In vivo studies of the early, peritoneal, cellular and free radical response in rats infected with Fasciola hepatica by flow cytometric analysis

Early recruitment of the peritoneal cell population was observed during migration of newly excysted juvenile flukes. The peritoneal lavages were examined for T cells, cytotoxic NK cells (CNK) and free radicals production of rats at an early stage of infection by Fasciola hepatica. Male Sprague–Dawley rats were infected with 50 metacercariae of F. hepatica and non-infected controls were euthanized 2, 4 and 7 days post infection (d.p.i.), respectively. The peritoneal fluid of experimental animals was analyzed by flow cytometry to estimate cell phenotypes. The peritoneal areas were infiltrated by inflammatory cells, particularly from numerous neutrophils, eosinophils and CD4+ lymphocytes, which were significantly higher for infected rats than non-infected. CNK cells dominated in the peritoneal fluid of infected rats as early as 2 d.p.i. However, after 4 d.p.i. there was a decreased level of CNK cells which may indicate a change from a cytotoxic natural killer (NK) to a regulatory NK response. The challenged group generated very high in vivo levels of inducible nitric oxide (NO) from eosinophils. Superoxide expression was very high in macrophages and neutrophils compared to the uninfected control. In conclusion, our studies suggest that early F. hepatica infection could directly affect lymphoid cells and generate a high in vivo NO production by eosinophils in the peritoneal cavity. Moreover juvenile flukes could stimulate the macrophages and neutrophils to generate H₂O₂ radicals. The host parasite interactions resulting from immune response regulation by effector cells and immune evasion are discussed.     

Vacuolization of target cells: response to microbial toxins

Summary Vacuolization as a marker of microbial activity on cells is a well-known reaction. The phenomenon involves the formation of vacuoles in the cytoplasm of target cells followed by lysis, especially after the action of different cytotoxins. In this review we summarize data on microbial toxic products causing vacuolization of target cells in the light of recent publications, on the basis of the widely described VacA cytotoxin of Helicobacter pylori.     

Puroindoline A-gene expression is involved in association of puroindolines to starch

Puroindolines largely influence cereal grain hardness. In order to understand how they exert this influence, we carried out a molecular analysis of the pina and pinb genes of many Italian wheat cultivars. On the basis of their pin genotypes they could be divided into three groups: Pina-D1a/Pinb-D1a; Pina-D1a/Pinb-D1b; and Pina-D1b/Pinb-D1a. Five cultivars from each group were chosen to be studied to examine the quantity of puroindolines associated with starch (friabilin) and the amount not associated with starch. In addition, the level of pina expression was measured using RT-PCR. Soft cultivars (Pina-D1a/Pinb-D1a) exhibited the highest level of expression of pina; among the hard cultivars, those with the Pina-D1a/Pinb-D1b genotype showed a lower level of expression, while those with the Pina-D1b/Pinb-D1a genotype did not express pina. Total puroindoline and friabilin content was then measured by flow cytometry. Soft Pina-D1a/Pinb-D1a cultivars displayed high puroindoline content that was primarily starch associated. Hard Pina-D1b/Pinb-D1a cultivars had very low puroindoline content with no puroindoline bound to starch. Hard Pina-D1a/Pinb-D1b cultivars were highly heterogeneous with respect to both the content of puroindolines and the level of association with starch. The accurate quantification of puroindolines in starch-bound and not starch-bound forms in association with molecular analysis, indicates that pina expression and presence controls the abundance of total puroindoline and its association with starch.Communicated by H.F. Linskens     

Proline metabolism in response to highest nitrogen dosages in green bean plants (Phaseolus vulgaris L. cv. Strike)

The objective of the present work was to determine what impact extremely high nitrogen dosages would have on proline metabolism in order to use this amino acid as a bioindicator of N status of green bean plants (Phaseolus vulgaris L. cv. Strike). In this effort, we identified the most favourable pathway of proline synthesis under our experimental conditions. The N was applied to the nutrient solution in the form of NH₄NO₃ at 5.4 mmol/L (N1, optimal level), 11.6 mmol/L (N2), 17.4 mmol/L (N3), and 23.2 mmol/L (N4). Our results indicate that the application of high N dosages inPhaseolus is characterized by the accumulation of NO₃⁻, NH₄⁺ and proline in root and foliar organs. However, although the enzymes in charge of proline biosynthesis, ornithine-δ-aminotransferase (OAT, EC 2.6.1.13) and Δ¹-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11/1.2.2.41) vary in behaviour depending on the N status, in our experiment, this amino acid appears to be synthesized mainly by the enzyme ornithine-δ-aminotransferase. This suggests predominance of the ornithine pathway over the glutamine pathway. Finally, under our experimental conditions, proline can be defined as a good indicator of N excess of green bean plants.     

