Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: a component of the P1/P2/P0 complex.

The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.     

Acidic ribosomal P proteins are phosphorylated in Trypanosoma cruzi.

Trypanosoma cruzi ribosomes from epimastigote forms were purified as determined by electron microscopy and isoelectrofocusing was used to analyse this purified ribosome fraction. Silver stained gels revealed that acidic proteins are present in at least 10 different isoforms, in accord with previous cloning studies. To detect phosphorylation, in vitro phosphorylation assays using the recombinant protein TcP2beta-mbp were carried out. The results showed that T. cruzi cytosolic fraction possesses protein kinase activity able to phosphorylate the recombinant protein. Purified ribosomes contain protein kinases that could also phosphorylate the recombinant protein TcP2beta-mbp. Labelling parasites with [(32)Pi] in a phosphate free medium demonstrated that ribosome proteins, recognised with a specific mouse antiserum against recombinant TcP2beta proteins, are phosphorylated in vivo. All these results suggest that in vivo phosphorylation of ribosome TcP2beta proteins are mediated by protein kinase(s) not yet identified.     

Valproate Increases Glutaminase and Decreases Glutamine Synthetase Activities in Primary Cultures of Rat Brain Astrocytes

Abstract: It has been proposed that hyperammonemia may be associated with valproate therapy. As astrocytes are the primary site of ammonia detoxification in brain, the effects of valproate on glutamate and glutamine metabolism in astrocytes were studied. It is well established that, because of compartmentation of glutamine synthetase, astrocytes are the site of synthesis of glutamine from glutamate and ammonia. The reverse reaction is catalyzed by the ubiquitous enzyme glutaminase, which is present in both neurons and astrocytes. In astrocytes exposed to 1.2 mM valproate, glutaminase activity increased 80% by day 2 and remained elevated at day 4; glutamine synthetase activity was decreased 30%. Direct addition of valproate to assay tubes with enzyme extracts from untreated astrocytes had significant effects only at concentrations of 10 and 20 mM, When astrocytes were exposed for 4 days to 0.3, 0.6, or 1.2 mM valproate and subsequently incubated with l -[U-¹⁴C]glutamate, label incorporation into [¹⁴C]glutamine was decreased by 11, 25, and 48%, respectively, and is consistent with a reduction in glutamine synthetase activity. Label incorporation from l -[U-¹⁴C]glutamate into [¹⁴C]aspartate also decreased with increasing concentrations of valproate. Following a 4-day exposure to 0.6 mM valproate, the glutamine levels increased 40% and the glutamate levels 100%. These effects were not directly proportional to valproate concentration, because exposure to 1.2 mM valproate resulted in a 15% decrease in glutamine levels and a 25% increase in glutamate levels compared with control cultures. Intracellular aspartate was inversely proportional to all concentrations of extracellular valproate, decreasing 60% with exposure to 1.2 mM valproate. These results indicate that valproate increases glutaminase activity, decreases glutamine synthetase activity, and alters Krebs-cycle activity in astrocytes, suggesting a possible mechanism for hyperammonemia in brain during valproate therapy.     

Immunocytochemical identification of murine and human megakaryocyte colonies in soft-agar cultures

Summary Murine megakaryocyte (MK) colonies in soft-agar cultures were immunocytochemically stained with platelet antiserum and an immuno-alkaline phosphatase procedure. Subsequently, cytochemical staining for acetylcholinesterase was used to confirm the specificity of the immunolabelling technique. The correlation of numbers of megakaryocyte colonies enumerated by independent observers was excellent. A comparable platelet antiserum directed against human platelet epitopes was utilized to identify human MK colonies in soft-agar cultures of human bone marrow. Using this method, we determined that the frequency of detectable human MK colonies in our agar culture system was maximal between days 10 and 12. The immunocytochemical staining technique we have developed for identification of MK colonies in soft-agar cultures yielded good cellular morphology and produced an intensely specific label against a clear background; it therefore facilitated accurate enumeration of MK colonies. This non-fluorescent method avoids dependence upon a non-permanent marker, and allows the simultaneous enumeration of positive and negative colonies.     

Near infrared spectral changes of cytochrome aa3 during potentiometric titrations.

