Use of disposable tubing in suction-assisted lipectomy.
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Equipment Design↗
Biomedical subjects
B Alvarez
Publications and source records attributed to B Alvarez.
Comparative intracellular cytokine production by in vitro stimulated T lymphocytes from human umbilical cord blood (HUCB) and adult peripheral blood (APB).
To date over 400 HUCB transplants have been reported from different centers. It has been suggested that there is a reduced graft-versus-host-disease (GVHD) with HUCB compared to bone marrow transplantation. Since cytokine production by a cell is an indication of the cells function it is important to determinate the differences between APB and HUCB with respect to production of these soluble factors. Our aim was to analyse the intracellular cytokine production by HUCB and APB T lymphocytes with and emphasize on their possible role in GVHD. Heparinized HUCB samples from 8 normal full-term deliveries and 10 normal blood donors were stimulated 4 hours at 37 degrees C and 5% CO2 with phorbol 12-myristate 13-acetate (PMA) and lonomycin in the presence of brefeldine. Afterwards cells were stained with CD3, CD4 or CD8 in different combinations. Finally, after cell permeabilization, cells were stained with Il-2, Il-4 or IFN-gamma. Data acquisition was performed on a FACScan flow cytometer. Compared to APB, HUCB T lymphocytes produced less Il-2, Il-4 and IFN-gamma. In HUCB, Il-2, Il-4 and IFN-gamma were produced predominantly by CD4+ T cells. In APB, Il-2 and Il-4 were also produced predominantly by CD4+ cells compared with CD8+ T lymphocytes, however, IFN-gamma was produced by both CD4+ and CD8+ T cells. These results indicate that there are clear differences in the cytokine profile between T cells in APB and HUCB.
Adult↗
The divergent effects of tumour necrosis factor-alpha on skeletal muscle: implications in wasting.
Tumour necrosis factor-alpha (TNF) is a pleiotropic cytokine that can have effects on many cell types, including skeletal muscle, the most abundant tissue (representing almost 45% of body weight), where many effects of this cytokine have been described. Thus, TNF receptors have been described in muscle tissue, and different investigations have revealed effects of the cytokine on membrane potential, glucose uptake and metabolism, amino acid transport and protein turnover. However, the results found are relatively divergent, therefore the main aim of the present review has been to clarify and reconcile some of the most contradictory studies concerning the effects of TNF on metabolism in skeletal muscle.
Amino Acids↗
Kinetics of peroxynitrite reaction with amino acids and human serum albumin.
An initial rate approach was used to study the reaction of peroxynitrite with human serum albumin (HSA) through stopped-flow spectrophotometry. At pH 7.4 and 37 degreesC, the second order rate constant for peroxynitrite reaction with HSA was 9.7 +/- 1.1 x 10(3) M-1 s-1. The rate constants for sulfhydryl-blocked HSA and for the single sulfhydryl were 5.9 +/- 0.3 and 3.8 +/- 0.8 x 10(3) M-1 s-1, respectively. The corresponding values for bovine serum albumin were also determined. The reactivity of sulfhydryl-blocked HSA increased at acidic pH, whereas plots of the rate constant with the sulfhydryl versus pH were bell-shaped. The kinetics of peroxynitrite reaction with all free L-amino acids were determined under pseudo-first order conditions. The most reactive amino acids were cysteine, methionine, and tryptophan. Histidine, leucine, and phenylalanine (and by extension tyrosine) did not affect peroxynitrite decay rate, whereas for the remaining amino acids plots of kobs versus concentration were hyperbolic. The sum of the contributions of the constituent amino acids of the protein to HSA reactivity was comparable to the experimentally determined rate constant, where cysteine and methionine (seven residues in 585) accounted for an estimated 65% of the reactivity. Nitration of aromatic amino acids occurred in HSA following peroxynitrite reaction, with nitration of sulfhydryl-blocked HSA 2-fold higher than native HSA. Carbon dioxide accelerated peroxynitrite decomposition, enhanced aromatic amino acid nitration, and partially inhibited sulfhydryl oxidation of HSA. Nitration in the presence of carbon dioxide increased when the sulfhydryl was blocked. Thus, cysteine 34 was a preferential target of peroxynitrite both in the presence and in the absence of carbon dioxide.
