Biomedical subjects

J Márquez

Publications and source records attributed to J Márquez.

At least 19 recordsLinked to original sources

PubMed1999

A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG, in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms.

A randomized blinded clinical trial was performed in 53 patients bitten by Bothrops sp. and Porthidium sp. in Antioquia and Chocó, Colombia, in order to compare the efficacy and safety of two antivenoms made of whole IgG obtained by either ammonium sulphate (monovalent anti-B. atrox) or caprylic acid (polyvalent) fractionation. Additionally, antivenoms were compared by electrophoretic and chromatographic analyses and anticomplementary activity in vitro. With a protocol of 2, 4 and 6 antivenom vials for the treatment of mild, moderate and severe envenomings, respectively, both antivenoms were equally efficient to neutralize the most relevant signs of envenoming and to clear serum venom levels in patients from the first hour and later on. Three patients with severe envenoming and initially treated with less than six vials on admission had persistent or recurrent venom antigenemia within 12-48 h. Monovalent antivenom fractionated by ammonium sulphate precipitation had higher amounts of protein aggregates and nonimmunoglobulin proteins than polyvalent antivenom fractionated by caprylic acid precipitation. Both antivenoms presented anticomplementary activity in vitro, being higher in the monovalent product. In agreement, monovalent antivenom induced a significantly higher incidence of early antivenom reactions (52%) than polyvalent antivenom (25%).

Adolescent↗

PubMed1999

Upregulation of glyceraldehyde-3-phosphate dehydrogenase mRNA in the spleen of tumor-bearing mice.

The influence of tumor implantation on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and stability was determined in the spleen of tumor-bearing mice. While GAPDH mRNA levels were not altered in skeletal muscle, kidney and liver from tumor-bearing mice, tumor implantation led to a 5.6-fold increase in the levels of splenic GAPDH mRNA. An enhanced message stability was observed in splenocytes from tumor-bearing animals, suggesting the involvement of post-transcriptional mechanisms in the selective GAPDH mRNA accumulation after tumor implantation. The GAPDH activity/glycolytic flux ratio was 18.5 in the spleen of healthy mice. Therefore, the three-fold increase in the glycolytic flux observed after tumor implantation could hardly justify the necessity for the upregulation of GAPDH.

Animals↗

PubMed1999

Subcutaneous fat necrosis of the newborn.

Subcutaneous fat necrosis of the newborn (SFN) is an uncommon disease that affects newborns who have suffered from tissue hypoxia during or following delivery. This disease appears during the first weeks of life. It consists of indurate, erythematous or purple-erythematous nodules and plaques in the skin. Histology of a biopsy specimen shows granulomatous necrosis in the subcutis with radial crystals in lipocytes and giant cells. Spontaneous resolution in a few weeks is usual, but the mobilization of calcium from the necrosed subcutis together with the action of some hormones may cause hypercalcemia and certain serious complications. A newborn female child developed SFN after dystocic delivery causing cerebral frontal lobe hemorrhage. The skin nodules resolved spontaneously in a few weeks and no complications were observed 1 year later.

Fat Necrosis↗

PubMed1998

Involvement of essential cysteine and histidine residues in the activity of isolated glutaminase from tumour cells.

The pH dependence of the phosphate-activated glutaminase isolated from Ehrlich tumour cells suggests a functional role for two prototropic groups with apparent pKa of 9.3 and 7.7 at the active site of the protein; these pKa values are compatible with cysteine and histidine residues, respectively. This possibility was investigated by chemical modification studies of the purified enzyme. N-Ethylmaleimide fully inactivated the purified glutaminase; the reaction order was very close to 1.0, suggesting that N-ethylmaleimide modifies glutaminase at a single essential site. Spectrophotometric studies of the isolated protein treated with diethyl pyrocarbonate indicate that two histidine residues are modified. Since glutaminase is loosely associated to the inner mitochondrial membrane, modification experiments were also carried out using mitochondrial membrane fractions. N-Ethylmaleimide and diethyl pyrocarbonate gave similar results in mitochondria membrane-bound enzyme to those obtained with purified enzyme. Glutamate, which behaves as a competitive inhibitor of the enzyme, partially protected the inactivation caused by N-ethylmaleimide in membrane-bound experiments. The results suggest the existence of a critical histidine residue(s) in the tumour glutaminase, and strongly support the notion that a cysteine residue, which is located at (or near) the active site, is involved in the catalytic mechanism as well.

Animals↗

PubMed1998

Early differential expression of two glutaminase mRNAs in mouse spleen after tumor implantation.

The influence of progressive tumor growth on phosphate-activated glutaminase (PAG) expression in splenocytes from mice bearing Ehrlich ascites carcinoma cells was investigated. Implantation of Ehrlich ascites tumor cells in the peritoneal cavity of mice led to a 2.3-fold stimulation of spleen PAG activity 48 h later. Four days after tumor implantation the glutaminase activity had returned to nearly basal value and remained at this level throughout the tumor development. Northern blot analysis indicated that two species of glutaminase mRNA were expressed in the spleen, which showed a differential expression pattern during the first 2 days after tumor implantation. The abundance of the transcript of higher electrophoretic mobility (approximately 3 kb) constantly increased over the first 2 days of tumor growth. The mRNA of lower electrophoretic mobility (approximately 6 kb) peaked at 12 h after tumor implantation and returned to control values at 48 h. These results demonstrate that tumor has the capability of altering glutaminase expression in the host spleen.

Animals↗

PubMed1998

Roughness feature of metaphase chromosome spreads and nuclei for automated cell proliferation analysis.

