The pentose phosphate pathway in Trypanosoma cruzi

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).     

Insertion of externally administered amyloid beta peptide 25-35 and perturbation of lipid bilayers

To understand the molecular basis and to prevent diseases such as Alzheimer's disease (AD), the targets of the triggering agent have to be elucidated. beta-Amyloid peptide (Abeta) is the major component of extracellular senile plaques characteristic of AD. For a very long time, the aggregated form of the Abeta was supposed to be responsible for the neurodegeneration that occurs in AD. Recently, the attention has been diverted to the monomeric or oligomeric forms of Abeta and their interaction with cellular targets. In our investigation, the physiological and medically important insertion of externally applied Abeta monomers into the bilayer of lipid vesicles is demonstrated. Abeta(25-35) has been localized in the region of the lipid alkyl chain, and it has a severe disordering effect on the lamellar order of the lipid bilayer. Both of these results are of biomedical relevance.     

Identification and characterisation of a cDNA sequence encoding a glutamic acid-rich protein specifically transcribed in Trichinella spiralis newborn larvae and recognised by infected swine serum

Presently, little is known of the mechanism by which Trichinella penetrates and modulates reprogramming of muscle cells. In light of evidence demonstrating strong protective characteristics of antigens derived from this stage, understanding this process may shed light on potential targets for effective abatement of infection. To this end, a PCR-derived cDNA expression library was constructed using 0.5 micro g of total RNA from Trichinella spiralis newborn larvae. The library consisted of >125000 insert-containing clones. Approximately 40-50 x 10(3) clones were screened immunologically using sera from pigs experimentally infected with 7000 Trichinella L1. Multiple clones reacting positively with the swine infection serum and encoding portions of a glutamic acid-rich protein were identified. Northern and Southern blots indicated at least two distinct genes that encoded the glutamic acid-rich proteins and that these genes were transcribed specifically in the newborn larvae stage. cDNA sequence data predicted open reading frames of 1497 and 1,716 bp generating proteins of 498 amino acids and 571 amino acids, respectively. Both sequences consisted of approximately 39% glutamic acid and 16% serine residues, and differed by the presence of a 219 bp fragment present in the 1716 bp sequence that was absent from the 1497 bp sequence. PCR data indicated that additional isoforms exist within this gene family that are different in length from those described above. In addition, it was found that more than one isoform can exist within a single worm and that this pattern can vary between individual worms within a population. Mouse antibodies to recombinant antigen localised the glutamic acid-rich proteins to the periphery of the developing stichocyte cells within the newborn larvae consistent with the hypothesis that the newborn larval antigens are secreted.     

The relationship of hydrophobic tubulin with membranes in neural tissue

Brain membrane preparations contain tubulin that can be extracted with Triton X-114. After the extract is allowed to partition, 8% of the total brain tubulin is isolated as a hydrophobic compound in the detergent-rich phase. Cytosolic tubulin does not show this hydrophobic behaviour since it is recovered in the aqueous phase. Membrane tubulin can be released by 0.1 M Na₂CO₃ treatment at pH11.5 in such a way that the hydrophobic tubulin is converted into the hydrophilic form. These results suggest that tubulin exists associated with some membrane component that confers the hydrophobic behaviour to tubulin. If the tissue is homogenized in microtubule-stabilizing buffer containing Triton X-100, the hydrophobic tubulin is isolated from the microtubule fraction. This result indicates that the hydrophobic tubulin isolated from membrane preparations belongs to microtubules thatin vivo are associated to membranes. Therefore, hydrophobic tubulin (tubulin-membrane component complex) can be obtained from membranes or from microtubules depending on the conditions of brain homogenization.Abbreviations TBS Tris-buffered saline - Mes 2-(N-morpholine) ethane sulfonic acid     

High resolution nanomechanical characterization of multi‐domain model membranes by fast Force Volume