Estrogen regulation of c-fos gene expression through phosphatidylinositol-3-kinase-dependent activation of serum response factor in MCF-7 breast cancer cells

17Beta-estradiol (E2) induces proliferation and c-fos gene expression in MCF-7 cells and both responses are partially blocked by wortmannin and LY294002 which are inhibitors of phosphatidylinositol-3-kinase (PI3-K). Analysis of the c-fos gene promoter shows that the effects of wortmannin and LY294002 are associated with inhibition of E2-induced activation through the serum response factor (SRF) motif within the proximal serum response element at -325 and -296. E2 activates constructs containing multiple copies of the SRF (pSRF) and a GAL4-SRF fusion protein; these responses are accompanied by PI3-K-dependent phosphorylation of Akt and inhibited by wortmannin/LY294002, the antiestrogen ICI 182780, but not by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD98059. Using a series of kinase inhibitors and dominant negative kinase expression plasmids, it was shown that the non-genomic activation of SRF by E2 was associated with src-ras-PI3-K pathway, thus, demonstrating hormonal activation of the SRE through src-ras activation of both PI3-K- and MAPK-dependent signaling pathways.     

Entamoeba invadens: Dynamics of DNA synthesis during differentiation from trophozoite to cyst

The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba invadens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the number of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0–8 h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commitment phase (8–24 h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucleated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24–72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be getting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucleated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens.     

Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes: application to viability assessment in waters

The CSE dye (Chemunex, Maisons-Alfort, France) was combined with an activity marker to improve bacterial activity assessment in natural waters. Its effectiveness to counterstain dead cells with permeabilised membranes was investigated on live and dead cells of a variety of strains from collections or isolated from the natural environment. Cells were killed by heat treatment. For all strains tested, the fluorescent dye showed an intense staining of killed cells having permeabilised membranes while no significant signal was detected when applied to live cells. Furthermore, the CSE dye had no toxicity on viable cells. Then, CSE was combined with the ChemChrome V6 dye (Chemunex) to assess the activity of bacterial cells in different waters. Both fluorescences were analysed simultaneously by solid-phase cytometry. The active cell counts were sometimes lower when both dyes were combined suggesting that CSE was able to counterstain cells having a residual esterase activity and compromised membranes. These cells were subtracted from the active cell counts determined with ChemChrome V6. In most samples, active cell counts were congruent with those determined by the direct viable count method.     

Modulation of the reductive metabolism of halothane by microsomal cytochrome b5 in rat liver

To study the modulation of the reductive metabolism of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) by microsomal cytochrome b₅, formation of 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), major reduced metabolites of halothane, was analyzed in vivo and in vitro. Rats were pretreated with both malotilate (diisopropyl-1,3-dithiol-2-ylidenemalonate) and sodium phenobarbital (malotilate-treated rats) or only with sodium phenobarbital (control rats). The microsomes of malotilate-treated rats had significantly more cytochrome b₅ than the controls, whereas the cytochrome P-450 content was not different between the two groups. At the end of 2-h exposure to 1% halothane in 14% oxygen, the ratio of CDE to CTE in arterial blood was significantly higher in malotilate-treated rats than in the controls. Under anaerobic conditions, the formation of CDE and the ratio of CDE to CTE were significantly greater in microsomal preparations of malotilate-treated rats than those of the controls. In a reconstituted system containing cytochrome P-450_PB purified from rabbit liver, addition of cytochrome b₅ to the system enhanced the formation of CDE and increased the ratio of CDE to CTE. These results suggested that cytochrome b₅ enhances the formation ratio of CDE to CTE by stimulating the supply of a second electron to cytochrome P-450, which might reduce radical reactions in the reductive metabolism of halothane.     

