Mechanism of mitochondrial carbamoyl-phosphate synthetase: synthesis and properties of active CO2, precursor of carbamoyl phosphate.

This paper demonstrates the formation of "active CO2" (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase. Absence of ammonia is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. Adenosine triphosphate (ATP) and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 = 0.75 s at 24 degrees C at neutral pH). In the absence of ammonia, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The "apparent KM'S" are approximately equal to those found for the adenosine triphosphate (ATPase) activity of the enzyme. The maximum level of CO2-P is limited by the amount of enzyme, and approximates 4 mol of intermediate/mol of enzyme. The unprotonated form of ammonia seems to be the species reacting with CO2-P to produce CP. The reaction of CO2-P and NH3 is very fast (rate constant kn = 8 x 10(4) M-1 S-1) and does not consume free ATP. Therefore, the 2 mol of ATP necessary for CP synthesis binds or reacts with the enzyme and/or CO2 prior to reaction with NH3. The reaction of CO2-P with NH3 also takes place in acetone under conditions at which the enzyme is not active, suggesting little or no assistance from enzyme catalysis or that a part of the catalytic site is "frozen" by the solvent in the active conformation. In the light of these and other findings, a new scheme is proposed for the mechanism of frog liver carbamoyl-phosphate synthetase and some considerations are made on the chemical nature of the intermediate and on the possible evolutionary significance of the reaction of CO2-P with NH3 in acetone.     

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medullapons or the cerebellum. Since G4 forms have been proposed to be present presynaptically, their age-related loss in those brain areas where acetylcholine plays an important role in neurotransmission may indicate an impairment of presynaptic mechanisms.     

Modular structure of the human lamin B2 replicator

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation.     

Micronuclei monitoring of fishes from Lake Paranoá, under influence of sewage treatment plant discharges

Fish micronuclei tests (MN) were used to evaluate the ability of wastewater from two municipal sewage treatment plants that empty into Lake Paranoá to cause genetic damage. There were no significant differences in the frequencies of micronuclei between the control and hypertrophic areas. In contrast, cyclophosphamide and mitomycin C, used to test the sensitivity of the biological assay, significantly increased the micronuclei counts in Tilapia rendalli, Oreochromis niloticus and Cyprinus carpio, T. rendalli was the most sensitive specie to both clastogens and C. carpio, the most resistant.     

Dietary Triacylglycerols with Palmitic Acid in the sn-2 Position Modulate Levels of N-Acylethanolamides in Rat Tissues

Background

Several evidences suggest that the position of palmitic acid (PA) in dietary triacylglycerol (TAG) influences different biological functions. We aimed at evaluating whether dietary fat with highly enriched (87%) PA in sn-2 position (Hsn-2 PA), by increasing PA incorporation into tissue phospholipids (PL), modifies fatty acid profile and biosynthesis of fatty acid—derived bioactive lipids, such as endocannabinoids and their congeners.

Study Design

Rats were fed for 5 weeks diets containing Hsn-2 PA or fat with PA randomly distributed in TAG with 18.8% PA in sn-2 position (Lsn-2 PA), and similar total PA concentration. Fatty acid profile in different lipid fractions, endocannabinoids and congeners were measured in intestine, liver, visceral adipose tissue, muscle and brain.

Results

Rats on Hsn-2 PA diet had lower levels of anandamide with concomitant increase of its congener palmitoylethanolamide and its precursor PA into visceral adipose tissue phospholipids. In addition, we found an increase of oleoylethanolamide, an avid PPAR alpha ligand, in liver, muscle and brain, associated to higher levels of its precursor oleic acid in liver and muscle, probably derived by elongation and further delta 9 desaturation of PA. Changes in endocannabinoids and congeners were associated to a decrease of circulating TNF alpha after LPS challenge, and to an improved feed efficiency.

Conclusions

Dietary Hsn-2 PA, by modifying endocannabinoids and congeners biosynthesis in different tissues may potentially concur in the physiological regulation of energy metabolism, brain function and body fat distribution.     

Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies

Background aimsBone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34⁺-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI.MethodsThe need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34⁺ cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34⁺ cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells.ResultsGene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14⁺ subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively.ConclusionsOur data suggest that combining basal and expanded BM-derived CD34⁺ cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.     

A step further in the analysis of human bile proteome

Bile was shown to collect proteins known as potential cancer biomarkers. Thorough proteomic analysis of bile is of particular interest to search for new, more sensitive and more specific, biomarkers of cancers affecting the biliary tract and surrounding organs, such as the pancreas and the liver. Therefore, extending the knowledge of the bile proteome is highly relevant, but this has proved technically difficult. In this study, we describe a strategy that circumvents problems related to the biochemical complexity of this sample and the presence of high concentrations of interfering substances. Bile collected from a patient suffering from a biliary stenosis caused by a pancreatic adenocarcinoma was fractionated by a differential centrifugation scheme, involving a stepwise increase in centrifugation speeds. Pellets and the final supernatant were further fractionated by polyacrylamide gel electrophoresis and proteins were in-gel digested prior to LC-MS/MS analysis. This approach allowed the identification of 445 unique proteins with at least two peptides (812 proteins if single-hit proteins were included), which represents a 3-fold increase in the knowledge of bile proteome. The subsequent literature comparison revealed that numerous biliary proteins identified in this sample were related to pancreas cancer. Immunoblot analysis of some known tumor markers revealed that they were preferentially associated with the soluble fraction rather than with pellets containing cellular components.