Charge effect on the photoinactivation of Gram-negative and Gram-positive bacteria by cationic meso-substituted porphyrins

Background  

In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms.     

Comparison of PCR and clinical laboratory tests for diagnosing <Emphasis Type="Italic">H. pylori</Emphasis> infection in pediatric patients

Background

Histology and/or culture are generally considered the gold standard for the detection of H. pylori infection. Especially in children, these tests may result in a false negative outcome because of patchy distribution of the organism in the stomach mucosa. We have developed a PCR assay utilizing nested primer pairs directed against a subunit of the H. pylori urease gene (ureA). As part of a prospective evaluation of diagnostic tests to aid in detecting H. pylori infection in children, the aim of this study was to compare our PCR and Western blot assays with results obtained from histologic examination of biopsy specimens, rapid urease tests, and an FDA approved serologic assay and published PCR results to determine if we could validate the assays for diagnostic use on our patient population.

Results

Gastric biopsy specimens obtained from 101 pediatric patients were evaluated for the presence of H. pylori using histologic techniques, rapid urease (CLOtest) test and the PCR assay. Serum samples from each patient were assayed using both ELISA and Western Blot for antibodies to H. pylori. A total of 32 patients tested were positive by at least one of the methods evaluated. Thirteen patients had positive histology, 13 had a positive CLOtest, and 17 patients had positive H. pylori PCR. Out of the 13 CLO positive patients, 12 were positive by histologic analysis and all 13 were positive by PCR. Results of serologic tests on the same population did not correlate well with other assays. Twenty-eight patients showed serologic evidence of H. pylori infection, of which 9 were both CLO and histology positive and 12 were positive by PCR. Of the seropositive patients, 26 were ELISA positive, 13 were positive by Western blot, and 11 by both serologic methods.

Conclusions

The results obtained suggest that our nested PCR assay has the specificity and sensitivity necessary for clinical application when compared to standard histologic examination and rapid urease test. In addition, we found the current commercially available approved ELISA method appears unable to accurately detect H. pylori in this population. The Western blot assay yielded better concordance with CLOtest and histology, but not as good as the nested PCR assay.

     

Differentiation of pre- and postsynaptic high affinity serotonin receptor binding sites using physico-chemical parameters and modifying agents

The two³H-labeled agonists [³H]8-hydroxy-2-(di-n-propylamino) tetralin ([³H]8-OH-DPAT) and [³H]serotonin ([³H]5-HT) have been used to examine the effects of physico-chemical parameters and modulatory agents on the high affinity 5-HT receptor binding sites in various regions of the rat central nervous system. Sites labeled by [³H]8-OH-DPAT and [³H]5-HT were differentially sensitive to changes in incubation demperature and pH, such that the optimal interaction of [³H]8-OH-DPAT with specific sites in the striatum was at 30°C and pH 7.4, whereas [³H]5-HT sites in the same region were most easily labeled at 2–23°C and pH 8.2. Micromolar concentrations of Mn²⁺ enhanced [³H]5-HT binding but inhibited markedly [³H]8-OH-DPAT binding to striatal membranes. In contrast, both [³H]5-HT and [³H]8-OH-DPAT binding were incerased by the cation in hippocampal membranes. Conversely, GTP reduced the binding of either ligand in the hippocampus but affected only [³H]5-HT binding in the striatum. Furthermore, N-ethylmaleimide inhibited equally [³H]5-HT and [³H]8-OH-DPAT binding to hippocampal membranes, but was markedly less potent against [³H]8-OH-DPAT binding to striatal     

Pharmacological Evidence for the Involvement of Calciuml Calmodulin in Serotonin 5-HT3 Receptor-Mediated Cation Permeability in NG 108-15 Cells

