Giant Pressure Dependence and Dimensionality Switching in a Metal-Organic Quantum Antiferromagnet.

We report an extraordinary pressure dependence of the magnetic interactions in the metal-organic system [CuF_{2}(H_{2}O)_{2}]_{2}pyrazine. At zero pressure, this material realizes a quasi-two-dimensional spin-1/2 square-lattice Heisenberg antiferromagnet. By high-pressure, high-field susceptibility measurements we show that the dominant exchange parameter is reduced continuously by a factor of 2 on compression. Above 18 kbar, a phase transition occurs, inducing an orbital re-ordering that switches the dimensionality, transforming the quasi-two-dimensional lattice into weakly coupled chains. We explain the microscopic mechanisms for both phenomena by combining detailed x-ray and neutron diffraction studies with quantitative modeling using spin-polarized density functional theory.

Influence of gemfibrozil on sulfate transport in human erythrocytes during the oxygenation-deoxygenation cycle.

The effects of gemfibrozil (GFZ), an antihyperlipidemic agent, on the anionic transport of the human red blood cells (RBC) during the oxygenation-deoxygenation cycle were examined. Gemfibrozil clearly plays a role in the modulation of the anionic flux in erythrocytes; in fact it causes a strong increment of anions transport when the RBCs are in the high-oxygenation state (HOS). Such an effect is remarkably reduced in the low-oxygenation state (LOS). With the aim of identifying the dynamics of fibrate action, this effect has been investigated also in human ghost and chicken erythrocytes. These latter, in fact, are known to possess a B3 (anion transporter or Band 3) modified at the cytoplasmic domain (cdb3) which plays a significant role in the metabolic modulation of red blood cells. The results were analyzed taking into account the well-known interactions between fibrates and both conformational states of hemoglobin i.e. the T state (deoxy-conformation) and the R state (oxy-conformation). The effect of gemfibrozil on anionic influx appears to be due to a wide interaction involving a "multimeric" Hb-GFZ-cdb3 macromolecular complex.

A two-dimensional electrophoresis preliminary approach to human hepatocarcinoma differentiation induced by PPAR-agonists.

Adopting biochemical and proteomic approaches, we investigated the effect of some PPAR-agonists, a new class of differentiating agents, on human hepatocellular carcinoma Hep-G2 cell line. Cancer differentiation was assayed by checking albumin, transferrin and alpha-fetoprotein synthesis. Cell metabolism was studied by NMR spectroscopy of cell culture supernatants and by evaluation of mitochondrial respiratory chain enzyme activities. The two dimensional electrophoresis approach was employed to analyze modifications in the expression of cellular proteins linked to cell phenotype differentiation in the attempt to identify potential diagnostic and prognostic biomarkers of hepatocellular carcinoma. Results indicate that PPAR-agonists are able to act as differentiating inducers in human hepatocellular carcinoma Hep-G2 cell line as well as to inhibit mitochondrial respiratory chain Complex I, provoking a selective derangement of cellular oxidative metabolism. Lastly, two dimensional electrophoresis showed interesting modifications in the pattern of expression of cellular proteins that confirm biochemical data (increase in albumin and transferrin, decrease of alpha-fetoprotein synthesis) and, moreover, emphasize the meaning of these data by the increase of spots indicatively ascribed to HSP70 and catalase.

Bezafibrate induces a mitochondrial derangement in human cell lines: a PPAR-independent mechanism for a peroxisome proliferator.

Bezafibrate is a hypolipidemic drug that belongs to the group of peroxisome proliferators because it binds to peroxisome proliferator-activated receptors type alpha (PPARs). Peroxisome proliferators produce a myriad of extraperoxisomal effects, which are not necessarily dependent on their interaction with PPARs. An investigation on the peculiar activities of bezafibrate could clarify some of the molecular events and the relationship with the biochemical and pharmacological properties of this class of compounds. In this view, the human acute promyelocytic leukemia HL-60 cell line and human rabdomiosarcoma TE-671 cell line were cultured in media containing bezafibrate and a number of observations such as spectrophotometric analysis of mitochondrial respiratory chain enzymes, NMR metabolite determinations, phosphofructokinase enzymatic analysis, and differentiation assays were carried on. Bezafibrate induced a derangement of NADH cytochrome c reductase activity accompanied by metabolic alterations, mainly a shift to anaerobic glycolysis and an increase of fatty acid oxidation, as shown by NMR analysis of culture supernatants where acetate, lactate, and alanine levels increased. On the whole, the present results suggest a biochemical profile and a therapeutic role of this class of PPARs ligands more complex than those previously proposed.

