Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

Identification of a Wee1–Like Kinase Gene Essential for Procyclic Trypanosoma brucei Survival

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.     

Effect of insulin on exocrine pancreatic secretion in healthy and diabetic anaesthetised rats

This investigation characterised the effects of exogenous insulin on exocrine pancreatic secretion in anaesthetised healthy and diabetic rats. Animals were rendered diabetic by a single injection of streptozotocin (STZ, 60 mg kg(-1) I.P.). Age-matched controls were injected citrate buffer. Rats were tested for hyperglycaemia 4 days after STZ injection and 7-8 weeks later when they were used for the experiments. Following anaesthesia (1 g kg(-1) urethane I.P.), laparotomy was performed and the pancreatic duct cannulated for collection of pure pancreatic juice. Basal pancreatic juice flow rate in diabetic rats was significantly (p < 0.001) increased whereas protein and amylase outputs were significantly (p < 0.001) decreased compared to control rats. Insulin (1 IU, I.P.) produced in healthy rats significant increases in pancreatic flow rate, amylase secretion and protein output compared to basal (p < 0.05). Insulin action also included a reduction in blood glucose (152.7 +/- 16.9 mg dl(-1), n = 6, prior to insulin and 42.0 +/- 8.4 mg dl(-1), n = 4, 100 min later). In fact, flow rate and glycaemia showed a strong negative correlation (p < 0.01, Pearson). Pretreatment with atropine (0.2 mg kg(-1), I.V.) abolished the effects of insulin on secretory parameters despite a similar reduction in glycaemia; in this series of experiments the correlation between flow rate and blood glucose was lost. In diabetic rats, insulin (4 IU, I.P.) did not modify exocrine pancreatic secretion. There was a fall in blood glucose (467.6 +/- 14.0 mg dl(-1), n = 10, prior to insulin and 386.6 +/- 43.6 mg dl(-1), n = 7, 120 min later). Rats, however, did not become hypoglycaemic. Similar results were observed in diabetic atropinized rats. The results of this study indicate that the effects of insulin on exocrine pancreatic secretion in anaesthetised healthy rats are mediated by hypoglycaemia-evoked vagal cholinergic activation.     

Plastic encapsulation of stabilized Escherichia coli and Pseudomonas putida

Escherichia coli and Pseudomonas putida dried in hydroxyectoine or trehalose are shown to be highly resistant to the organic solvents chloroform and acetone, and consequently, they can be encapsulated in a viable form in solid plastic materials. Bacteria are recovered by rehydration after physical disruption of the plastic. P. putida incorporated into a plastic coating of maize seeds was shown to colonize roots efficiently after germination.     

Relative value of light and food as synchronizers of liver phosphorylase circadian rhythm.

The role played by light and feeding schedules on the circadian rhythm of glycogen content and phosphorylase activity of the liver has been studied. In one experiment, mice were subjected to a regimem of constant darkness during 21 days and compared with mice kept in 12 hrs of light alternating with 12 hrs of darkness. Both groups received food and water ad libitum. Liver glycogen content as well as phosphorylase activity showed, with slight differences, similar circadian variations. In a second experiment, mice under similar lighting conditions (LD 12:12), with water access ad libitum, were divided into two groups; one was offered food ad libitum while the other group recieved food from 0700 to 1800 only. This experiment allowed up to compare two different schedules of food intake; ad libitum, normal schedule (from 1800 to 0600) and reversed schedule (from 0700 to 1800). A complete reversal of the circadian rhythm was observed after 21 days in the group with the reverted feeding schedule. We conclude that food can function as the primary synchronizer in spite of the lighting regimen.     