Metabolic fluxes and l-lysine synthesis by Corynebacterium glutamicum in relation to cellular total reducing activity

The total reducing activity (TRA) of cells was used to estimate the physiological activity of Corynebacterium glutamicum under conditions of l-lysine synthesis. This was estimated as the rate of reduction of 2,3,5- triphenyltetrazolium chloride by intact cells. TRA of cells was linearly correlated with the intracellular concentrations of RNA and the bacterial growth rate. It was concluded that this activity reflected the rate of energy generation in cells. A decrease in TRA of growing cells was related to an increase in bacterial lysine synthesis activity. Alteration in metabolic pathway functioning and an increase in the intracellular concentrations of lysine precursors favoured an increase in the rate of lysine synthesis by bacterial cells when cellular TRA was decreasing.     

Seasonal variation of energy metabolism in ghost crab Ocypode quadrata at Siriú Beach (Brazil)

The aim of this study was to analyze the seasonal variations of carbohydrate and lipid metabolism of the ghost crab Ocypode quadrata (Fabricius, 1787) on a sandy beach in the southern region of Brazil. Crabs and hemolymph samples were collected monthly in the field. Hepatopancreas, gills, gonads and claw muscles were used for glycogen determination. In males, blood glucose levels increased in the summer and in the winter. The glycogen values increased significantly in the hepatopancreas in the winter, but remained constant in the muscle, gonads and gills. In females, hemolymph glucose levels, glycogen values in the hepatopancreas and in the gills remained constant throughout the year; however, muscular glycogen increased in the spring and gonad glycogen decreased in the summer. Hemolymph triglyceride levels of males and females and total cholesterol of males decreased significantly in the spring. In females, a significant increase of total cholesterol levels was found in the winter. The findings suggest that in O. quadrata lipids seem to be an important reserve of energy used during reproduction, both in males and females, while glycogen may be used during periods of intense activity or fasting.     

The significance of hydrogenase activity for the energy metabolism of green algae: anaerobiosis favours ATP synthesis in cells of Chlorella with active hydrogenase

In cells of the green alga Chlorella fusca, which contain active hydrogenase(s), the concentration of ATP, NADH and NADPH were measured during a 5 h period of anaerobiosis in the dark and upon subsequent illumination with high light intensities (770 W/m²), conditions which favour optimal hydrogen photoproduction.ATP concentrations were also determined in cells of Chlorella fusca, whose hydrogenase was inactivated prior to illumination, and in cells of Chlorella vulgaris which do not contain hydrogenase. In the dark, the ATP concentration increased slightly during anaerobiosis in cells with active hydrogenase. This increase in ATP concentration was accompanied by an increase of NADH and a decrease of NADPH content.Upon illumination, the ATP content increased in cells with an active hydrogenase, whereas the NADH content decreased. The rate of phosphorylation was twice that observed in cells without active hydrogenase.This ATP synthesis in the light was not inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (10 mol/l) nor by carbonylcyanide-3-chlorophenyl-hydrazone (CCCP) (1 mol/l) but was diminished by 500 mol/l dibromothymoquinone (DBMIB) and 6 mol/l carbonylcyanide-3-chlorophenyl-hydrazone (CCCP).It was concluded that an active hydrogenase can support ATP production under anaerobic conditions in the dark as well as in the light. NADH might serve in vivo as electron donor for a fermentative production of hydrogen in the light.Possible mechanisms underlying ATP production under anaerobiosis and hydrogen productive conditions are discussed.Abbreviations CCCP Carbonylcyanide-3-chlorophenyl-hydrazone - DBMIB dibromothymoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide-p-trifluormethoxyphenyl-hydrazone - HEPES N-2-hydroxyethylpiperazin-N-2-ethan-sulfonic acid - PSI II, photosystem I, II respectively - PQ plastoquinone     

Red/far-red light modulates phospholipase D activity in oat seedlings: relation of enzyme photosensitivity to photosynthesis

Phospholipase D (PLD) activity was found to be higher in etiolated oat seedlings than in green seedlings. White and red (R) light exposure inhibited PLD activity in etiolated seedlings. Far-red light eliminated R-light-induced decrease in PLD activity, indicating phytochrome participation in observed photomodulation. Inhibitor of electron transport in chloroplast 3-(3,4-dichlorophenyl)-1,1-dimethylurea stimulated and glucose suppressed PLD activity in green and etiolated oat seedlings, respectively. These results suggest that PLD activity in oat seedling is regulated by light with involvement of phytochrome photoreceptor, and associated with photosynthesis process.     

