Lello Zolla

miR-143 regulates hexokinase 2 expression in cancer cells

Tumor cells activate pathways that facilitate and stimulate glycolysis even in the presence of adequate levels of oxygen in order to satisfy their continuous need of molecules, such as nucleotides, ATP and fatty acids, necessary to support their rapid proliferation. Accordingly, a variety of human tumors are characterized by elevated expression levels of the hexokinase 2 isoform (HK2). Although different molecular mechanisms, including genetic and epigenetic mechanisms, have been suggested to account for the altered expression of HK2 in tumors, the potential role of microRNAs (miRNAs) in the regulation of HK2 expression has not been evaluated. Here, we report that miR-143 inhibits HK2 expression via a conserved miR-143 recognition motif located in the 3′-untranslated region (3′UTR) of HK2 mRNA. We demonstrate that miR143 inhibits HK2 expression both in primary keratinocytes and in head and neck squamous cell carcinoma (HNSCC)-derived cell lines. Importantly, we found that miR-143 inversely correlates with HK2 expression in HNSCC-derived cell lines and in primary tumors. We also report that the miRNA-dependent regulation of hexokinase expression is not limited to HK2 as miR-138 targets HK1 via a specific recognition motif located in its 3′UTR. All these data unveil a new miRNA-dependent mechanism of regulation of hexokinase expression potentially important in the regulation of glucose metabolism of cancer cells.

Interaction of hemoglobin with salts: Effects on the functional properties of human hemoglobin

Abstract Oxygen isotherms of human hemoglobin measured in distilled water and in solutions of different inorganic salts in the concentration range from below 10−3 m to above 1·5 m at neutral pH indicate that the oxygen affinity decreases with increasing salt concentration in the lower range of ionic strength; above the physiological range, there is in most cases a further decrease in oxygen affinity, but this varies with the nature of the salt and, in some instances, the affinity goes through a maximum. The effect of cations, which is opposite to that of anions, operates primarily in the higher concentration range; i.e. above 0·1 m. This effect is especially large for Li+, Ca2+ and Mg2+. The alkaline Bohr effect depends strongly on anion concentration, being displaced towards higher pH values and being reduced in magnitude as chloride concentration is increased. On the other hand, the acid Bohr effect, observed below pH 6, appears to be independent of chloride concentration from 6 × 10−2 m to 2 m. The overall heat of oxygenation has been determined for the isoionic protein as well as at different concentrations of chloride and phosphate. The average intrinsic heat of reaction of hemoglobin with oxygen in solution is found to be −14·6 kcal/mol of O2.

Storing red blood cells with vitamin C and N-acetylcysteine prevents oxidative stress-related lesions: a metabolomics overview

BACKGROUND Recent advances in red blood cell metabolomics have paved the way for further improvements of storage solutions. MATERIALS AND METHODS In the present study, we exploited a validated high performance liquid chromatography-mass spectrometry analytical workflow to determine the effects of vitamin C and N-acetylcysteine supplementation (anti-oxidants) on the metabolome of erythrocytes stored in citrate-phosphate-dextrose saline-adenine-glucose-mannitol medium under blood bank conditions. RESULTS We observed decreased energy metabolism fluxes (glycolysis and pentose phosphate pathway). A tentative explanation of this phenomenon could be related to the observed depression of the uptake of glucose, since glucose and ascorbate are known to compete for the same transporter. Anti-oxidant supplementation was effective in modulating the redox poise, through the promotion of glutathione homeostasis, which resulted in decreased haemolysis and less accumulation of malondialdehyde and oxidation by-products (including oxidized glutathione and prostaglandins). DISCUSSION Anti-oxidants improved storage quality by coping with oxidative stress at the expense of glycolytic metabolism, although reservoirs of high energy phosphate compounds were preserved by reduced cyclic AMP-mediated release of ATP.

Effect of mercuric ions on human erythrocytes. Relationships between hypotonic swelling and cell aggregation.

This study was undertaken to verify the hypothesis that the haemolytic effect of mercuric ions on human erythrocytes is strongly decreased under swelling conditions (relative to isotonic suspensions). In fact, interaction of Hg2+ with swollen erythrocytes yields a rapid and cooperative cell aggregation, a phenomenon that appears to prevent penetration of mercuric ions into the cells and, accordingly, to avoid any haemolytic effect induced by the Hg2+ entrance. Since in vivo erythrocytes undergo big shape changes (swelling being a kind of shape modification) related to mechanical or (in some animals) osmotic stresses, the reported observations turn out to be also of some relevance for the understanding of certain toxicological effects of mercuric ions.

Kinetic and thermodynamic parameters for oxygen binding to the allosteric states of panulirus interruptus hemocyanin

The temperature dependence of the oxygen binding equilibria and kinetics of Panulirus interruptus hemocyanin has been analyzed within the context of the two-state allosteric model. Oxygenation of the T-state is characterized by a more negative value of DeltaH than that of the R-state; therefore, cooperative effects in oxygen binding to P. interruptus hemocyanin are thermodynamically governed by favorable entropy changes. The allosteric transition in the unliganded derivative shows an enthalpy-entropy compensation effect. The activation enthalpies for oxygenation and deoxygenation of the T-state are larger than those for the R-state, while the activation entropies are favorable for the T-state and unfavorable for the R-state. Thus, the activation free energies for oxygen binding to the T- and R-states are similar, while for the deoxygenation reaction DeltaG++ is smaller for the T-state. The analysis reported confirms the applicability of the Monod-Wyman-Changeux two-state allosteric model to P. interruptus hemocyanin and yields a complete thermodynamic characterization of oxygen binding under both equilibrium and dynamic regimes.