Formation and stabilization of diacetyl and acetoin concentration in fully grown cultures ofStreptococcus lactis subsp. diacetylactis

Summary The effects of pH and temperature on diacetyl and acetoin concentration and diacetyl:acetoin ratio evolution of non-growing cells ofStreptococcus lactis subsp. diacetylactis CNRZ 124 were studied. A cooling down to 10°C allowed the cells to retain 6 to 10 times as much diacetyl. A large reduction of acetoin was promoted at pH 7 whereas a twice increase was observed at pH 4,8. As a result of these variations, the ratio diacetyl: acetoine showed an opposite evolution according to the pH.     

Influence of citrate on the diacetyl and acetoin production by fully grown cells ofStreptococcus lactis subsp.diacetylactis

The effects of citrate on diacetyl and acetoin level by fully grown cells ofStreptococcus lactis subsp.diacetylactis CNRZ 124 were studied. In the absence of citrate, diacetyl synthase as well as acetolactate synthase and acetoin and diacetyl reductases exhibited a basal activity confirming their constitutive nature. However, when initial citrate concentration ranged from 8.8 to 59 mM, the enzyme levels increased in the same way, indicating no saturation rate of citrate metabolism. These results were reflected by a similar enhancement in acetoin and diacetyl production. When citrate was added in fed-batch conditions, its utilization by the fully grown cells led to a twofold increase in diacetyl yield over batch conditions.     

TGGE analysis of the diversity of ammonia-oxidizing and denitrifying bacteria in submerged filter biofilms for the treatment of urban wastewater

The spatial and temporal diversity of the bacterial community-forming biofilms in a pilot-scale submerged biofilter used for the treatment of urban wastewater was analyzed by a temperature-gradient gel electrophoresis (TGGE) approach. TGGE profiles based on partial sequence of the 16S rRNA gene showed that the community composition of the biofilms remained fairly stable along the column system and during the whole time of operation of the biofilter (more than 1 year). Community-profiling based on the amplification and separation of partial ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) genes demonstrated that ammonia-oxidizing and denitrifying bacteria coexisted in both the anoxic and the aerated parts of the system. Several amoA and nosZ bands separated by TGGE were reamplified and sequenced, in order to further analyze the composition of these microbial communities in the biofilm. Phylogeny inferred from amoA/AmoA revealed the prevalence of Nitrosomonas species with five sequences affiliated to Nitrosomonas oligotropha, six sequences affiliated to Nitrosomonas europaea, and three sequences that showed only 75.7–76.1% identity of the DNA sequence with the closest described species (Nitrosomonas nitrosa). According to literature, this low identity value is indicative of previously undiscovered species. Eighteen new partial nosZ sequences were obtained which were mostly related to nosZ of gamma-proteobacteria (Pseudomonas) or clustered in the periphery of previously known denitrifying alpha-proteobacteria (Bradyrhizobium and Azospirillum).     

Carbonate Precipitation of Bacterial Strains Isolated from Sediments and Seawater: Formation Mechanisms

This article presents a research study on carbonate formation in solid and liquid media by Thalassospira sp., Halomonas sp., Bacillus pumilus, and Pseudomonas grimontii, four bacterial strains isolated from sediments and deep seawater. As part of this study, we analyzed carbonic anhydrase activity, pH, adsorption of calcium and magnesium ions, and total organic and inorganic carbon. The geochemical program PHREEQC was also used to calculate the mineral saturation indexes in all the cultures. The minerals formed were studied with X-ray diffraction, X-ray dispersive energy microanalysis, and scanning electron microscopy. In addition, all four bacterial strains were found to induce carbonate precipitation and to have carbonic anhydrase activity. Sterile control experiments did not precipitate carbonate. In solid M1 and B4 media, all of the strains precipitated magnesium calcite, whereas in the liquid media, they precipitated different percentages of magnesium calcite, aragonite, and monohydrocalcite. In both cases, small amounts of amorphous precipitates were also produced. This article discusses carbonate formation and the possible role played by metabolic activity, bacterial surfaces and carbonic anhydrase in this process. Finally, the results obtained lead to a hypothesis regarding the importance of carbonate precipitation for the survival of bacteria populations in certain habitats.