Cytotoxicity of a new endodontic filling material.

In vitro cell cultures have been widely used as a means of evaluating cytotoxicity of root canal filling materials. Following ANSI/ADA spec. no. 41, the aim of the present study was to investigate the biological compatibility of a new sealer (FibreFill) and compare it with some commercially available endodontic sealers (Bioseal and Acroseal). Mouse 3T3 fibroblasts were seeded and cultured and subsequently extracts of the cements were added. After 24 hours incubation, the cellular vitality of fibroblasts was evaluated by the neutral red uptake test (NRU), which measures the membrane permeability. Data were collected and statistically analysed. Results showed that all tested materials exhibited mild cytotoxic effects, which are compatible with normal clinical use, and no statistically significant difference was noted between FibreFill and the other tested materials. Therefore, selection amongst these sealers should be based on other factors.

O-raffinose-polymerised haemoglobin. A biochemical and pharmacological profile of an oxygen carrier.

Haemoglobin-based oxygen carriers (HBOCs) represent an interesting class of blood substitutes which are undergoing advanced clinical trials. The therapeutic goal of these compounds is to avoid or reduce blood transfusion in different surgical and medical situations of acute haemoglobin deficiency. Their main advantages include availability in large volumes, storage for prolonged periods, rapid administration (without typing and cross matching) and sterilisation by pasteurisation. Their main known disadvantages are reduced circulation half-life, haemodynamic and gastrointestinal perturbations, probably related to nitric oxide (NO) scavenging, free radical induction, and alterations of biochemical and haematological parameters (increases in liver enzymes levels, platelet aggregation). Cell-free o-raffinose cross-linked and oligomerised human haemoglobin (O-r-poly-Hb) (Hemolink, Hemosol, Canada) is a modified haemoglobin with molecular weight ranging from 32- > 500 kDa. Its affinity for oxygen appears lower than normal blood and an n (Hill coefficient) value of about 1 indicates a very low degree of co-operativity. Probably related to the low O2 affinity value and to the high molecular weight, O-r-poly-Hb has been shown to induce lesser haemodynamic perturbations than other first generation modified haemoglobins. This HBOC is in Phase III clinical trials in cardiac and orthopaedic surgery for perioperative haemodilution, at doses from 25 g (250 ml)-100 g (1000 ml).

Bivalirudin: a new generation antithrombotic drug.

Bivalirudin (Angiomax, The Medicines Company) is a synthetic 20 amino acid peptide rationally designed on the basis of structural studies of hirudin, a naturally occurring anticoagulant. Bivalirudin represents a new class of anticoagulant drugs that directly inhibits thrombin, a key component in blood clot formation and extension. With its high binding affinity and specificity for thrombin, bivalirudin acts directly on thrombin, rather than via other clotting factors. The compound has a variety of potential uses as an alternative to heparin in the management of cardiovascular disease and related medical procedures i.e., unstable angina (UA), myocardial infarction (MI) and percutaneous transluminal coronary angioplasty (PTCA).

Prinomastat, a hydroxamate-based matrix metalloproteinase inhibitor. A novel pharmacological approach for tissue remodelling-related diseases.

Prinomastat (formerly AG3340, Agouron Pharmaceuticals, Inc.) is a potent, selective oral inhibitor of matrix metalloproteinase-2, -9, -13 and -14. This peculiar selectivity should represent an advantage for prinomastat in terms of efficacy/tolerability. The drug has been shown to inhibit tumour growth and angiogenesis in a variety of preclinical models, including cancer of colon, breast, lung and intriguingly in melanoma and glioma models. Moreover, the combination of prinomastat and several chemotherapeutic agents was shown to induce additive effects. The drug is currently in Phase III clinical trials for patients with non-small cell lung cancer in combination with paclitaxel and carboplatin, as well as in advanced hormone refractory prostate cancer in combination with mitoxantrone. The most common side effects are musculoskeletal pain and stiffness. These side effects generally cease with treatment interruption. Finally, considering the pathophysiology of MMPs, Agouron is exploring the utility of prinomastat in ophthalmology and dermatology.

Bezafibrate as differentiating factor of human myeloid leukemia cells.

