed healing. These results suggest that the induction of the immune response to P. gingiualis is a CD4(+) T-cell-dependent mechanism and that CD4(+) T cells are important in the healing process.

The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard Cr-51-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few ( greater than or equal to6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers ( greater than or equal to 25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51 Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies. (C) 2002 Elsevier Science B.V. All rights reserved.