Molecular characterization of the alpha-glucosidase activity in Enterobacter sakazakii reveals the presence of a putative gene cluster for palatinose metabolism

Enterobacter sakazakii is considered an opportunistic pathogen for premature infants and neonates. Although E. sakazakii has been isolated from various types of food, recontaminated dried infant formula has been epidemiologically identified as the major source of infection. Amongst others, alpha-glucosidase activity is one of the most important biochemical features, which differentiates E. sakazakii from other species in the family Enterobacteriaceae and has therefore been used as a selective marker in the development of differential media. However, it has been shown, that methods based on this biochemical feature are prone to producing false-positive results for presumptive E. sakazakii colonies due to the presence of this enzymatic activity in other species of the Enterobacteriaceae. Therefore, elucidation of the molecular basis responsible for the biochemical feature in E. sakazakii would provide novel targets suitable for the development of more specific and direct identification systems for this organism. By applying the bacterial artificial chromosome (BAC) approach, along with heterologous gene expression in Escherichia coli, the molecular basis of the alpha-glucosidase activity in E. sakazakii was characterized. Here we report the identification of two different alpha-glucosidase encoding genes. Homology searches of the deduced amino acid sequences revealed that the proteins belong to a cluster of gene products putatively responsible for the metabolism of isomaltulose (palatinose; 6-O-alpha-d-glucopyranosyl-d-fructose). The glycosyl-hydrolyzing activity of each protein was demonstrated by subcloning the respective open reading frames and screening of E. coli transformants for their ability to hydrolyze 4-methyl-umbelliferyl-alpha-d-glucoside. Analysis at the protein level revealed that both enzymes belong to the intracellular fraction of cell proteins. The presence of the postulated palatinose metabolism was proven by growth experiments using this sugar as a sole carbon source.     

Plasma glucocorticoid concentrations in free-ranging platypuses (Ornithorhynchus anatinus): response to capture and patterns in relation to reproduction

Energy demands in the platypus are likely to increase in the breeding season, which occurs from winter to early spring. Glucocorticoids, which play a major role in energy mobilisation, were measured in consecutive blood samples from free-ranging adults at approximately monthly intervals throughout the year. Glucose and free fatty acids were also measured in some samples. Plasma concentrations of glucocorticoids rose significantly during the first 30 min after capture, accompanied by a rise in free fatty acids, but no corresponding increase in glucose concentrations. We observed a strong pattern in plasma glucocorticoids in samples collected within 15 min of capture (indicative of pre-disturbance concentrations) in different phases of the annual reproductive cycle, with significantly higher levels in both males and females in the breeding season compared to the non-breeding season. These data, and the decline in tail fat stores that occur towards the end of the mating period (around October), suggest that platypus experience high-energy demands during this phase of reproduction. Plasma glucocorticoid concentrations in females sampled during the lactation period (October-February) were relatively low, and similar to those in females sampled in the non-breeding, non-lactation period (March-June). The latter requires further investigation as results may have been influenced by sampling limitations.     

Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale

The coevolution of ticks and the pathogens that they transmit has ensured their mutual survival. In these studies, we used a functional genomics approach to characterize tick genes regulated in response to Anaplasma marginale infection. Differentially regulated genes/proteins were identified by suppression-subtractive hybridization and differential in-gel electrophoresis analyses of cultured IDE8 tick cells infected with A. marginale. Nine of 17 of these genes were confirmed by real-time RT-PCR to be differentially regulated in ticks and/or IDE8 tick cells in response to A. marginale infection. RNA interference was used for functional studies. Six genes, which encode putative selenoprotein W2a, hematopoietic stem/progenitor cells protein-like, proteasome 26S subunit, ferritin, GST, and subolesin control, were found to affect A. marginale infection in IDE8 tick cells. Four genes, which encode putative GST, salivary selenoprotein M, vATPase, and ubiquitin, affected A. marginale infection in different sites of development in ticks. The results of these studies demonstrated that a molecular mechanism occurs by which tick cell gene expression mediates the A. marginale developmental cycle and trafficking through ticks.     

The metabolism of ethylene to ethylene oxide in cultivars of Vicia faba: Relationship with waterlogging resistance

The ability of cultivars of Vicia faba L. to metabolize ethylene and the resistance of such cultivars to waterlogging was assessed. Those cultivars showing the highest resistance to waterlogging also had the ability to rapidly metabolize ethylene. It is suggested that a causal relationship may exist between the two processes.     

The response of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> CH34 to spaceflight in the international space station

The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the ‘International Space Station’ (ISS), strain CH34 was grown for 10–12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.     

