Superoxide dismutase, catalase and glutathione peroxidase activities in the lymphoid organs and skeletal muscles of rats treated with dexamethasone

The effect of dexamethasone (a synthetic glucocorticoid) on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase and glutathione peroxidase) of the lymphoid organs (mesenteric lymph nodes (MLN), spleen and thymus) was investigated. For comparison with non-immune tissues, skeletal muscles (soleus and gastrocnemius (GC) were also studied. As an indication of the occurrence of lipid peroxidation, the content of thiobarbituric acid reactant substances (TBARs) was also determined. Dexamethasone treatment decreased the TBARs content of the lymphoid organs and raised it in the GC and soleus muscles. The activity of Cu/Zn-SOD was reduced in all tissues. However, the activity of Mn-SOD was decreased in the MLN and soleus muscle only. The activity of catalase was reduced in the MLN and thymus and raised in the spleen and GC and soleus muscles. The imposed treatment raised the activity of GPX in the MLN, thymus and spleen and reduced it in GC and soleus muscles. These data led us to postulate that the mechanism for the therapeutic effect of glucocorticoids as antiinflammatory and immunosuppressive agents might include modification of antioxidant enzyme activities.     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

NBD-cholesterol incorporation by rat macrophages and lymphocytes: a process dependent on the activation state of the cells

The time-course of incorporation of NBD-cholesterol by macrophages (Ma) and lymphocytes (LY) obtained from untreated and thioglycollate-injected (thio) rats was investigated. NBD-cholesterol incorporation was also examined in Ma obtained from untreated rats and stimulated in vitro by lipopolysaccharide (LPS) and phorbol-myristate acetate (PMA). The same measurement was performed in LY from untreated rats stimulated by addition of LPS and concanavalin A (Con A) into the culture medium. Thio-treated Ma showed high fluorescence intensity after 1 h of incubation with NBD-cholesterol. Ma submitted concomitant to LPS and NBD-cholesterol showed low fluorescence intensity, as well as Ma stimulated with PMA. Ma from untreated and LPS pre-treated rats showed a similar time-course of incorporation. LY from thio-treated rats showed lower incorporation of NBD-cholesterol in comparison to LY from untreated rats. Incorporation was reduced when LPS was added concomitantly with NBD-cholesterol. On the other hand, LY pre-treated with LPS for 48 h showed a very high incorporation of NBD-cholesterol. Con A treatment did not cause a significant effect on NBD-cholesterol incorporation. The findings presented herein led us to conclude that the uptake of NBD-cholesterol by Ma and LY is markedly affected by the activation state of the cells.     

Genotype and allele frequencies of polymorphic cytochromes P450 CYP1A2 and CYP2E1 in Mexicans

CYP1A2 and CYP2E1 are two of the main cytochrome P450 isoforms involved in the metabolism of commonly used drugs and xenobiotic compounds considered to be responsible for or possible participants in the development of several human diseases. Individual susceptibility to developing these pathologies relies, among other factors, on genetic polymorphism which depends on ethnic differences, as the frequency of mutant genotypes varies in different human populations. Thus the aim of this study was to investigate the frequency of CYP1A2 5'-flanking region and CYP2E1 Rsa I/Pst I polymorphisms in Mexicans by PCR-RFLP methods. The DNA of 159 subjects was analysed and mutant allele frequencies of 30% for CYP2E1 Rsa I/Pst I sites and 43% for CYP1A2 5'-flanking region were found. These frequencies are higher than those previously reported for other human populations.     

Overexpression of human DNA polymerase mu (Pol mu) in a Burkitt's lymphoma cell line affects the somatic hypermutation rate

DNA polymerase μ (Pol μ) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol μ protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol μ in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol μ to mutation of G and C residues during SHM. In vitro analysis of Pol μ misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol μ to induce transient template/primer misalignments.     

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.     

Influence of liposomal local anesthetics on platelet aggregation in vitro

We assessed the effect of local anesthetics (LA) from different families such as esters (benzocaine), linear aminoamides (lidocaine) and cyclic aminoamides (bupivacaine) on the platelet aggregation induced by ADP. Liposomal formulations of the three LA, prepared with egg phosphatidylcholine:cholesterol alpha-tocopherol, were also tested. The three LA were able to inhibit platelet aggregation induced by ADP, in the following order: bupivacaine > lidocaine > benzocaine. After encapsulation into liposomes the inhibitory effect increased for all anesthetics studied, showing that aggregation tests could be used to assess the toxicity of new drug formulations.     

Stereoselectivity in the placental transfer and kinetic disposition of racemic bupivacaine administered to parturients with or without a vasoconstrictor

The aim of the present study was to investigate the stereoselectivity in the kinetic disposition and the transplacental distribution of bupivacaine in term parturients during labor. Maternal age ranged from 18-37 years and fetal gestational age from 37.6-41.5 weeks. Healthy parturients (n = 23) received epidural 0.5% racemic bupivacaine alone (group A) or combined with epinephrine (group B). Maternal venous blood was sampled at regular intervals until 8 h after drug administration and umbilical venous blood was obtained at delivery. Bupivacaine enantiomers were determined in plasma samples by HPLC using a Chiralcel(R) OD-R column and a UV detector. One- or two-compartment models were fitted to data and differences between the (+)-(R) and (-)-(S) enantiomers were compared with the paired Wilcoxon test (P< 0.05). The influence of epinephrine was evaluated using the unpaired Mann-Whitney test (P< 0.05). The disposition of bupivacaine in maternal plasma was stereoselective, with higher V(d/f) (140.60 vs. 132.81 L for group A and 197.86 vs. 169.46 L for group B) and C(l/f) (29.00 vs. 25.43 L/h for group A and 33.15 vs. 26.39 L/h for group B) and lower t(1/2)beta (3.24 vs. 3.30 h for group A and 4.36 vs. 4.45 h for group B) being observed for (+)-(R)-bupivacaine. The combined administration of epinephrine resulted in higher V(d/f) (197.86 vs. 140.60 L for (+)-(R) and 169.46 vs. 132.81 L for (-)-(S)) and t(1/2)beta values (4.36 vs. 3.24 h for (+)-(R) and 4.45 vs. 3.30 h for (-)-(S)). The transplacental distribution of bupivacaine was stereoselective only when bupivacaine was administered without epinephrine (group B), with a higher cord blood/maternal blood ratio being observed for (-)-(S)-bupivacaine (0.40 vs. 0.35). Chirality 16:65-71, 2004.     

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.