Bezafibrate belongs to the class of fibric acid derivatives usually used as antihyperlipidemia agents. From the biochemical point of view, these drugs show intriguing properties which leads one to think they may promote a differentiation process in tumour cells. This new pharmacological activity of fibrates could partially depend on the induction of an oxidative stress. To test this hypothesis, the effect of bezafibrate, as well as of clofibric acid and gemfibrozil, on growth, functional and cytochemical characteristics of human leukaemia-derived cell lines HL-60, U-937 and K-562 has been studied in some details. The results show that bezafibrate, gemfibrozil and clofibric acid, do induce differentiation in human myeloid leukaemia cell lines as indicated by several differentiation markers. Moreover fibrates, in dose dependent manner, significantly alter the cell cycle distributions, mainly leading to G0/G1 phase increment and G2/M phase reduction. The differentiating activity of fibrates could have significant implications both for the pharmacotoxicological profile of this class of compounds and for the pathophysiology of neoplastic disease.

Brief communication: Resistance to Falciparum malaria in alpha-thalassemia, oxidative stress, and hemoglobin oxidation.

A recent survey conducted on Vanuatu Island suggests that resistance to Plasmodium falciparum in alpha-thalassemic individuals may have an immunological basis. This study is important since it seems to undermine the current idea that red-cell genetic defects give protection against falciparum malaria by reducing intraerythrocytic growth and development of the parasite. However, the mechanisms underlying these clinical and genetic observations are not yet fully understood. Based on a review of the relevant literature, we first show that the model based on the interaction between hemoglobin (Hb) and membrane components may provide a molecular basis for the involvement of the immune response in genetic adaptation to malaria. Second, we discuss the main evolutionary implications of the model. Finally, we suggest two approaches by which anthropological studies could provide a useful way of testing the model: 1) analysis of the interactions of malaria-resistance genes with genetic polymorphisms which affect the erythrocyte redox status and 2) study of the antimalarial effects of natural products (introduced as a part of a diet or for traditional antimalarial therapy) capable of interfering with the Hb/membrane interaction.

Bucindolol (Bristol-Myers Squibb Co).

Bucindolol is a beta1 and beta2 adrenoceptor antagonist under development by Intercardia as a potential treatment for hypertension and congestive heart failure (CHF). Intercardia, Knoll AG and the NIH are conducting phase III trials for these indications. Intercardia is a subsidiary of Interneuron formed to acquire 80% of Cardiovascular Pharmacology and Engineering Consultants (CPEC) which includes the rights to bucindolol. CPEC was founded by researchers who had studied bucindolol in CHF for Bristol-Myers Squibb which ceased development of the compound in 1989 and licensed it to CPEC.

Impaired reactive oxygen metabolism of phagocytic leukocytes in NIDDM patients. A role for non-enzymatic glycosylation of collagen.

Non-enzymatic glycosylation (NEG) of collagen has been previously shown to significantly influence the reactive oxygen metabolism (ROM) of phagocytic cells in healthy subjects. Considering the role of NEG in the pathophysiology of diabetes, we have further analysed the oxidative metabolism of polymorphonuclear cells (PMNs) and monocytes in 23 patients with non-insulin dependent diabetes mellitus in order to better elucidate a possible pathogenic role of NEG of the extracellular matrix in long-term complications of diabetes. Experiments were performed in triplicate on native-collagen and glycated-collagen coated vials, using a chemiluminescence (CL) assay. Results show that PMNs from diabetic patients display a significant increased basal and zymosan-induced CL activity with respect to controls that are not related to the glycation state of the substrate. Conversely, the CL activity of monocytes induced by zymosan shows a decrease in diabetic patients with respect to healthy volunteers (p < 0.05). Moreover, monocyte CL was reduced by the glycated matrix, both in healthy volunteers and in diabetic subjects (p < 0.05 and p < 0.01, respectively). These data highlight a complex role of phagocytic leukocytes in the pathophysiology of extracellular matrix alterations secondary to NEG that are typically present in clinical conditions such as diabetes or ageing.

Glycated human hemoglobin (HbA1c): functional characteristics and molecular modeling studies.

