Two-photon excitation studies of m-THPC photosensitizer and photodynamic activity in an epithelial cell line.

A femtosecond laser delivering pulses of wavelength 800nm and 124fs duration at rates of 1kHz has been used to investigate the two-photon excited fluorescence in the photosensitizer m-THPC. The scaling of fluorescence amplitude with laser power and fluorescence sidelight imaging are found to support a predominantly two-photon excitation mechanism. A value for the two-photon cross-section of δ=1.8×10(-57)m(4)s is derived by comparing the fluorescence signals excited by wavelengths of 800 and 400nm. Preliminary results demonstrating the two-photon induced PDT activity of m-THPC in an epithelial cell line are also reported.

Recovery of thermophilic campylobacters from pond water and sediment and the problem of interference by background bacteria in enrichment culture.

The aim of this study was to address problems in the determination of thermophilic campylobacters in turbid pond water and sediment. Thirty sets of three samples of pond water (volumes 10, 100, 1000 ml) or sediment (0.1, 1.0, 5.0 ml) were examined for the presence of thermophilic campylobacters. The different volumes of pond water were processed by membrane filtration followed by selective enrichment. The samples of sediment were subjected directly to selective enrichment. Presumptive isolates were confirmed by Gram stain, cell morphology, presence of oxidase and catalase, growth under microaerobic but not aerobic conditions, and PCR. Confirmed Campylobacter species were recovered only from 10 and 100 ml samples of water and from 0.1 and 1.0 ml samples of sediments. The 1000 ml samples of water and 5.0 ml samples of sediment never gave positive isolates. PCR indicated that the confirmed isolates were all either Campylobacter jejuni or C. coli. Enrichment cultures from 1000 ml filtrations contained the highest number of background bacteria. It is suggested that the processing of large volumes of turbid environmental water samples or of sediment is counterproductive and may not yield positive Campylobacter cultures. This is probably due to antagonistic effects of large numbers of background bacteria out-competing campylobacters during the enrichment stage. Pilot studies to establish appropriate volumes of pond water or sediment samples should be undertaken before routine determination of campylobacters is begun.

Trichomonas vaginalis requires traces of oxygen and high concentrations of carbon dioxide for optimal growth.

The effects of O2 and CO2 on the growth in culture of Trichomonas vaginalis strain C1-NIH were investigated. Growth under pre-purified N2 in the absence of CO2 supplementation gave a doubling time of 4.4 h; when traces of O2 (less than 0.25 microM) were present, the doubling time was 3.5 h. Organisms grew most rapidly (doubling time 2.3 h) with traces of O2 (less than 0.25 microM) and with the CO2 level controlled at 5 mM. The balance of fermentation products from maltose was greatly influenced by supplied gases. Under strictly anaerobic conditions at 5 mM CO2, equimolar glycerol and lactate accounted for more than 95% of the measured products, whereas lower CO2 increased acetate production. Under microaerobic conditions (O2 less than 0.25 microM) acetate was the major product when CO2 was limited to that evolved endogenously; again 5 mM CO2 favoured glycerol and lactate production. Activities of key enzymes measured in cell-free extracts (pyruvate:ferredoxin oxidoreductase, hydrogenase, glycerol kinase, malate dehydrogenase (decarboxylating) and lactate dehydrogenase) altered with growth conditions commensurately with observed changes in metabolic flux patterns. These results suggest that T. vaginalis is optimally adapted to conditions it experiences in situ in the vagina (traces of O2, high CO2).

Effects of inhibitors on the oxygen kinetics of Nippostrongylus brasiliensis.