Abstract: In NG 108–15 clonal cells, extracellular application of micromolar concentrations of serotonin [5-hydroxy-tryptamine (5-HT)] and substance P induces the opening of a cation permeability monitored by the influx of [¹⁴C]-guanidinium. The serotoninergic component of this cation permeability Is linked to 5-HT₃ receptor activation, whereas the substance P component probably involves an “N-terminal-dependent substance P receptor.” In this study, [¹⁴C]guanidinium influx triggered by 1 μM 5-HT plus 10 μM substance P was shown to be insensitive to tetrodotoxin, verapamil, diltiazem, nimodipine, and ω-conotoxin, as expected from a process independent of voltage-sensitive sodium and calcium channels. In contrast, [¹⁴C]guanidinium influx was inhibited by millimolar concentrations of extracellular calcium and by the chelation of intracellular calcium by bis-O-aminophenoxyethanetetraacetic acid. The inhibition by extracellular calcium apparently involved a competition between the divalent cation and [¹⁴C]guanidinium for the same channel. When NG 108–15 cells were exposed to X537A, an ionophore that specifically induces release of calcium from intracellular stores, [¹⁴C]guanidinium uptake was markedly increased even in the absence of 5-HT and/or substance P. Conversely, [¹⁴C]guanidinium influx due to the latter substances could be reversibly and dose-dependently blocked by various drugs that possess calmodulin-antagonizing properties. These results strongly suggest that the cation permeability opened by 5-HT and substance P in NG 108–15 cells involves a calcium/calmodulin-dependent process. However, as the phosphodiesterase inhibitor isobutylmethylxanthine, the nitric oxide synthase inhibitor A/monomethylarginine, the protein kinase C inhibitor staurosporine, and the protein kinase C activator 12-O-tetradeca-noylphorbol 13-acetate did not alter [¹⁴C]guanidinium influx in NG 108–15 cells exposed to 5-HT and substance P., this process probably does not involve the calcium-dependent nitric oxide pathway and protein kinase C activation.     

Multiparameter analysis of clastogenic factors, pro-oxidant cytokines, and inflammatory markers in HIV-1-infected patients with asymptomatic disease, opportunistic infections, and malignancies.

HIV-1-infected patients are in chronic oxidative stress and clastogenic factors (CFs) are present in their plasma. CFs from patients with HIV are formed via superoxide anion radical and stimulate further superoxide production. The pathophysiolgic significance and the exact composition of the circulating clastogenic material in patients with HIV is unknown. Cytokines, such as tumor necrosis factor-alpha (TNF-alpha), are increased in the plasma of patients with HIV and TNF-alpha shows clastogenic activity in vitro. The aim of this clinical study was to compare levels of CF in HIV-1-positive patients with asymptomatic disease, opportunistic infections, and malignancies with those in HIV-1-negative control groups and to correlate CF activity with CD4+ T cell numbers, the cytokines (TNF-alpha, interleukin-2 [IL-2], IL-6), and the inflammatory markers (C-reactive protein [CRP], neopterin, granulocyte elastase). CFs were significantly increased in all HIV-1-positive patients and in HIV-1-negative patients with malignant tumors. HIV-1-positive patients with Kaposi's sarcoma showed the highest CF activity in their plasma (p < 0.08). CFs appear very early in HIV infection, and they correlate negatively with CD4+ T cells, which are an indicator of disease activity. The presence of CF in the plasma of HIV-infected patients is not a general response to a viral infection because these factors are not increased in HIV-1-negative patients with viral infection (zoster). CFs are not specific for the HIV-1 infection; they also occur in HIV-1-negative patients with malignant tumors. There was a tendency towards a positive correlation (p < 0.14) between CF and TNF-alpha but there was no positive correlation of CF with IL-2, IL-6, CRP, elastase, and neopterin levels. This indicates that TNF-alpha may be among the components of CF in HIV-1-infected patients. In addition, other unidentified components may contribute to the clastogenic activity of the plasma or the composition of CF may vary from patient to patient. Further clinical studies with larger sample populations are necessary to analyze the composition of CF in HIV-1-positive patients.     

Superoxide and hydrogen peroxide production by macrophages of New Zealand black mice

Resident peritoneal macrophages from New Zealand Black (NZB) mice release O2- and H2O2 after adherence to a plastic surface without any chemical or particulate stimulant. This phenomenon is age dependent and more pronounced in animals with sever autoimmune disease. Significant differences were observed between the high and low breakage NZB sublines (HB and LB), which were previously developed by selective matings on the basis of chromosome breakage rates. The LB subline differs significantly from the HB subline with respect to autoimmune hemolytic anemia and tumor incidence. When the macrophages were stimulated with the tumor promoter TPA, the number of "responders" was higher in the HB than in the LB subline and correlated with the degree of splenomegaly, that is, with the severity of the disease. A negative response to agonist stimulation and very low spontaneous production of active oxygen species was observed in NZW and Swiss mice, which is the normal finding for resident macrophages according to data from the literature. The increased superoxide and hydrogen peroxide production by macrophages of NZB mice is discussed with respect to autoimmune disease and cancer.     