Clinical Metabolomics: the next stage of clinical biochemistry

Centuries of scientific advances have paved the way for the relatively recent great strides in clinical biochemistry, a field which mainly relies upon biochemical analyses of various body fluids, prime amongst which are urine1, blood and cerebrospinal fluid2. Technological innovation, through the introduction of cutting edge instrumentation has enabled decades of substantial improvements in the field of standard analytical chemistry in the clinical setting. At its dawn, clinical biochemistry relied on rudimental approaches, such as in the case of Richard Bright’s (1789–1858) test for proteinuria in cases of suspected renal disease, in which a candle flame was used to heat urine in a tablespoon3. Only minor technological improvements could date back to the early twentieth century. As reported by Olukoga et al.4, the equipment of a clinical pathology laboratory within a 200-bedded American hospital in 1920 listed “a centrifuge, a urinometer, two monocular microscopes, two small substage microscope lights, a Bunsen burner, a Dubosq colorimeter, a basal metabolic rate machine, an electro-cardiograph, a microtome, a knife, a paraffin bath, a few antisera and an assortment of test tubes, beakers and pipettes”.

Reaction of carbon monoxide with hemocyanin: Stereochemical effects of a non-bridging ligand

Abstract The carbon monoxide binding equilibria and kinetics of a number of molluscan and arthropodal hemocyanins have been investigated employing the visible luminescence of the carbon monoxide-copper complex. Proteins from both phyla, in oligomeric and monomeric form, bind carbon monoxide non-co-operatively; the reaction is largely enthalpy driven is associated with a small unfavourable entropy change. Molluscan hemocyanins display a carbon monoxide affinity (p50 = 1 to 10mm Hg) higher than that of arthropodal hemocyanins (p50 = 100 to 700mm Hg), and only Panulirus interruptus hemocyanin, among those studied here, exhibits a small Bohr effect. The observed differences in equilibrium constant are kinetically reflected in differences in the carbon monoxide dissociation rate constant, which ranges from 20 to 70 s−1 for molluscan hemocyanins and from 200 to 9000 s−1 for arthropodal hemocyanins; on the other hand the differences in the combination rate constants between the two phyla are considerably smaller. A comparison of the equilibrium and kinetic results shows some discrepancies between the two sets of data, suggesting that carbon monoxide binding may be governed by a complex mechanism. The correlation between the ligand binding properties and the stereochemistry of the active site is discussed in the light of the knowledge that, while oxygen is bound to both copper atoms in a site, carbon monoxide is a “non-bridging” ligand, being bound to only one of the metals.

Encapsulation of proteins into human erythrocytes : a kinetic investigation

Moderate osmotic shocks of human erythrocytes by hypotonic dialysis (0.06 mosmol/kg) induce cell swelling and formation of pores, without causing apparent lysis. Using 125I-labeled macromolecules of different molecular weight and net charge, we followed the kinetics and efficiency of their encapsulation into erythrocytes. After a 20-30 min period of cell dialysis, macromolecules of up to 50 kDa begin diffusing into the swollen cells by a process which can be described by a first-order two-compartment kinetics. Adsorption to the external cell surface was insignificant, while adsorption to the inner membrane surface was substantial (15-20%) only for positively charged proteins, at physiological pH. After resealing, pores of a 12-14 kDa cut-off might remain open allowing some release of entrapped material (20-30%), depending on the final cytocrit, while the remaining might be associated with inner membrane or cytosolic components. Although the method of hypotonic dialysis is known to affect minimally the biophysical and immunological properties of red blood cell membranes, the interaction of encapsulated material with cell constituents would need to be further assessed when considering red cells as macromolecular carriers.

Hemocyanin from Palinurus elephas: general properties and effects of heavy metals

Abstract The structural and functional properties of hemocyanin from the lobster Palinurus elephas indicate that this protein is similar to that of Panulirus interruptus , as expected on the basis of the phylogenetic relatedness of the two species. The process of (re)association of subunits into hexamers has been studied by ultracentrifuge and stopped-flow techniques; complete reassociation requires both neutral pH and presence of calcium ions and appears to be a relatively fast process (time-scale of 1 min). The effect of three heavy metals (Hg, Cd and Cr) on the structural and functional properties of this protein has been investigated in order to ascertain whether hemocyanin may be a target of metal poisoning. At low concentrations the three metals are effective in modifying (reversibly) the functional properties of the protein, although each metal induces different modifications. Very high concentrations of Cr and Hg, and long incubation times result in the removal of copper from the active site, while Cd seems unable to do so.

Root effect of Panulirus interruptus hemocyanin.

Panulirus interruptus hemocyanin exhibits a progressive decrease in oxygen affinity and a parallel loss of cooperativity with decrease in pH, resulting in an apparent loss of the oxygen-binding capacity of the protein. For a characterization of this system, oxygen-binding curves have been determined over the complete range of oxygen saturation, applying a special technique which involves high-pressure spectrophotometry. Although the oxygen-binding behavior as a function of pH is complex and cannot be described within the frame of a simple two-state Monod-Wyman-Changeux model, the observed Root effect is clearly related to a progressive stabilization of a low oxygen affinity state of the protein and functional heterogeneity is not apparent.