A minor hemoglobin component of human red blood cell hemolysate, HbA1c, is the result of the non-enzymatic reaction of glucose with the alpha-amino groups of the valine residues at the N-terminus of the beta-chains of human hemoglobin. In this paper, the effect of protons, chloride and 2,3-diphosphoglycerate (DPG) on the functional properties of HbA1c has been investigated in some details. Moreover, the structural modifications induced on the native molecule by the sugar moieties, studied by computer modeling, do agree with the observed functional alterations. In particular, the functional results indicate that: (a) the low-affinity conformation (or T-state) of HbA1c is destabilized by the chemical modification per se; (b) the Bohr effect is reduced with respect to that of native HbA0; (c) the affinity of the T-state of HbA1c for 2,3-diphosphoglycerate is about 2.6 x lower than that of the corresponding conformational state of HbA0, while the R-state is less affected with, the affinity being 1.7 x lower. At the structural level, computer modeling studies show that the two sugar moieties are asymmetrically disposed within the 2,3-diphosphoglycerate binding site. In addition, molecular mechanics and dynamics calculations concerning the interaction with 2,3-diphosphoglycerate indicate that while in HbA0 the effector can assume two different stable orientations, in glycated Hb only one orientation is possible. All together, the results show that glycation of the Val 1 residues of both beta-chains does not impair the binding of DPG but imposes a different mode of binding by changing the internal geometry of the complex and the surface distribution of the positive electrostatic potential within the binding pocket.

The priming effect of gemfibrozil on reactive oxygen metabolism of phagocytic leucocytes. An intriguing side effect.

Gemfibrozil is an antihyperlipidaemia agent used in therapy to reduce the occurrence of coronary heart disease. Considering the biochemical and pharmacological peculiarities of this class of drugs, we investigated the influence of a single oral therapeutic dose of gemfibrozil on the reactive oxygen metabolism of phagocytic leucocytes. Analysis was carried out adopting a chemiluminescence assay. Results clearly indicate that gemfibrozil, acting as a primer, significantly enhances the induced reactive-oxygen-species (ROS) production by overall blood phagocytes (increment of Stimulation Index (SI) = +52% with respect to time 0 values; P < 0.01), by polymorphonuclear leucocytes (increment of SI = +28% with respect to control values; P < 0.01) and by monocytes (increment of SI = +83% with respect control values; P < 0.001), when these cells are stimulated by phorbol myristate acetate. This iatrogenic derangement of ROS metabolism could explain, at least in part, the occurrence of some side effects that occur in treatment with fibrates.

Monocyte oxygenation activities are significantly influenced by non-enzymatic glycosylation of collagen.

The pathophysiology of the acquired extracellular matrix alterations, like non-enzymatic glycosylation (NEG) of proteins secondary to diabetes or ageing, is not well characterized, particularly considering its relationship with leukocytes. We have analysed the influence of collagen NEG on the fundamental function of phagocytic cells, i.e. the production of reactive oxygen species (ROS). To this aim, we considered the activity of polymorphonuclear leukocytes and monocytes incubated in the presence of both non-glycated and glycated collagen. ROS production was monitored by chemi-luminescence (CL), a standardized and sensitive assay of phagocytes oxidative metabolism. All experiments were performed in triplicate on collagen-coated and glycated collage-coated vials. Results showed that PMNs ROS metabolism appeared unrelated to the glycation state of the substrate. Conversely, data regarding zymosan-induced CL by monocytes indicated a significant and intriguing decrease in reactive oxygen metabolism, which appeared greatly compromised by the glycation state of the matrix (monocytes in collagen, 197.4 +/- 31.2 vs. monocytes in glycated collagen, 138.0 +/- 20.4; p < 0.001 expressed as counts/cell/60 min). These data highlight the different role of polymophonuclear and monocytic phagocytes in the pathophysiology of the acquired extracellular matrix alteration secondary to non-enzymatic glycosylation (NEG) of proteins.

Oxygen transport by fetal bovine hemoglobin.

The functional properties of fetal bovine hemoglobin have been studied as a function of temperature, chloride and 2,3-diphosphoglycerate (DPG) concentration. The fetal bovine erythrocyte has six times the concentration of the allosteric modulator DPG compared with the adult cell, and yet the oxygen affinity of the fetal hemoglobin still exceeds that of the adult molecule at the respective physiological concentration of DPG and at physiological temperature. We find that the allosteric modulator strongly affects the enthalpy of oxygen for the fetal hemoglobin but not for the adult protein. We propose that this may be an important mechanism for the exchange of heat from mother to fetus. In particular, under stripped conditions the oxygen affinity of fetal bovine Hb is considerably higher than that of the adult hemoglobin. Due to the higher DPG concentration that characterizes fetal bovine erythrocytes this difference is almost abolished in the presence of the respective physiological concentration of DPG and at 20 degrees C. However, on going from 20 degrees C to 37 degrees C, the difference in O2 affinity between the two hemoglobins is restored, as it should if oxygen has to be transferred from maternal to fetal blood, by virtue of the lower overall heat of oxygenation (delta H) displayed by fetal Hb when in the presence of DPG at physiological concentration. This behavior is reminiscent of that of human fetal Hb and outlines the role of temperature and of its interplay with heterotropic ligands in the modulation of hemoglobin function to fully meet the physiological needs of the organism.