Physicochemical Properties of Serotonin 5-HT3 Binding Sites Solubilized from Membranes of NG 108-15 Neuroblastoma-Glioma Cells

Specific binding sites with pharmacological properties typical of serotonin 5-HT3 receptors were identified in membranes of the murine hybridoma cell line NG 108-15, using [3H]zacopride as a ligand. Optimal solubilization of these sites (yield, 50%) could be achieved using the detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) at 24 mM plus 0.5 M NaCl in 25 mM Tris-HCl, pH 7.4. Specific [3H]zacopride binding to soluble sites in the 100,000-g CHAPS extract was saturable and showed characteristics (Bmax = 425 +/- 81 fmol/mg of protein; KD = 0.19 +/- 0.02 nM) closely related to those of membrane-bound sites (Bmax = 932 +/- 183 fmol/mg of protein; KD = 0.60 +/- 0.03 nM). Determination of association (k+1 = 0.17 nM min-1) and dissociation (k-1 = 0.02 min-1) rate constants for the soluble sites gave a KD value of 0.12 nM, a result consistent with that calculated from saturation studies. As assessed from the displacement potencies (IC50) of 10 different drugs, the pharmacological profile of [3H]zacopride specific binding sites was essentially the same (r = 0.99) in the CHAPS-soluble extract and in cell membranes, although some increase in the affinity for 5-HT3 antagonists (zacopride, ICS 205-930, and MDL 72222) and decrease in the affinity for 5-HT3 agonists (2-methyl-5-hydroxytryptamine and phenylbiguanide) were noted for the soluble sites. Sucrose density gradient sedimentation of the CHAPS-soluble extract gave a Svedberg coefficient of 12S for the material with [3H]zacopride specific binding capacity. Chromatographic analyses using Sephacryl S-400 and wheat germ agglutinin-agarose columns indicated marked enrichment (by 2.5- and 10-fold, respectively) in [3H]zacopride specific binding activity in the corresponding eluates compared with the starting soluble extract, a finding suggesting that both steps are of potential interest for the partial purification of solubilized 5-HT3 receptors. Two soluble materials with apparent molecular masses of approximately 600 and approximately 36 kDa were found to bind [3H]zacopride specifically in the Sephacryl S-400 eluate. Interestingly, molecular mass determination by radiation inactivation of [3H]zacopride binding sites in frozen NG 108-15 cells gave a value of approximately 35 kDa.     

Hippocampal Membranes Contain a Neurotrophic Activity That Stimulates Cholinergic Properties of Fetal Rat Septal Neurons Cultured Under Serum-Free Conditions

Primary cultures of fetal rat septal neurons were used to identify a membrane-associated cholinergic neurotrophic activity. Under serum-free culture conditions, approximately 98% of the septal cells are neurons, and approximately 6% of the neurons are cholinergic as determined immunocytochemically. Crude membranes prepared from rat hippocampal homogenates stimulate choline acetyltransferase (ChAT) activity in treated septal neurons. The membrane-associated trophic activity is apparent at lower protein concentrations than activity present in the soluble fraction and is unevenly distributed in various brain regions; it is highest in hippocampus and striatum and negligible in cerebellum. Membrane trophic activity is developmentally regulated, is heat and trypsin sensitive, and increases the rate of expression of ChAT in septal neurons. Upon gel filtration chromatography of a high-salt membrane extract, trophic activity elutes as a broad peak in the 500 kilodalton (kD) molecular mass range. Stimulation of septal neuronal ChAT activity by either crude membranes or partially purified preparations is not inhibited by antibodies against nerve growth factor (NGF), and its maximal activity is additive to maximally active doses of NGF. The results indicate that hippocampal membranes contain cholinergic neurotrophic activity which may be important for the development of septal cholinergic neurons.     

Chromatographic Analyses of the Serotonin 5-HT1A Receptor Solubilized from the Rat Hippocampus

Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 microM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mM MnCl2, and 50 micrograms/ml of cholesteryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2-4 days at 4 degrees C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethylaminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1A binding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.