Endogenous respiration of the parasitic nematode Nippostrongylus brasiliensis and the succinate oxidase activity of isolated mitochondria were partially inhibited by antimycin A; the remaining respiratory activity was sensitive to salicylhydroxamic acid (SHAM). Sub-millimolar concentrations of SHAM markedly stimulated respiration by 60% in whole N. brasiliensis and isolated mitochondria; stimulation by SHAM was not observed in the presence of antimycin A. Little change in the relative fluxes of electrons through the classical, antimycin A-sensitive pathway and the alternative SHAM sensitive pathway was observed between low and high O2 concentrations; this may suggest that the O2 affinities of both pathways are similar. O2 dependence of respiration showed O2 thresholds above which respiration decreases; in the absence of inhibitors whole N. brasiliensis and isolated mitochondria had threshold values around 60 microM O2. Increased O2 threshold values were observed in the presence of SHAM and antimycin A. The apparent Km values for O2 of whole N. brasiliensis and isolated mitochondria were 31 +/- 2 microM O2 and 3.5 +/- 0.2 microM O2 respectively; this difference in apparent Km values may reflect the presence of O2 gradients in the whole worm. The Km and O2 inhibition threshold values observed for whole N. brasiliensis are in good agreement with the proposed range of O2 concentrations thought to exist within the worm's natural environment. H2O2 production was detected in respiring uncoupled mitochondria, but H2O2 could not be detected in the medium surrounding whole N. brasiliensis. SHAM-stimulated respiration was accompanied by increased H2O2 production which was prevented by the addition of antimycin A.

Giardia lamblia produces alanine anaerobically but not in the presence of oxygen.

Proton nuclear magnetic resonance was used to follow glucose metabolism in Giardia lamblia. Under strictly anaerobic conditions this organism produces equimolar ethanol and alanine as well as CO2 and some acetate. Aerobically the production of both alanine and ethanol are inhibited and more acetate and CO2 are formed. These changes in the balance of products are reversible over the range 0-46 microM O2. In the presence of 46 microM O2, alanine was not detectable. The O2-sensitivity of alanine production may highlight the necessity for redox-balancing reactions in an organism exposed in situ to fluctuating concentrations of O2.

The effects of oxygen on fermentation in Giardia lamblia.

Detailed study of the effects of oxygen on the carbohydrate metabolism of Giardia lamblia revealed that low concentrations of oxygen (< 0.25 microM) produced profound alterations in the carbon balance of this organism. Although this concentration of oxygen could not be detected by mass spectrometry, a marked stimulation of ethanol production was observed. Associated with this was an inhibition of alanine production and oxidation of the intracellular NAD(P)H pool. Higher concentrations of oxygen inhibited ethanol production and further reduced levels of alanine. These results suggest that this stimulation is due to changes in carbon flux. Analysis of cell and medium hydrolysates after the growth of trophozoites in [U-14C]glucose suggests that G. lamblia does not synthesise detectable levels of labelled amino acids, except alanine and to a lesser extent valine, from this sugar. Trophozoites of G. lamblia have both glutamate dehydrogenase and alanine aminotransferase activity. As glutamate is taken up from the medium, it is suggested that glutamate dehydrogenase and alanine aminotransferase cooperate to convert pyruvate to alanine, with the concomitant oxidation of NAD(P)H.

Respiration in the filarial nematode Brugia pahangi.

Glucose-supported O2 uptake in the filarial nematode Brugia pahangi was partially inhibited by antimycin A (30-40%), with the remaining activity being sensitive to o-hydroxydiphenyl or salicylhydroxamic acid (SHAM). The production of CO2 by B. pahangi in the presence of D-glucose was stimulated by O2; the stimulation of CO2; the stimulation of CO2 production was sensitive to antimycin A. The O2 dependencies of respiration showed that the apparent O2 affinity for B. pahangi was diminished in the presence of antimycin A; O2 thresholds for inhibition of respiration were observed which showed that the alternative electron transport pathway was less sensitive to inhibition at elevated O2 concentrations. H2O2 production and its excretion could be detected in whole B. pahangi; higher rates were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The effects of inhibitors on H2O2 production suggest two sites of H2O2 production, one associated with the classical antimycin A-sensitive pathway, the other with the alternative respiratory pathway. The similarity in the O2 dependencies of H2O2 production and respiration may indicate that H2O2 production is involved in O2-mediated toxicity. Succinate and malate respiring sub-mitochondrial particles of B. pahangi produced O2.- radicals at a site on the antimycin A-sensitive respiratory pathway. Inhibition of the alternative electron pathway by SHAM was unusual; sub-millimolar concentrations markedly stimulated respiration, H2O2 production and O2.- production by 30, 20 and 25%, respectively, whereas higher concentrations (greater than 2.5 mM) inhibited respiration by 75% and H2O2 and O2.- production by up to 85%.