Plasma membrane is a complex structure, mainly composed by lipids and proteins, which plays a pivotal role in cell metabolism by regulating its selective permeability to ions and molecules. According to the “raft hypothesis”, lipids in the bilayer are not forming a structurally passive solvent, but are rather organized in specific domains, which present different structural and functional characteristics. The mechanical properties of the lipid part of plasma membrane have been recently characterized through Atomic Force Microscopy, by analyzing the features of force vs distance curves collected on supported lipid bilayers (SLBs). In case of lipid domains sizing from tens to hundreds of nanometers, which mimic in a good way the lateral organization of real membranes, a high lateral resolution and a large number of curves are often required for properly expressing the complexity of the system, with a consequent exponential growth of acquisition and processing time. In this paper we propose a method, based on a recently developed high speed Force Volume technique and on home‐built data processing software, for the mechanical characterization of nanostructured SLBs. With our software we have been able to process data set composed by tens of thousands of curves, collected with a spatial resolution ranging from 8 to 40 nm/pixel. Multiparametric maps and distribution histograms produced by our analysis allowed identifying a specific behavior for each lipid phase in the investigated model membranes, even in presence of nanosized features. Copyright © 2015 John Wiley & Sons, Ltd.     

Simplification of the tetracyclic SIRT1-selective inhibitor MC2141: coumarin- and pyrimidine-based SIRT1/2 inhibitors with different selectivity profile

In this report we describe the synthesis and biological characterization of two series of sirtuins' inhibitors (SIRTi), designed as simplification products of the previously reported SIRT1-selective inhibitor MC2141 (4). In the first series (5a-t) we report a number of 2-substituted-1,2-dihydrobenzo[f]chromen-3-ones with a marked selectivity for the inhibition of SIRT2 over SIRT1. Some of such derivatives showed also high pro-apoptotic (5i and 5l) and/or cytodifferentiating (5d, 5i, and 5o) properties in a human leukemia cell line (U937). The second group of SIRTi (6a-q) is characterized by some analogues of cambinol (3), a well known SIRTi active against the Burkitt lymphoma. Such compounds, differently from the unselective prototype, are endowed with a selective inhibition of SIRT1 over SIRT2, and, in some cases (6j, 6k, and 6q), are more efficient than 3 to induce apoptosis in U937 cells.     

Influence of pH, temperature, and urea molar flowrate on Arthrospira platensis fed-batch cultivation: a kinetic and thermodynamic approach

Arthrospira platensis was cultivated photoautotrophically at 6.0 klux light intensity in 5.0-L open tanks, using a mineral medium containing urea as nitrogen source. Fed-batch experiments were performed at constant flowrate. A central composite factorial design combined to response surface methodology (RSM) was utilized to determine the relationship between the selected response variables (cell concentration after 10 days, X(m), cell productivity, P(X), and nitrogen-to-cell conversion factor, Y(X/N)) and codified values of the independent variables (pH, temperature, T, and urea flowrate, K). By applying the quadratic regression analysis, the equations describing the behaviors of these responses as simultaneous functions of the selected independent variables were determined, and the conditions for X(m) and P(X) optimization were estimated (pH 9.5, T = 29 degrees C, and K = 0.551 mM/day). The experimental data obtained under these conditions (X(m) = 749 mg/L; P(X) = 69.9 mg/L.day) were very close to the estimated ones (X(m) = 721 mg/L; P(X) = 67.1 mg/L.day). Additional cultivations were carried out under the above best conditions of pH control and urea flowrate at variable temperature. Consistently with the results of RSM, the best growth temperature was 29 degrees C. The maximum specific growth rates at different temperatures were used to estimate the thermodynamic parameters of growth (DeltaH* = 59.3 kJ/mol; DeltaS* = -0.147 kJ/mol.K; DeltaG* = 103 kJ/mol) and its thermal inactivation (DeltaH(D) (o) = 72.0 kJ/mol; DeltaS(D) (o) = 0.144 kJ/mol.K; DeltaG(D) (o) = 29.1 kJ/mol).