Singular value decomposition (SVD) was used to deconvolute the spectral changes occurring in the near infrared region during potentiometric titrations of cytochrome aa3. Overall oxidized minus reduced difference spectra revealed a broad absorbance feature centered near 830 nm with an apparent Em near 250 mV. However, SVD did not isolate any spectral species with an absorbance centered near 830 nm. It was found that the spectral changes occurring in the wavelength region from 650 to 950 nm were associated mainly with cytochromes a and a3. It was concluded that the absorbance at 830 nm should not be used as an independent measure of the concentration of CuA in cytochrome aa3.     

Pulmonary perfusion in the prone and supine postures in the normal human lung.

Prone posture increases cardiac output and improves pulmonary gas exchange. We hypothesized that, in the supine posture, greater compression of dependent lung limits regional blood flow. To test this, MRI-based measures of regional lung density, MRI arterial spin labeling quantification of pulmonary perfusion, and density-normalized perfusion were made in six healthy subjects. Measurements were made in both the prone and supine posture at functional residual capacity. Data were acquired in three nonoverlapping 15-mm sagittal slices covering most of the right lung: central, middle, and lateral, which were further divided into vertical zones: anterior, intermediate, and posterior. The density of the entire lung was not different between prone and supine, but the increase in lung density in the anterior lung with prone posture was less than the decrease in the posterior lung (change: +0.07 g/cm(3) anterior, -0.11 posterior; P < 0.0001), indicating greater compression of dependent lung in supine posture, principally in the central lung slice (P < 0.0001). Overall, density-normalized perfusion was significantly greater in prone posture (7.9 +/- 3.6 ml.min(-1).g(-1) prone, 5.1 +/- 1.8 supine, a 55% increase; P < 0.05) and showed the largest increase in the posterior lung as it became nondependent (change: +71% posterior, +58% intermediate, +31% anterior; P = 0.08), most marked in the central lung slice (P < 0.05). These data indicate that central posterior portions of the lung are more compressed in the supine posture, likely by the heart and adjacent structures, than are central anterior portions in the prone and that this limits regional perfusion in the supine posture.     

Ribonucleoside Triphosphate-Dependent Pyrophosphate Exchange of Reovirus Cores

Reovirus cores catalyze a ribonucleoside triphosphate (rNTP)-dependent pyrophosphate exchange reaction in the presence of all four rNTP species. When rNTP species are tested individually, only guanosine-5'-triphosphate supports pyrophosphate exchange.     

Chloroplast Aldolase is Controlled by a Nuclear Gene

Variant chloroplast fructose 1,6-diphosphate aldolases were found in Pisum sativum when 10 commercial varieties were examined for electrophoretically distinct species of chloroplast triose phosphate isomerase, phosphoglyceric acid kinase, glyceraldehyde 3-phosphate dehydrogenase, and aldolase. When reciprocal crosses are made, both aldolases appear in individuals in the F(1) generation. Backcrossing gives offspring having aldolases characteristic of the homozygous or of the heterozygous parent; the inheritance is therefore not maternal but Mendelian. Clearly this chloroplast reductive pentose phosphate cycle enzyme is under nuclear gene control in P. sativum.     

Robustness of norm-driven cooperation in the commons

Sustainable use of common-pool resources such as fish, water or forests depends on the cooperation of resource users that restrain their individual extraction to socially optimal levels. Empirical evidence has shown that under certain social and biophysical conditions, self-organized cooperation in the commons can evolve. Global change, however, may drastically alter these conditions. We assess the robustness of cooperation to environmental variability in a stylized model of a community that harvests a shared resource. Community members follow a norm of socially optimal resource extraction, which is enforced through social sanctioning. Our results indicate that both resource abundance and a small increase in resource variability can lead to collapse of cooperation observed in the no-variability case, while either scarcity or large variability have the potential to stabilize it. The combined effects of changes in amount and variability can reinforce or counteract each other depending on their size and the initial level of cooperation in the community. If two socially separate groups are ecologically connected through resource leakage, cooperation in one can destabilize the other. These findings provide insights into possible effects of global change and spatial connectivity, indicating that there is no simple answer as to their effects on cooperation and sustainable resource use.