Intracellular pH regulation in U-2 OS human osteosarcoma cells transfected with P-glycoprotein

The molecular mechanisms responsible for intracellular pH regulation in the U2-OS osteosarcoma cell line were investigated by loading with 2',7'-bis(2-carboxyethyl)-5(6) carboxyfluorescein ester and manipulation of Cl(-) and Na(+) gradients, both in HEPES- and HCO(3)(-)/CO(2)-buffered media. Both acidification and alkalinisation were poorly sensitive to 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, inhibitor of the anion exchanger, but sensitive to amiloride, inhibitor of the Na(+)/H(+) exchanger. In addition to the amiloride-sensitive Na(+)/H(+) exchanger, another H(+) extruding mechanism was detected in U-2 OS cells, the Na(+)-dependent HCO(3)(-)/Cl(-) exchanger. No significant difference in resting pH(i) and in the rate of acidification or alkalinisation was observed in clones obtained from U-2 OS cells by transfection with the MDR1 gene and overexpressing P-glycoprotein. However, both V(max) and K' values for intracellular [H(+)] of the Na(+)/H(+) exchanger were significantly reduced in MDR1-transfected clones, in the absence and/or presence of drug selection, in comparison to vector-transfected or parental cell line. NHE1, NHE5 and at a lower extent NHE2 mRNA were detected in similar amount in all U2-OS clones. It is concluded that, although overexpression of P-glycoprotein did not impair pH(i) regulation in U-2 OS cells, the kinetic parameters of the Na(+)/H(+) exchanger were altered, suggesting a functional relationship between the two membrane proteins.     

Endogenous ghrelin is an orexigenic peptide acting in the arcuate nucleus in response to fasting

Ghrelin, a circulating growth-hormone releasing peptide derived from stomach, stimulates food intake through neuropeptide Y (NPY) neurons of the arcuate nucleus in the hypothalamus (ARC). We examined the effect of ghrelin microinjected into the ARC and the influence of intracerebroventricular (i.c.v.) pretreatment with a GHRH or NPY receptor antagonist on ghrelin-induced food intake in free-feeding male rats. Ghrelin (0.1-1 microg) stimulated food intake in a dose-dependent manner, and this effect was reduced by 55-60% by the Y(5) NPY receptor antagonist (10 microg i.c.v.), but not by the GHRH receptor antagonist MZ-4-71 (10 microg i.c.v.). We also evaluated the effects of passive ghrelin immunoneutralization by the microinjection of anti-ghrelin immunoglobulins (IgGs) intracerebroventricularly or directly into the ARC on food intake in free-feeding and fasted male rats. i.c.v. administration of anti-ghrelin IgGs decreased cumulative food intake over 24 h, whereas microinfusion of anti-ghrelin IgGs into the ARC induced only a short-lived (2 and 6 h) effect. Collectively, these data would indicate that centrally derived ghrelin has a major role in the control of food intake in rats and, in this context, blood-born ghrelin would be effective only in relation to its ability to reach the ARC, which is devoid of blood-brain barrier.     

2'-Deoxyadenosine causes apoptotic cell death in a human colon carcinoma cell line

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.     

Truncated hemoglobins and nitric oxide action

Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO3-.     

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.     

A skeletochronological study of growth,longevity, and age at sexual maturity in a population of<Emphasis Type="Italic">Rana latastei</Emphasis> (Amphibia,Anura)

Longevity and age at sexual maturity in an Italian population ofRana latastei were studied by skeletochronology performed on the phalanges. Frogs collected in 1998 and 1999 by drift fences and pitfall traps were marked by toe-clipping. After marking, individuals were released and the cut phalanges were processed for skeletochronological analysis. The maximum age so far recorded was 3 years in males and 4 years in females. The smallest male and female that were sexually mature on the basis of histological analysis of the gonads were 36 and 35 mm snout vent length (SVL), respectively. In both sexes, most individuals were estimated to breed shortly after emergence from their first overwintering. Among the European Brown Frogs,Rana latastei appears to be one of the shortest-lived and one of the first to reach sexual maturity.     

Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme

Macrophage-generated oxygen- and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis 'truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxification, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three-dimensional structure of oxygenated trHbN, solved at 1.9 A resolution, displays the two-over-two alpha-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal alpha-helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for approximately 28 A through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O2 stereochemistry and the protein matrix tunnel may promote O2/NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.     

The Clonopsis gallica puzzle: Mendelian species, polyploid parthenogens with karyotype re-diploidization and clonal androgens in Moroccan stick insects (Phasmida)

The stick insect Clonopsis gallica is a morphologically stable obligate parthenogen, ranging from North Africa to southern Europe, with a variable karyotype (54–57 chromosomes), considered diploid or triploid. Clonopsis gallica origin and relationships with Clonopsis algerica and Clonopsis maroccana remained unknown. Our Moroccan samples shared the same egg and body patterns and, in addition to C. gallica (2 n  = 54, XX), comprised the new bisexual Clonopsis sp. 1 (2 n  = 35/36, X0/XX) and the new all-female Clonopsis sp. 2 (2 n  = 72, XX), apparently forming a numerically polyploid series with re-diplodized karyotypes; C. gallica could thus be a triploid hybrid in origin. Furthermore, two strains of ameiotic males with 2 n  = 35 (X0) or 53 (X0) were found, which would maintain themselves as clonal androgens. We could conclude that in stick insects body and karyotype 'phenotypes' are considerably independent, thus preventing a sound taxonomy. Generally, the high heterozygosity of hybrids allows parthenogens to persist for long, particularly when hybrid polyploids are produced, as backcrosses of the hybrids to the parental species allow repeated incorporations of genetically diversified genomes. The distribution range of Clonopsis parthenogens appears to realize a sound instance of geographical parthenogenesis.