A comparative analysis of microscopy and PCR-based detection methods for blood parasites

We compared information obtained by both microscopy and nested mitochondrial cytochrome b PCR in determining prevalence of haemosporidian infections in naturally infected birds. Blood samples from 472 birds of 11 species belonging to 7 families and 4 orders were collected in Europe, Africa and North America. Skilled investigators investigated them using the PCR-based screening and microscopic examination of stained blood films. The overall prevalence of haemosporidian infections, which was determined combining results of both these methods, was 60%. Both methods slightly underestimated the overall prevalence of infection, which was 54.2% after the PCR diagnostics and 53.6% after microscopic examination. Importantly, both these tools showed the same trends of prevalence of Haemoproteus spp. (21% by PCR and 22% by microscopy), Plasmodium spp. (17% and 22%) and Leucocytozoon spp. (30% and 25%) in the same sample, testifying that microscopy is a reliable tool in determining patterns of distribution of blood haemosporidian parasites in naturally infected birds. We encourage using optical microscopy in studies of blood parasites in parallel to the now widely employed molecular methods. Microscopy is relatively inexpensive and provides valuable information about directions how molecular methods can be further improved and most effectively applied, especially in the field studies of parasites. Importantly, blood films, which are used for microscopic examination, should be of good quality; they should be examined properly by skilled investigators. In spite of relatively long duration of microscopy of each sample, such examination provides opportunities for simultaneous determination and verification of taxonomically different parasites. Presently, different PCR protocols must be used for the detection of parasites belonging to different genera; this is expensive and time-consuming.     

Neuron–Astrocyte Interactions,Pyruvate Carboxylation and the Pentose Phosphate Pathway in the Neonatal Rat Brain

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-¹³C]glucose and [1,2-¹³C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-¹³C]glucose and were sacrificed 30 min later. Brain extracts were analysed using ¹H- and ¹³C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-¹³C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-¹³C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-¹³C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.     

Comparative phylogeography of Ponto-Caspian mysid crustaceans: isolation and exchange among dynamic inland sea basins

The distributions of many endemic Ponto-Caspian brackish-water taxa are subdivided among the Black, Azov and Caspian Sea basins and further among river estuaries. Of the two alternative views to explain the distributions, the relict school has claimed Tertiary fragmentation of the once contiguous range by emerging geographical and salinity barriers, whereas the immigration view has suggested recolonization of the westerly populations from the Caspian Sea after extirpation during Late Pleistocene environmental perturbations. A study of mitochondrial (COI) phylogeography of seven mysid crustacean taxa from the genera Limnomysis and Paramysis showed that both scenarios can be valid for different species. Four taxa had distinct lineages related to the major basin subdivision, but the lineage distributions and depths of divergence were not concordant. The data do not support a hypothesis of Late Miocene (10-5 Myr) vicariance; rather, range subdivisions and dispersal from and to the Caspian Sea seem to have occurred at different times throughout the Pleistocene. For example, in Paramysis lacustris each basin had an endemic clade 2-5% diverged from the others, whereas Paramysis kessleri from the southern Caspian and the western Black Sea were nearly identical. Species-specific ecological characteristics such as vagility and salinity tolerance seem to have played important roles in shaping the phylogeographic patterns. The mitochondrial data also suggested recent, human-mediated cryptic invasions of P. lacustris and Limnomysis benedeni from the Caspian to the Sea of Azov basin via the Volga-Don canal. Cryptic species-level subdivisions were recorded in populations attributed to Paramysis baeri, and possibly in P. lacustris.     

The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells

5-Aminolevulinic acid (ALA), a precursor of the endogenous photosensitizer protoporphyrin IX, is used in the photodynamic therapy (PDT) of cancer. Sub-lethal ALA-PDT (1-min irradiation with 370-450 nm blue light, 0.6 mW/cm(2) after 2-h incubation with 1 mM ALA) has been earlier shown to change cell morphology and to inhibit both trypsin-induced detachment of cultured cancer cells from the plastic substrata and cell attachment to the bottom of the plastic well plates. In the present study, we found that such treatment of human adenocarcinoma WiDr cells grown in dense colonies stimulated the formation of actin cortex between cells in the colonies and increased the number of actin stress fibres in some, but not in all, cells. However, ALA-PDT did not change the microtubular cytoskeleton in these cells. A similar treatment of glioblastoma D54Mg cells, which grow separately and communicate by protrusions, caused loss of fibrillar actin structures in growth cones, retraction of protrusions, and surface blebbing in some cells. The application of the cytoskeleton inhibitors cytochalasin D, colchicine or taxol showed that the inhibition of trypsin-induced detachment of photosensitized WiDr cells was related to ALA-PDT-induced changes in actin and microtubular cytoskeleton. Some signal transduction processes are suggested to be involved in ALA-PDT-induced changes in cytoskeleton, cell shape, and adhesion.     

Is 1-hydroxypyrene a reliable bioindicator of measured dietary polycyclic aromatic hydrocarbon under normal conditions?