Amino Acids↗
Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection.
The cellular immune response to a European isolate of porcine reproductive and respiratory syndrome (PRRS) virus in animals recovered from the experimental infection has been studied in vitro. Peripheral blood mononuclear cells (PBMC) from these pigs proliferated specifically when they were stimulated with PRRS virus. This response was not detectable until 4 weeks after inoculation and remained for more than 3 months. Addition of blocking monoclonal antibodies to the cultures showed that this proliferation was mainly dependent on CD4(+) cells with the participation of SLA-class II molecules. T-cell cultures established by stimulating responding cells with PRRS virus and maintained in culture for up to 3 weeks showed an increase of CD8(+) CD4(+) and CD4(-) CD8(+) subsets within activated cells, gated according to their light scatter parameters, whereas CD4(+) CD8(-) cells declined along the time in culture. Within the activated cells, those expressing the TcR gammadelta receptor also increased, being most of them also positive for the CD8 marker. By RT-PCR, T-cells responding to the virus showed a Th1 type cytokine production pattern. During the culture period the cytotoxic activity against K-562 cells increased from 15 to 35% of specific lysis. This cellular immune response may play a relevant role in the clearance of PRRS virus and the recovery of the infection.
Animals↗
Mechanism for the increased skeletal muscle protein degradation in the obese Zucker rat.
Obese (fa/fa) Zucker rats showed a decreased protein content in skeletal muscle compared with their lean counterparts. This was associated with both a decrease in the fractional rate of protein synthesis and an enhanced fractional rate of protein degradation in skeletal muscle, as studied by pre-loading body proteins with (14)C-bicarbonate. The increased protein degradation could be the result of the clearly enhanced expression for several transcripts of the ubiquitin genes in skeletal muscle. The results suggest that the increased protein degradation in the skeletal muscle of the obese animals may be due to increased activity of the ubiquitin-dependent proteolytic system.
Journal Article↗
Naming times for the Snodgrass and Vanderwart pictures in Spanish.
We present new Spanish norms for object familiarity and rated age of acquisition for 140 pictures taken from Snodgrass and Vanderwart (1980), together with data on visual complexity, image agreement, name agreement, word length (in syllables and phonemes), and five measures of word frequency. The pictures were presented to a group of 64 Spanish subjects, and oral naming latencies were recorded. In a multiple regression analysis, age of acquisition, object familiarity, name agreement, word frequency, and word length made significant independent contributions to predicting naming latency.
Adult↗
In the rat, tumor necrosis factor alpha administration results in an increase in both UCP2 and UCP3 mRNAs in skeletal muscle: a possible mechanism for cytokine-induced thermogenesis?
Since the discovery of the new members of the UCP (uncoupling protein) family, UCP2 and UCP3, very few studies have dealt with the regulation of their expression. Bearing this in mind, administration of a single intravenous injection of TNF-alpha (100 microg/kg body weight) to rats resulted in a significant increase in UCP2 (242%) and UCP3 (113%) gene expression in skeletal muscle. The results suggest a possible role for UCP2 and UCP3 in the increase of energy expenditure associated with cytokine treatment.
Animals↗
Different cytokines modulate ubiquitin gene expression in rat skeletal muscle.
Intravenous administration of different cytokines caused important changes in the expression of ubiquitin genes in skeletal muscle. Tumour necrosis factor-alpha caused a 2.2- and 1.9-fold increase in the expression of the 2.4 and 1.2 kb transcripts, respectively. Administration of interferon-gamma also caused a 2.2- and 1.8-fold increase in the 2.4 and 1.2 kb transcripts, respectively. While administration of leukaemia inhibitory factor and interleukin-6 resulted in no changes in ubiquitin gene expression, interleukin-1 administration also caused an increase in both ubiquitin gene transcripts (2.8- and 1.9-fold for the 2.4 and 1.2 kb transcripts, respectively). The results suggest that some of the cytokine effects on the ubiquitin system gene expression could be related to the enhanced skeletal muscle proteolysis found during cancer cachexia and other pathological states.