Waterfowl and the bacteriological quality of amenity ponds.

This study investigated the impact of waterfowl on the bacteriological quality of village ponds in East Yorkshire, north-east England. Water and sediment samples were collected from ponds with and without resident ducks and geese; faecal indicator and potentially pathogenic bacteria were assayed by membrane filtration and by selective enrichment. Escherichia coli, faecal streptococci and, to a degree, Clostridium perfringens were more abundant in ponds with waterfowl; Salmonella was isolated in June-August from the sediment of a pond with waterfowl. The results suggested that the bacteriological quality of village ponds might be adversely affected by waterfowl. All water samples from ponds with waterfowl had faecal indicators at higher concentrations than EU requirements for bathing waters. Although these ponds are not bathing waters we suggest skin contact and accidental ingestion of water should be avoided.

Metabolic changes in Giardia intestinalis during differentiation.

The oxygen uptake rate and metronidazole (MTZ) sensitivity in Giardia spp. cysts is greatly reduced from that in trophozoites. Thus, this project was undertaken to assess when in the encystation process these phenomena occur. Oxygen uptake rates approximately doubled (from approximately 4.9 to 8.3 microM O2 min(-1) 10(-6) cells) during the first 5 hr into encystation. This increase was followed by a marked decrease to 2.3 microM O2 min(-1) 10(-6) by 12 hr. By 50 hr into encystation, oxygen uptake was 0.7 microM O2 min(-1) 10(-6) cells. Glucose stimulated oxygen uptake by 89% in trophozoites but did not demonstrably stimulate oxygen uptake in cells after 12 hr into encystment. Deoxy-D-glucose uptake dropped by more than an order of magnitude in encysting cells compared to nonencysting cells. In contrast, aspartate uptake remained relatively constant regardless of whether cells were encysting or not. This suggests that there is a change in the parasite's ability to transport glucose during cyst formation; a similar change in the parasite's ability to transport aspartate was not observed after 40 hr into encystation. MTZ inhibited oxygen uptake by 77% in trophozoites, but there was no detectable inhibition of oxygen uptake 8 hr after trophozoites were transferred to encystation medium. We propose that this resistance to MTZ may be due to a change in metabolic flux away from the pyruvate ferredoxin oxidoreductase pathway. Oxygen uptake by noninduced cysts increased exponentially during the 30 min following the induction of excystation. Likewise, MTZ sensitivity returned within 15 min after the induction of excystation, and by 30 min into excystation full sensitivity had returned.

A pre-enrichment step is essential for detection of Campylobacter sp. in turbid pond water.