Effects of gemfibrozil on the oxygen transport properties of erythrocytes.

1. In the present study we have investigated the effects of the relatively low plasma concentrations of gemfibrozil (GFZ) found in clinical practice on the oxygen dissociation curve (ODC) of erythrocytes. 2. ODCs were measured at 30 degrees C and 37 degrees C and at pH 7.4: a) both on HbA solution and erythrocytes incubated in vitro with gemfibrozil and clofibric acid; b) on erythrocytes from healthy volunteers treated with a single oral dose of gemfibrozil. 3. These experiments showed a significant drug-induced shift of the ODC towards lower O2 affinity values without any significant modification of metabolic parameters of erythrocytes such as intracellular pH and intraerythrocytic levels of ATP and DPG. 4. In our experimental conditions gemfibrozil appears to lower both in vitro and in vivo, the partial pressure of oxygen required to give 50% of the haemes saturated with oxygen (P50) of erythrocytes from the control value of 24 +/- 0.5 mm Hg to 29 +/- 0.5 mm Hg (mean +/- s.d.; P < 0.02 by ANOVA). 5. These data clearly indicate that therapeutic doses of gemfibrozil may influence the oxygen transport properties of red cells. This effect could have relevant pharmacological and toxicological implications.

The multiple functions of hemoglobin.

The aim of this review is to focus and discuss several parallel biological functions of hemoglobin besides its basic function of oxygen transport. In light of the information present in the literature the following possible physiological roles of hemoglobin are discussed: (1) hemoglobin as molecular heat transducer through its oxygenation-deoxygenation cycle, (2) hemoglobin as modulator of erythrocyte metabolism, (3) hemoglobin oxidation as an onset of erythrocyte senescence, (4) hemoglobin and its implication in genetic resistance to malaria, (5) enzymatic activities of hemoglobin and interactions with drugs, and (6) hemoglobin as source of physiological active catabolites.

Functional alterations in adult and fetal hemoglobin Sassari Asp-alpha 126(H9)-->His. The role of alpha 1 alpha 2 contact.

The effects of pH, organic phosphates (2,3-diphosphoglycerate), and temperature on the functional properties of both adult and fetal hemoglobin Sassari alpha (Asp-126-->His) have been studied. The functional properties of the adult variant are characterized by the following: (i) an oxygen affinity higher than that of normal HbA in all the experimental conditions used; (ii) a dramatic reduction of homotropic interactions (n50 very close to unity); and (iii) a significant decrease of the effect of 2,3-diphosphoglycerate, which is 35% lower than that observed on HbA. The fetal variant shows an increased oxygen affinity compared with normal HbF and an almost abolished heme-heme interaction. The molecular basis of these functional differences is discussed in terms of the possible role played by the substitution of alpha (Asp-26-->His) on the stability of the R state of the molecule due to a decreased interaction at the level of alpha 1 alpha 2 contact.

Synergistic modulation by chloride and organic phosphates of hemoglobin from bear (Ursus arctos).

The oxygen binding properties of hemoglobin (Hb) from brown bear (Ursus arctos) have been studied focussing on the effect of heterotropic ligands, and the behaviour has been compared with that of human HbA, taken as a prototype of mammalian Hbs. It has been observed that in bear Hb chloride ions and 2,3-diphosphoglyceric acid (Gri(2,3)P2) can modulate the oxygen affinity in a synergistic way such that their individual effect is enhanced whenever they are both present in saturating amounts. The thermodynamic analysis of such a feature indicates that in bear Hb there are two classes of chloride binding sites, one acting synergistically with Gri(2,3)P2 and another one, which likely overlaps with the organic phosphate interaction cleft, and is therefore fully operative only in the absence of Gri(2,3)P2. The behaviour of the last site is similar to that observed in human HbA, where the effect of Cl- and Gri(2,3)P2 is mutually exclusive. The interaction energy between chloride and Gri(2,3)P2 synergistic binding sites appears to be O2-linked so that the interplay may have a relevant physiological role in modulating the oxygen transport in brown bear. This behaviour is associated with a marked pH-dependence of the oxygenation enthalpy in bear Hb, such that under acidotic and hypercloruremic conditions, oxygen supply to peripheral tissues could be maintained essentially unaltered even under low temperature conditions.