Structural characterization of photosynthetic cells in an amphibious sedge, Eleocharis vivipara, in relation to C3 and C4 metabolism

Eleocharis vivipara Link is a unique amphibious leafless sedge. The terrestrial form has Kranz anatomy and the biochemical traits of C₄ plants while the submerged form develops structural and biochemical traits similar to those of C₃ plants. The structural features of the culms, which are the photosynthetic organs, of the two forms were examined and compared. The culms of the terrestrial form have mesophyll cells and three bundle sheaths which consist of three kinds of cell, namely, the innermost Kranz cells that contain large numbers of organelles, the middle mestome sheath cells that lack chloroplasts, and the outermost parenchyma sheath cells that contain chloroplasts. The culms of the submerged form had a tendency towards reduction in numbers and size of Kranz cells and vascular bundles, as compared to the terrestrial form, and they had spherical mesophyll cells that were tightly packed without intercellular spaces inside the epidermis. The submerged form had a higher ratio of cross-sectional area of mesophyll cells plus parenchyma sheath cells to that of Kranz cells than the terrestrial form. The difference was mainly due to a decrease in the number and the size of the Kranz cells and to a marked increase in the size of the mesophyll cells and the parenchyma sheath cells in the submerged form, as compared to the terrestrial form. The Kranz cells of the terrestrial form had basically the structural characteristics of plants of the NAD-malic enzyme type, with the exception of the intracellular location of organelles. The Kranz cells of the submerged form included only a few organelles, and the percentage of organelles partitioned to the Kranz cells was significantly smaller in the submerged form than in the terrestrial form. In addition, the size of chloroplasts of the Kranz cells was 60–70% of that of the terrestrial form. These structural differences between the two forms may be related to the functional differences in their mechanisms of photosynthesis.Abbreviations KC Kranz cell - MC mesophyll cell - PSC parenchyma sheath cell - NAD-ME NAD-malic enzyme - VB vascular bundle This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology).     

布鲁氏菌粗糙型RA343株与光滑型A19株免疫小鼠后特异性T细胞和抗体动态变化分析

【背景】布鲁氏菌病（简称布病）严重威胁着人类健康和畜牧业的发展,使用毒力弱、免疫原性好、不干扰血清学诊断的疫苗是防控布病的有效措施。【目的】比较布鲁氏菌粗糙型RA343株与光滑型A19株免疫小鼠后机体特异性T细胞和抗体的动态变化,评价粗糙型RA343株的免疫效果。【方法】采用6周龄雌性BALB/c小鼠,共分为3组,RA343株免疫组和A19株免疫组各15只小鼠,空白对照组5只小鼠。免疫组的每只小鼠经腹股沟皮下注射0.1 mL （含菌量为1×10⁸ CFU）菌液。免疫后1、2和3周各免疫组及空白对照组分别剖杀5只小鼠,无菌取脾脏,一部分脾脏研磨分离淋巴细胞,用流式细胞术检测小鼠布鲁氏菌特异性CD4⁺T细胞与CD8⁺T细胞所占比例的变化趋势;另一部分脾脏称重后分离布鲁氏菌RA343株和A19株,评估RA343株和A19株在小鼠脾脏中的定殖情况。在剖杀小鼠的同时取外周血分离血清,用ELISA方法检测免疫后小鼠血清中IgM和IgG抗体的消长规律。采用GraphPad Prism 8.0软件作图并进行统计学分析。【结果】与A19免...     

Infection with the protozoan parasite Bonamia ostreae modifies in vitro haemocyte activities of flat oyster Ostrea edulis

Bonamia ostreae is an intracellular protozoan parasite, infecting haemocytes of the European flat oyster Ostrea edulis. Oyster defence mechanisms mainly rely on haemocytes. In the present study in vitro interactions between parasites and flat oyster haemocytes were investigated using flow cytometry and light microscopy.Haemocyte parameters including: non specific esterase activity, reactive oxygen species (ROS) production and phagocytosis were monitored using flow cytometry after 2 h cell incubation with live and dead B. ostreae. Two ratios of parasites per haemocyte were tested (5:1 and 10:1), haemocytes alone were used as controls and the experiment was carried out three times. Flow cytometry revealed a decrease of non specific esterase activities and ROS production by haemocytes after incubation with live parasites, while there was little difference in phagocytosis activity when compared with controls. Similarly, dead parasites induced a decrease in haemocyte activities but to a lesser extent compared to live parasites. These results suggest that B. ostreae actively contributes to the modification of haemocyte activities in order to ensure its own intracellular survival.