This work aimed to detect Campylobacter species from naturally contaminated turbid pond water by PCR. A total of 16 water samples were collected from a turbid village pond. Four methods of DNA extraction were applied to centrifuge pellets from eight 100 ml pond water samples prior to attempted detection of Campylobacter by PCR without an enrichment step. These methods were (1) Tris-HCl and sodium dodecyl sulfate followed by phenol:chloroform:isoamylalcohol extraction followed by treatment with DNA clean up kit, (2) proteinase K, (3) Chelex® 100, and (4) boiling. The other eight pond water samples (10 ml and 100 ml) were filtered and filters were incubated overnight in Preston enrichment broth. The centrifuge pellets obtained from enrichment cultures were treated by proteinase K for DNA extraction. Primers CF03 and CF04 for the flagellin genes (flaA and flaB) of Campylobacter jejuni and Campylobacter coli were used for amplifying the extracted DNA. The DNA extracted from eight-100 ml pond water samples that were not subject to selective enrichment was never amplified with primers CF03 and CF04, hence Campylobacter was not detected. In contrast, the DNA that was from samples that were subjected to a selective enrichment step in Preston broth prior to PCR assay always gave amplified bands of 340-380 bp, therefore the presence of Campylobacter was confirmed. Detection of campylobacters from naturally contaminated, turbid, environmental water may not be feasible by direct PCR assay because of low numbers and the presence of high concentration of humic matter and other PCR inhibitors. The enrichment of water samples in selective broth, however, facilitated PCR detection of Campylobacter probably by increasing cell number and by diluting PCR inhibitors.

Acanthamoeba can be differentiated by the polymerase chain reaction and simple plating assays.

Acanthamoeba are opportunistic pathogens with invasive and noninvasive species. For clinical purposes it is important to differentiate potentially pathogenic from nonpathogenic isolates. For the rapid and sensitive identification of Acanthamoeba at the genus level, we used a polymerase chain reaction (PCR)-based method which detected as few as five cells. Further, we tested nine isolates of Acanthamoeba for their ability to produce cytopathic effects (CPE) on corneal epithelial cells. On the basis of the results, Acanthamoeba were divided into pathogenic or nonpathogenic groups. However, because CPE assays are not available to every diagnostic laboratory, we developed a simple plating assay based on osmotolerance which correlated well with the CPE assays. Pathogenic Acanthamoeba showed growth on higher osmolarity (agar plates containing one molar mannitol), while growth of nonpathogens was inhibited on these plates. In conclusion, we have developed methods for the rapid identification and differentiation of Acanthamoeba.

Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.

Nippostrongylus brasiliensis and Ascaridia galli: characterization of peroxisomes.

A method is described for the isolation of peroxisomes from mitochondria-enriched fractions obtained from both species of nematodes. The distributions of these organelles are characterized after density gradient centrifugation in sucrose or Percoll by urate oxidase and catalase activities. The possession of peroxisomes may be part of an important defence mechanism in parasites.

Oxygen uptake in cysts and trophozoites of Giardia lamblia.

Oxygen uptake in cysts and trophozoites of the parasitic protozoan Giardia lamblia was examined. Both showed oxygen uptake activity, but that of cysts was only 10% to 20% that of trophozoites. Oxygen dependence of oxygen uptake in cysts and trophozoites showed oxygen maxima above which oxygen uptake decreased. The oxygen concentration at which the oxygen uptake rate was greatest was higher for trophozoites than for cysts. The effect of various inhibitors on cyst and trophozoite oxygen uptake suggested that flavoproteins and quinones play some role in oxygen uptake. The substrate specificities and the effect of inhibitors on G. lamblia trophozoites were similar to those observed for G. muris. Metronidazole, the drug most commonly used in treatment of giardiasis, inhibited oxygen uptake and motility in trophozoites; however, it had no obvious effect on either oxygen uptake or excystation in cysts. Menadione, a redox cycling naphthaquinone, first stimulated, then completely inhibited, oxygen uptake in cysts and trophozoites; a complete loss of cyst viability and trophozoite motility was also observed. The effect of menadione on G. lamblia may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.

Haemoprotein terminal oxidases in the nematodes Nippostrongylus brasiliensis and Ascaridia galli.

