The role of pH dynamics and the Na+/H+ antiporter in the etiopathogenesis and treatment of cancer. Two faces of the same coin--one single nature

Looked at from the genetic point-of-view cancer represents a daunting and, frankly, confusing multiplicity of diseases (at least 100) that require an equally large variety of therapeutic strategies and substances designed to treat the particular tumor. However, when analyzed phenotypically cancer is a relatively uniform disease of very conserved 'hallmark' behaviors across the entire spectrum of tissue and genetic differences [D. Hanahan, R.A. Weinberg, Hallmarks of cancer, Cell 100 (2000) 57-70]. This suggests that cancers do, indeed, share common biochemical and physiological characteristics that are independent of the varied genetic backgrounds, and that there may be a common mechanism underlying both the neoplastic transformation/progression side and the antineoplastic/therapy side of oncology. The challenge of modern oncology is to integrate all the diverse experimental data to create a physiological/metabolic/energetic paradigm that can unite our thinking in order to understand how both neoplastic progression and therapies function. This reductionist view gives the hope that, as in chemistry and physics, it will possible to identify common underlying driving forces that define a tumor and will permit, for the first time, the actual calculated manipulation of their state. That is, a rational therapeutic design. In the present review, we present evidence, obtained from a great number of studies, for a fundamental, underlying mechanism involved in the initiation and evolution of the neoplastic process. There is an ever growing body of evidence that all the important neoplastic phenotypes are driven by an alkalization of the transformed cell, a process which seems specific for transformed cells since the same alkalinization has no effect in cells that have not been transformed. Seen in that light, different fields of cancer research, from etiopathogenesis, cancer cell metabolism and neovascularization, to multiple drug resistance (MDR), selective apoptosis, modern cancer chemotherapy and the spontaneous regression of cancer (SRC) all appear to have in common a pivotal characteristic, the aberrant regulation of hydrogen ion dynamics [S. Harguindey, J.L. Pedraz, R. García Ca?ero, J. Pérez de Diego, E.J. Cragoe Jr., Hydrogen ion-dependent oncogenesis and parallel new avenues to cancer prevention and treatment using a H+-mediated unifying approach: pH-related and pH-unrelated mechanisms, Crit. Rev. Oncog. 6 (1) (1995) 1-33]. Cancer cells have an acid-base disturbance that is completely different than observed in normal tissues and that increases in correspondence with increasing neoplastic state: an interstitial acid microenvironment linked to an intracellular alkalosis.     

On the mechanism of Rhodotorula gracilis D-amino acid oxidase: role of the active site serine 335

Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.     

A new semi-automated measurement technique based on X-ray pictures for ankle morphometry

Knowledge of human joint morphology is important in orthopaedic surgery and in prosthesis design. The literature on quantitative morphological analysis of the ankle joint is particularly scarce. A semi-automated radiographic measurement method was developed to collect morphological measures of the ankle joint. The method was based on standard lateral and antero-posterior X-ray pictures of the ankle joint. These were then scanned and analysed by means of specialized software designed for the purpose, which requires minimal operator contribution. Accuracy of the method was experimentally assessed by in vitro direct measurements. Intra- and inter-operator variability was also tested. Accuracy was assessed to be within 1mm for most measurements. Repeatability was not affected by operator skill and was within 2mm. The newly proposed method was applied successfully on 15 male adult subjects and relevant results are reported. The method allows ankle morphology to be analysed in a large number of subjects providing reliable data for anthropometric statistics.     

Biochemical and immunochemical evidences for the presence of lipoxygenase in plant mitochondria

In this paper, both biochemical and immunochemical evidence for the presence of lipoxygenase (LOX) in plant mitochondria is presented. Highly purified pea (Pisum sativum L., cv. Alaska) mitochondria show LOX activity, evaluated as conjugated diene formation, oxygen consumption, and hydroperoxide formation. Both 9- and 13-hydroperoxy-octadecadienoic acids are produced by the oxidation of linoleic acid. LOX activity is particularly evident in swollen mitochondria; it is inhibited by nordihydroguaiaretic acid, a pea anti-LOX B antibody, and has two pH optima (6.0 and 7.5). A mitochondrial protein of approximately 97 kDa cross-reacts with a pea seed anti-LOX B antibody. This reaction is detectable in both soluble (matrix fraction) and membrane-bound (submitochondrial particles) proteins. Considering that pea mitochondria were extracted from actively growing stems that were differentiating tube elements, it is suggested that the presence of LOX in these organelles may be related to their degradation linked to xylem differentiation.     