Animals↗
Skeletal muscle UCP2 and UCP3 gene expression in a rat cancer cachexia model.
Rats bearing the Yoshida AH-130 ascites hepatoma showed an increased expression of both uncoupling protein-2 (UCP2) (194%) and UCP3 (189%) mRNA levels in skeletal muscle 7 days after tumour inoculation. Interestingly, an even greater increase was observed in mRNA for both UCP2 (278%) and UCP3 (797%) in the pair-fed animals, suggesting that the increase in gene expression was the result of the anorexia associated with tumour burden. The results constitute the first report of UCP2 and UCP3 gene expression during cancer cachexia and agree to their possible role in the increase of energy expenditure associated with tumour growth.
Adipose Tissue↗
Formation of spin trap adducts during the decomposition of peroxynitrite.
Peroxynitrite-mediated one-electron oxidations may be an important event in its cytotoxic mechanisms, and yet, free radical formation in the presence of peroxynitrite is difficult to study by EPR-spin trapping because adducts from most spin traps are destroyed by the oxidant. This led to some controversy with regard to the interpretation of experiments in the presence of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), an adequate spin trap to study most of free radicals. In this report we reexamined peroxynitrite-mediate formation of spin-trap adducts. Kinetic studies and EPR experiments with water labeled with 17O are in agreement with the reaction of DMPO with a highly reactive intermediate derived from peroxynitrite to produce the DMPO-hydroxyl radical adduct by a mechanism not involving the oxidation of DMPO to a cation radical followed by water addition. The results cannot discriminate between two mechanisms of DMPO-hydroxyl radical formation, either spontaneous peroxynitrite homolysis to the hydroxyl radical or DMPO-assisted peroxynitrite homolysis. The formation of DMPO adducts during peroxynitrite-mediated oxidation of dimethyl sulfoxide, ethanol, and formate occurs through free radical mechanisms as confirmed by studies of oxygen consumption and product formation. Accordingly, spin-trapping experiments in the presence of 3,5-dibromo-4-nitrosobenzenesulfonic acid, a spin trap that is more resistant to nitrogen dioxide, led to the detection of the methyl and the beta-hydroxyethyl radical during peroxynitrite-mediated oxidation of dimethyl sulfoxide and ethanol, respectively. Oxidation of these hydroxyl radical scavengers to detectable radicals favors the hypothesis that the hydroxyl radical is produced during peroxynitrite homolysis. Bicarbonate was able to modulate peroxynitrite-mediated one-electron oxidations.
Free Radicals↗
Serous gland dimorphism in the skin of Melanophryniscus stelzneri (Anura: Bufonidae).
Two serous gland types (I and II) in the skin of the Argentine toad Melanophryniscus stelzneri were discovered using light and electron microscopy. Glands of the two types differ in several traits: features of the products (both mature and immature), organelles involved in biosynthesis, and paths of serous maturation. No consistent differences, however, were detected between the myoepithelial sheaths encircling the secretory units. Type I glands manufacture vesicles containing a single dense body with a repeating substructure and conform to the fundamental secretory line of bufonid skin, a secretory line involved in biosynthesis of steroids. Type II glands synthesize granules of varying densities and seem to belong to a line of glands that secrete proteinaceous products. The occurrence of the two serous gland types in Melanophryniscus stelzneri is discussed in a comparison with current literature on the morphofunctional characteristics of anuran poison glands, which perform both regulative and defensive roles. It is suggested that di- or polymorphism in serous glands is an adaptive trait that allows differential release of active molecules on the body surface.