1. Mitochondria isolated from the gut-dwelling nematodes Nippostrongylus brasiliensis and Ascaridia galli (muscle and gut + reproductive tissue) were examined for cytochromes, and it was observed that N. brasiliensis and A. galli muscle tissue mitochondria contained a-, b- and c-type cytochromes, but their stoichiometries were quite different (1:2:1.9 and 1:11.4:13.6 respectively); A. galli gut + reproductive-tissue mitochondria, however, only contained b and c cytochromes, in a ratio of 1:0.8. 2. CO difference spectra showed the presence of CO-reacting b-type cytochrome(s) in all three types of mitochondria; the fast-reacting species comprised 30, 44 and 39% of the total in N. brasiliensis, A. galli muscle and A. galli gut + reproductive-tissue mitochondria respectively. 3. Cytochrome aa3 was observed in N. brasiliensis mitochondria and in those from A. galli muscle, but was below the level of detectability (less than 0.005 nmol/mg of protein) for A. galli gut + reproductive-tissue mitochondria. 4. Photochemical action spectra for the reversal of CO inhibition of the endogenous respiration of whole worms (at 24 microM- and 40 microM-O2 respectively for N. brasiliensis and A. galli) gave maxima at 598 and 542-543 nm, corresponding to the alpha- and beta-absorption maxima of cytochrome aa3, and at 567 nm (b-type cytochrome) for both worms. These results suggest that cytochrome aa3 is the major functional oxidase in N. brasiliensis, whereas the CO-reacting b-type cytochrome dominates in A. galli.

The O2-dependence of respiration and H2O2 production in the parasitic nematode Ascaridia galli.

1. Respiration in the parasitic nematode worm Ascaridia galli was inhibited at O2 concentrations in excess of 255 microM, and an apparent Km,O2 of 174 microM was determined. 2. Mitochondria-enriched fractions isolated from the tissues of A. galli have much lower apparent Km,O2 values (approx. 5 microM). They produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 3. Antimycin A inhibited respiration in muscle tissue mitochondria by 10%, but had no effect on respiration in gut + reproductive tissue mitochondria; the major portion of respiration in both types of mitochondria could be attributed to an alternative electron-transport pathway. 4. o-Hydroxydiphenyl, an inhibitor of alternative electron-transport pathways, inhibits respiration by 98% and completely inhibits the production of H2O2 in gut-plus-reproductive-tissue mitochondria; respiration and H2O2 production in muscle tissue mitochondria were inhibited by 90 and 86% respectively. 5. Another inhibitor of alternative electron transport, salicylhydroxamic acid, had the same effect as o-hydroxydiphenyl on H2O2 production and respiration in gut-plus-reproductive-tissue mitochondria. However, its effect on muscle tissue mitochondria was complex; a low concentration (0.35 mM) stimulated H2O2 production, whereas 3 mM inhibited respiration by 87% and prevented H2O2 production completely. 6. The similarities between the apparent Km,O2 values for H2O2 production and respiration in muscle mitochondria and in gut-plus-reproductive-tissue mitochondria suggests that the site of H2O2 production on the alternative electron-transport chain is cytochrome 'o'. 7. These results are discussed in relation to potential O2 toxicity in A. galli.

Respiration in the cysts and trophozoites of Giardia muris.

Cysts and trophozoites of the parasitic protozoon Giardia muris both showed respiratory activity but respiration in cysts was only 10 to 20% that of trophozoites. The O2 dependence of respiration in cysts and trophozoites showed O2 maxima above which respiration decreased. The O2 concentration at which the respiration rate was greatest was higher for cysts than trophozoites. The effects of various inhibitors on cyst and trophozoite respiration suggested that flavoproteins and quinones play some role in respiration. The substrate specificities and the effects of inhibitors on G. muris trophozoites were similar to those observed for Giardia lamblia. Metronidazole, the drug most commonly used in the treatment of giardiasis completely inhibited respiration and motility in trophozoites; however, it had no effect on either respiration or viability in cysts. Menadione, a redox cycling naphthoquinone, stimulated then completely inhibited respiration in cysts and trophozoites; a complete loss of cyst viability or trophozoite motility was also observed. The effects of menadione on G. muris may indicate that redox cycling compounds have potential as chemotherapeutic agents for the treatment of giardiasis.