Free radical biology - terminology and critical thinking

Deoxyribonucleoside kinases, which catalyse the phosphorylation of deoxyribonucleosides, are present in several copies in most multicellular organisms and therefore represent an excellent model to study gene duplication and specialisation of the duplicated copies through partitioning of substrate specificity. Recent studies suggest that in the animal lineage one of the progenitor kinases, the so-called dCK/dGK/TK2-like gene, was duplicated prior to separation of the insect and mammalian lineages. Thereafter, insects lost all but one kinase, dNK (EC 2.7.1.145), which subsequently, through remodelling of a limited number of amino acid residues, gained a broad substrate specificity.     

Defining the p53 DNA-binding domain/Bcl-x(L)-binding interface using NMR

We have determined the first de novo position of the secondary quinone Q_B in the Rhodobacter sphaeroides reaction center (RC) using phases derived by the single wavelength anomalous dispersion method from crystals with selenomethionine substitution. We found that in frozen RC crystals, Q_B occupies primarily the proximal binding site. In contrast, our room temperature structure showed that Q_B is largely in the distal position. Both data sets were collected in dark-adapted conditions. We estimate that the occupancy of the Q_B site is 80% with a proximal: distal ratio of 4:1 in frozen RC crystals. We could not separate the effect of freezing from the effect of the cryoprotectants ethylene glycol or glycerol. These results could have far-reaching implications in structure/function studies of electron transfer in the acceptor quinone complex because the above are the most commonly used cryoprotectants in spectroscopic experiments.     

FAD transport and FAD-dependent protein thiol oxidation in rat liver microsomes

The transport of FAD and its effect on disulfide bond formation was investigated in rat liver microsomal vesicles. By measuring the intravesicular FAD-accessible space, we observed that FAD permeates across the microsomal membrane and accumulates in the lumen. Rapid filtration experiments also demonstrated the uptake and efflux of the compound, which could be inhibited by atractyloside and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. FAD entering the lumen promoted the oxidation of protein thiols and increased the intraluminal oxidation of glucose-6-phosphate. These findings support the notion that, similar to yeast, free FAD may have a decisive role in the mechanism of oxidative protein folding in the endoplasmic reticulum lumen of mammalian cells.     

Mechanisms of soluble beta-amyloid impairment of endothelial function

Alzheimer's disease (AD) has been recently associated with vascular risk factors. beta-amyloid peptides (AbetaP), the main component of senile plaques typical of AD, circulate in soluble globular form in bloodstream. Interestingly, AbetaP is able to induce endothelial dysfunction, and this effect may represent the link between vascular and neuronal pathophysiological factors involved in AD. We aimed to clarify the molecular mechanisms underlying globular AbetaP-induced vascular toxicity. Using several methodological approaches, we have observed that in vascular tissues globular AbetaP is unable to induce oxidative stress, one of the mechanisms hypothesized involved in beta-amyloid toxicity. More important, we have demonstrated that globular AbetaP is able to localize on vascular endothelium, where it inhibits eNOS enzymatic activity. In particular, AbetaP enhances eNOS phosphorylation on threonine 495 and serine 116 and reduces acetylcholine-induced phosphorylation on serine 1177. Such an effect depends on a PKC signaling pathway, as suggested by its phosphorylation on serine 660. In fact, selective inhibition of the calcium-dependent group of PKC is able to rescue beta-amyloid-induced alteration of eNOS phosphorylation, NO production, and endothelial vasorelaxation. The activation of these Ca(2+)-dependent pathways is probably due to the ability of AbetaP to evoke Ca(2+) leakage from inositol 1,4,5-triphosphate receptors on endoplasmic reticulum. Our data demonstrate that globular AbetaP-induced endothelial NO dysfunction can be attributed to an alteration of intracellular Ca(2+) homeostasis, which could lead to the activation of calcium-dependent group of PKC with a consequent change of the eNOS phosphorylation pattern. These mechanisms could contribute to shed further light on the toxic effect of beta-amyloid in vascular tissues.