Five healthy volunteers consumed similar amounts of identical foods for 5 consecutive days. The concentration of pyrene and of benzo(a)pyrene was determined in each of the 15 meals by a short analytical method that included sample saponification, solvent extraction, and HPLC analysis. The volunteers also provided three daily total volume 8-h urine samples for the duration of the study for the assessment of 1-hydroxypyrene, a biomarker of pyrene and polycyclic aromatic hydrocarbon (PAH) exposure. Mean recoveries were 83 and 75%, respectively, for pyrene and benzo(a)pyrene in food. Daily dietary pyrene doses varied from 0.7 to 3 microg. Excluding two outliers consisting of meals containing charbroiled pork and beef, pyrene content in the meals estimated from the published literature data was correlated to the measured pyrene, but overestimated the actual concentration by ca. 70%. Despite the identical ingested doses of pyrene, there was a 50-76% (coefficient of variation) interindividual variability in the daily-excreted amount of 1-hydroxypyrene. Urinary excretion of this metabolite was not correlated with ingested dose of pyrene under the normal feeding conditions used in this study. Bioavailability, enzymatic polymorphism, and differences in enterohepatic cycling of the metabolite may contribute to the observed variability. It was calculated that dietary pyrene intake accounts for between 87.5 and 99.8% of the sum of dietary and inhalation intake. From the presented data, unless the above-mentioned factors are taken into account, 1-hydroxypyrene might not be a reliable bioindicator of ingested pyrene (PAHs) under normal feeding conditions.     

The effect of folic acid on porphyrin synthesis in tumors and normal skin of mice treated with 5-aminolevulinic acid or methyl 5-aminolevulinate.

5-Aminolevulinic acid (ALA) or its derivative methyl 5-aminolevulinate (MAL) combined with folic acid was applied in nude mice bearing human colon adenocarcinoma. The aim of the study is to see whether folic acid may increase biosynthesis of porphyrins in tumor tissue after systemic or topical administration of ALA or MAL. The production of porphyrins was determined by spectrofluorometric measurements with an optical fibre probe. It was found that the porphyrin production after i.p injection of 200 mg kg(-1) ALA or MAL was significantly increased by i.p injection of 100 mg kg(-1) folic acid. However, in the case of topically applied 20% ALA, folic acid had no effect. In the case of topically applied 20% MAL, folic acid (i.p or topically applied) reduced the porphyrin synthesis. This might be used for the protection of normal skin against photosensitization. The effects of folic acid were similar in tumors and normal skin. Two mechanisms may explain the results: enhancement of the efficiency of the rate-limiting enzyme porphobilinogen deaminase by folic acid or interference of folic acid with the transport of ALA and MAL to and into the cells synthesizing porphyrins in the tissues. The present data seem to favour the latter mechanism. Folic acid may have a role as an adjuvant in photodynamic therapy with systemically administered ALA and its derivatives.     

Chlorpromazine,clozapine and olanzapine inhibit anionic amino acid transport in cultured human fibroblasts

Summary. We report here that chlorpromazine, a first generation antipsychotic drug, inhibits anionic amino acid transport mediated by system X⁻ _AG (EAAT transporters) in cultured human fibroblasts. With 30 μM chlorpromazine, transport inhibition is detectable after 3 h of treatment, maximal after 48 h (>60%), and referable to a decrease in V_max. Chlorpromazine effect is not dependent upon changes of membrane potential and is selective for system X⁻ _AG since transport systems A and y⁺ are not affected. Among antipsychotic drugs, the inhibitory effect of chlorpromazine is shared by two dibenzodiazepines, clozapine and olanzapine, while other compounds, such as risperidon, zuclopentixol, sertindol and haloperidol, are not effective. Transport inhibition by clozapine and olanzapine, but not by chlorpromazine, is reversible, suggesting that the mechanisms involved are distinct. These results indicate that a subset of antipsychotic drugs inhibits EAAT transporters in non-nervous tissues and prompt further investigation on possible alterations of glutamate transport in peripheral tissues of schizophrenic patients.     

Centriole movements in mammalian epithelial cells during cytokinesis

Background  

In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and α-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules.     

Impaired glutamine metabolism in NMDA receptor hypofunction induced by MK801

Paradoxically, glutamate receptor antagonists have neurotoxic and psychotogenic properties in addition to their neuroprotective potential during excessive glutamate release. In the present study the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK801 was used to examine glial-neuronal interactions in NMDA receptor hypofunction. Rats were given a subanesthetic dose of MK801 together with [1-13C]glucose and [1,2-13C]acetate, and brains were removed 20 min later. Analyses of extracts from cingulate, retrosplenial plus middle frontal cortices (CRFC) and temporal lobe were performed using HPLC and 13C and 1H nuclear magnetic resonance spectroscopy. Hypofunction of the NMDA receptor induced similar changes in both brain areas investigated; however, the changes were most pronounced in the temporal lobe. Generally, only labeling from [1-13C]glucose was affected by MK801. In CRFC and temporal lobe amounts of both labeled and unlabeled glutamine were increased, whereas those of aspartate were decreased. In the CRFC the decrease in labeling of aspartate was greater than the decrease in concentration, leading to decreased 13C enrichment. In temporal lobe, not in CRFC, increased concentrations of glutamate, GABA, succinate, glutathione and inositol were detected together with increased labeling of GABA and succinate from [1-13C]glucose. 13C Enrichment was decreased in glutamate and increased in succinate. The results point towards a disturbance in glutamate-glutamine cycling and thus interaction between neurons and glia, since labeling of glutamate and glutamine from glucose was affected differently.