PI-3-kinase/NF-kappaB mediated response of Jurkat T leukemic cells to two different chemotherapeutic drugs, etoposide and TRAIL

Jurkat T leukemic cells respond to Etoposide, antineoplastic agent which targets the DNA unwinding enzyme, Topoisomerase II, and TNF-Related-Apoptosis-Inducing-Ligand (TRAIL), 34 kDa transmembrane protein, which displays minimal or no toxicity on normal cells and tissues, not only disclosing the occurrence of apoptosis but also a kind of resistance. A similar rate of viability upon the exposure to these two drugs up to 24 h has been evidenced, followed by the occurrence of a rescue process against TRAIL, not performed against Etoposide, along with an higher number of dead cells upon Etoposide exposure, in comparison with TRAIL treatment. These preliminary results let us to speculate on the possible involvement of PI-3-kinase in TRAIL resistance disclosed by surviving cells (20%), may be phosphorylating Akt-1 and, in parallel, IkappaB alpha on both serine and tyrosine residues. On the other hand, in Etoposide Jurkat exposed cells Ser 32-36 phosphorylation of IkappaB alpha is not sufficient to overbalance the apoptotic fate of the cells, since Bax increase, IAP decrease, and caspase-3 activation determine the persistence of the apoptotic state along with the occurrence of cell death by necrosis. Thus, the existence of a balance between apoptotic and rescue response in 20% of cells surviving to TRAIL suggests the possibility of pushing it in favor of cell death in order to improve the yield of pharmacological strategies.     

Near infrared spectroscopy as a tool for the determination of eumelanin in human hair

Eumelanins are brown-black pigments present in the hair and in the epidermis which are acknowledged as protection factors against cell damage caused by ultraviolet radiation. The quantity of eumelanin present in hair has recently been put forward as a means of identifying subjects with a higher risk of skin tumours. For epidemiological studies, chromatographic methods of determining pyrrole-2,3,5-tricarboxylic acid (PTCA; the principal marker of eumelanin) are long, laborious and unsuitable for screening large populations. We suggest near infrared (NIR) spectroscopy as an alternative method of analysing eumelanin in hair samples. PCTA was determined on 93 samples of hair by means of oxidizing with hydrogen peroxide in a basic environment followed by chromatographic separation. The same 93 samples were then subjected to NIR spectrophotometric analysis. The spectra were obtained in reflectance mode on hair samples which had not undergone any preliminary treatment, but had simply been pressed and placed on the measuring window of the spectrophotometer. The PTCA values obtained by means of HPLC were correlated with the near infrared spectrum of the respective samples. A correlation between the PTCA values obtained by means of HPLC and the PTCA values obtained from an analysis of the spectra was obtained using the principal component regression (PCR) algorithm. The correlation obtained has a coefficient of regression (R(2)) of 0.89 and a standard error of prediction (SEP) of 13.8 for a mean value of 108.6 ng PTCA/mg hair. Some considerations about the accuracy of the obtained correlation and the main sources of error are made and some validation results are shown.     

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.     

Production of tumor necrosis factor alpha by Treponema pallidum, Borrelia burgdorferi s.l., and Leptospira interrogans in isolated rat Kupffer cells

Stimulation of isolated rat Kupffer cells by viable Leptospira interrogans, Treponema pallidum and Borrelia garinii elicited cellular responses resulting in the release of different tumor necrosis factor alpha (TNF-alpha) levels, depending on the spirochetes. L. interrogans induced TNF-alpha levels higher than those achieved with B. garinii and T. pallidum (in this order), but lower than the levels achieved with lipopolysaccharide (LPS). In contrast to L. interrogans, pretreatment of borreliae and treponemes with polymyxin B did not substantially diminish the ability of B. garinii and T. pallidum to stimulate Kupffer cells. Purified T. pallidum lipoproteins TpN47, TmpA, TpN15-TpN17, and B. garinii OspA induced TNF-alpha responses comparable to that achieved by LPS. This response was almost insensitive to the action of polymyxin B.     

Dynamics of polymorphism of acidocalcisomes in<Emphasis Type="Italic"> Leishmania</Emphasis> parasites

Growth of Leishmania mexicana amazonensis promastigotes in different culture media resulted in structurally and chemically different acidocalcisomes. When grown in SDM-79 medium, the promastigotes showed large spherical acidocalcisomes of up to 1.2 m diameter distributed throughout the cell. X-ray microanalysis and elemental mapping of the organelles showed large amounts of oxygen, phosphorus, sodium, potassium, magnesium, calcium, and zinc. Immunofluorescence microscopy using antisera raised against a peptide sequence of the vacuolar-type proton pyrophosphatase of Arabidopsis thaliana that is conserved in the Leishmania enzyme, indicated localization in acidocalcisomes. When cells were transferred to Warrens medium, the acidocalcisomes transformed from spherical into branched tubular organelles. The labeling pattern of the vacuolar proton-pyrophosphatase, considered as a marker for the organelle, changed accompanying the structural changes of the acidocalcisomes, and the enzyme showed an apparently lower proton-transporting activity when measured in digitonin-permeabilized promastigotes. X-ray microanalysis and elemental mapping of these structures revealed the additional presence of iron. Together, the results reveal that the morphology and composition of acidocalcisomes are greatly influenced by the culture conditions.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Enthalpy/entropy compensation phenomena in the reduction thermodynamics of electron transport metalloproteins

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwalds approach for the interpretation of H/S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E° values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.     

Protein stability and mutations in the axial methionine loop of a minimal cytochrome<Emphasis Type="Italic"> c</Emphasis>

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.Abbreviations Bpcytc soluble fragment of cytochrome c₅₅₃ from Bacillus pasteurii - GdmCl guanidinium chloride - I75A Ile75 to Ala mutant - I75V Ile75 to Val mutant - P72A Pro72 to Ala mutant - P72G Pro72 to Gly mutant - Q68K Gln75 to Lys mutant - WT wild type