Animals↗
Slowing of peroxynitrite decomposition in the presence of mannitol and ethanol.
The kinetics of peroxynitrite decomposition in the presence of the hydroxyl radical scavengers mannitol and ethanol were studied by stopped-flow spectrophotometry. Mannitol and ethanol decreased the rate of peroxynitrite decomposition in a concentration-dependent manner, following a hyperbolic function. The decreases in peroxynitrite decay rates were observed all throughout the pH range 5.8 to 8.0. In the presence of 100 mM mannitol or ethanol, the first-order rate constant for peroxynitrite decomposition changed from 1.25 +/- 0.01 s-1 at 25 degrees C, to values of 0.83 +/- 0.01 s-1 and 0.95 +/- 0.01 s-1, respectively. One explanation for this decrease in the rate of peroxynitrite decay with mannitol and ethanol could be a stabilizing effect of the substrate by hydrogen bonding with peroxynitrite, analogous to what has been recently proposed for hydrogen peroxide (Alvarez, B., Denicola, A. and Radi, R. Chem. Res. Toxicol. 8:859-869; 1995). In this sense, kinetic data fitted a mechanism implying fast equilibria between peroxynitrite anion and peroxynitrous acid with the substrates to form the corresponding complexes. The equilibrium constants of complex dissociation were estimated to be (6.7 +/- 0.9) x 10(-3) M and (9.6 +/- 1.5) x 10(-3) M for mannitol and ethanol, respectively. When bonded to mannitol or ethanol, peroxynitrous acid could ionize, too, or decompose at a slower rate than in the absence of substrate, in part to a reactive intermediate which performs oxidations. While mannitol and ethanol inhibit oxidation and nitration processes that occur through the reaction of secondary reactive intermediates of peroxynitrite with target molecules, up to 0.5 M mannitol or ethanol failed to inhibit cysteine oxidation by peroxynitrite at pH 7.4 and 25 degrees C. Thus, the formation of stabilizing complexes would not divert the reaction yield of direct, second order reactions such as thiol oxidation, but highlights the importance of hydrogen bonding and solvent effects on peroxynitrite stability.
Buffers↗
Protein turnover in skeletal muscle of the diabetic rat: activation of ubiquitin-dependent proteolysis.
Induction of experimental insulin-deficiency by a single administration of streptozotocin to rats resulted in substantial changes in heart and skeletal muscle size and protein content. This was accompanied by a marked loss of total body (carcass) nitrogen and raised concentrations of circulating branched-chain amino acids. These changes were related to alterations in protein turnover in skeletal muscle. Thus, the diabetic animals showed changes in both the fractional protein rates of synthesis (decreased by 37%) and degradation (increased by 141%). The increased protein degradation observed in the muscle of the diabetic animals was associated only with an increase in the expression of the genes controlling ubiquitin-dependent proteolysis. It may be suggested that the hormonal changes associated with the diabetic state play an important role in the regulation of the activity of the ubiquitin-dependent proteolytic system in skeletal muscle, highlighting the major role of this system in the diabetes-related cachexia.
3-Hydroxybutyric Acid↗
Pathways of peroxynitrite oxidation of thiol groups.
Peroxynitrite mediates the oxidation of the thiol group of both cysteine and glutathione. This process is associated with oxygen consumption. At acidic pH and a cysteine/peroxynitrite molar ratio of < or = 1.2, there was a single fast phase of oxygen consumption, which increased with increasing concentrations of both cysteine and oxygen. At higher molar ratios the profile of oxygen consumption became biphasic, with a fast phase (phase I) that decreased with increasing cysteine concentration, followed by a slow phase (phase II) whose rate of oxygen consumption increased with increasing cysteine concentration. Oxygen consumption in phase I was inhibited by desferrioxamine and 5,5-dimethyl-1-pyrroline N-oxide, but not by mannitol; superoxide dismutase also inhibited oxygen consumption in phase I, while catalase added during phase II decreased the rate of oxygen consumption. For both cysteine and glutathione, oxygen consumption in phase I was maximal at neutral to acidic pH: in contrast, total thiol oxidation was maximal at alkaline pH. EPR spin-trapping studies using N-tert-butyl-alpha-phenylnitrone indicated that the yield of thiyl radical adducts had a pH profile comparable with that found for oxygen consumption. The apparent second-order rate constants for the reactions of peroxynitrite with cysteine and glutathione were 1290 +/- 30 M-1.S-1 and 281 +/- 6 M-1.S-1 respectively at pH 5.75 and 37 degrees C. These results are consistent with two different pathways participating in the reaction of peroxynitrite with low-molecular-mass thiols: (a) the reaction of the peroxynitrite anion with the protonated thiol group, in a second-order process likely to involve a two-electron oxidation, and (b) the reaction of peroxynitrous acid, or a secondary species derived from it, with the thiolate in a one-electron transfer process that yields thiyl radicals capable of initiating an oxygen-dependent radical chain reaction.
Catalase↗
The metabolic basis of cancer cachexia.
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Adipose Tissue↗
The increased skeletal muscle protein turnover of the streptozotocin diabetic rat is associated with high concentrations of branched-chain amino acids.
Experimental streptozotocin-induced diabetes resulted in important changes in body weight which were associated with abnormalities in water and food intake. In addition, diabetic rats showed a clear muscle atrophy involving a decrease in both skeletal muscle size and protein content. This was accompanied by a marked loss of total carcass nitrogen. These changes were related to important alterations in protein turnover in skeletal muscle. Thus, the diabetic animals showed changes in the fractional protein rates of both synthesis (decreased by 37%) and degradation (increased by 140%). The increased protein degradation observed in the muscle of the diabetic animals was associated with important changes in the concentration of both circulating and muscle amino acids. Interestingly, the diabetic animals did not show important changes in either liver or kidney protein turnover rates, in spite of having a clear increase (over 50%) in kidney mass. In addition, and although the total amino acid concentration was not affected by the diabetic state, the chemically induced diabetic animals showed important elevations of branched-chain amino acids (leucine, isoleucine, and valine) in both blood and skeletal muscle. Similarly, important decreases in the blood concentrations of glutamate+glutamine, alanine, glycine, proline, serine, and threonine were also observed. These observations reinforce the idea of the association between muscle protein wasting, increased protein turnover, and alterations in branched-chain amino acids previously proposed by our group.
Amino Acids, Branched-Chain↗
Monoclonal antibodies specific for porcine monocytes/macrophages: macrophage heterogeneity in the pig evidenced by the expression of surface antigens.
Macrophages are widely distributed in most tissues of the body, where they play important roles in host defense and repair of tissue damage. In this report we describe the production and characterization of a panel of six monoclonal antibodies (mAb) against porcine macrophages and their use for phenotyping tissue macrophages. All mAbs were produced by immunizing mice with porcine alveolar macrophages. Three of them (2A10/11, 3B11/11 and 3F7/11) react mainly with macrophages and, at a lower extent, blood monocytes, whereas the others (1E12/11, 2C12/10 and 4E9/11) also recognize granulocytes. Antigens recognized by these antibodies could be characterized by Western blot and/or immunoprecipitation, with the exception of that one recognized by 2C12/10. By their behavior in SDS-PAGE under reducing and nonreducing conditions, all seem to be single polypeptides, whose apparent molecular weight under reducing conditions are: 1E12/11 and 3B11/11 larger than 204 kDa; 2A10/11, 150 kDa; 4E9/11, 125-170 kDa; and 3F7/11, 135 kDa. Immunohistochemical analyses of both lymphoid and non-lymphoid organs using these mAbs reveal important antigenic heterogeneity among tissue macrophages. These mAbs are, therefore, useful tools for the study of porcine macrophage maturation and differentiation and for determining their heterogeneity both in normal and pathological conditions.
Animals↗