As a step towards automation of mitotic index estimation for cell proliferation studies, a roughness feature of surface-intensity images is introduced: the mean depth-width ratio of extrema (MDWRE). This feature allows identification of variable-shaped metaphases and interphase nuclei in the presence of many artefacts (one metaphase per hundreds of nuclei and thousands of artefacts). The texture of the cytological objects (seen as rough surfaces) is quantified by scanning, in one dimension, the lines contained in a closed contour. MDWRE proves to be suitable for image magnifications by a factor of as low as ten, making faster scanning of slides possible. The use of this feature gives +14%, +65%, +133% and +133% better performance figures than classical textural features derived from co-occurrence matrices, such as contrast, energy, entropy and angular second moment, respectively, and +51% better than the relative extrema density (RED). The MDWRE per object and the shape of the histogram of the depth-width ratio of grey-level roughs have been shown to be very useful as textural features for the classification of metaphase images.

Cell Culture Techniques↗

PubMed1997

Early tumor effect on splenic Th lymphocytes in mice.

Tumors are characterized by their ability to avoid the host immune system. Ehrlich ascites tumor cells were used to investigate the early alterations of the host immune system after tumor inoculation. The results show that frequencies of splenic Th lymphocytes were drastically reduced during tumor growth, reaching a minimum only two days after tumor inoculation. The frequency of splenic CD4+ lymphocytes expressing IFN-gamma was significantly increased, although the total number was unchanged, suggesting that there was no net induction of Th1-type response. Splenic macrophages were increased, in both frequency and cell number, after four days of tumor growth. The same pattern was observed when mice were inoculated with cell free ascitic fluid. TGF-beta precursors were detected in tumor cells as well as in ascitic fluid. The data suggest that tumor actively interacts with host immune system by means of tumor cell secreted factors.

Animals↗

PubMed1997

Polyamine contents of human breast cancer cells treated with the cytotoxic agents chlorpheniramine and dehydrodidemnin B.

The cytotoxic agents chlorpheniramine and dehydrodidemnin B decreased the cell growth of estrogen receptor-negative human breast cancer cells MDA-MB231 and estrogen receptor-positive MCF-7, after 48 h treatment. Both agents reduced ornithine decarboxylase activity, but polyamine levels were increased in MDA-MB231 cells treated with dehydrodidemnin B. MCF-7 cells when treated with dehydrodidemnin B showed significant increases in spermidine and spermine contents. The results suggest that besides other effects, the cytotoxicity of DDB could be explained in part by the over-accumulation of spermidine and spermine.

Antineoplastic Agents↗

PubMed1997

Submitochondrial localization and membrane topography of Ehrlich ascitic tumour cell glutaminase.

The intramitocondrial localization of the phosphate-activated glutaminase from Ehrlich cells has been examined by a combination of techniques, including: mitochondria subfractionation studies, chemical modification with sulfhydryl group reagents of different permeability, enzymatic digestion in both sides of the inner mitochondrial membrane, and immunological studies. Using alkaline extraction at high ionic strength, hypoosmotic shock and freezing-thawing cycle techniques, the enzyme was found in the particulate fraction. On the contrary, glutaminase activity was labile when subfractionation was carried out by digitonin/lubrol method; Western blot analysis localized the inactive enzyme in the matrix fraction. In addition, glutaminase was fully inactivated when mitoplasts were incubated with phospholipase A2 and phospholipase C. The enzyme also showed a non-linear Arrhenius plot with a break at 24 degrees C. The membrane-impermeant thiol reagents mersalyl and p-chloromercuriphenylsulfonic acid do not inhibit glutaminase activity in freeze-thawed mitochondria and mitoplasts, but N-ethylmaleimide, which is membrane permeant, strongly inhibited the enzyme. However, mersalyl and p-chloromercuriphenylsulfonic acid were effective inhibitors when the alkylation was performed on the matrix side of mitoplasts or using detergent-solubilized enzyme. Furthermore, trypsin digestion of mitoplasts was only effective inactivating glutaminase when the proteolysis was carried out on the matrix side of the vesicles. Enzyme-linked immunosorbent assay of the soluble and membrane fractions obtained in the preparation of submitochondrial particles, revealed that most of the enzyme was solubilized, but in the inactive form. Phase separation with Triton X-114 rendered most of the protein in the aqueous phase. These results taken together discard a transmembrane localization for the protein, whereas they are consistent with anchorage of glutaminase on the matrix side of the inner mitochondrial membrane, the matrix portion of the enzyme being relevant for its function.

4-Chloromercuribenzenesulfonate↗

PubMed1997

[The absence of interference between GSM mobile telephones and implantable defibrillators: an in-vivo study. Groupe Systèmes Mobiles].

INTRODUCTION AND OBJECTIVES: The electromagnetic field created by mobile telephones can cause pacemaker dysfunction. Although implantable cardioverter defibrillators are also susceptible to electromagnetic interference, few studies have addressed this issue and compatibility with the GSM mode has not been tested. This study was developed to detect possible "in vivo" interference between GSM mobile telephones and implantable cardioverter defibrillators. MATERIAL AND METHODS: The study group is composed of 30 patients with 8 different models of defibrillators. Twenty six had endocardial leads and 4 epicardial. Three GSM mobile phones were used: Siemens S3 COM and Motorola 6200 in all cases and Ericsson GA 318 in one. The tests were performed under continuous electrocardiographic monitoring. All therapies were deactivated and sensitivities were set to maximal parameters. The telephones were positioned in close contact to the defibrillator can and precordium, in two different angles. Three situations were evaluated: calling, established contact for 15 seconds and ringing. The protocol was repeated during pacing to assess the possibility of pacemaker mode inhibition. RESULTS: No cases of electromagnetic interference were observed. One patient presented non-sustained ventricular tachycardia episodes during the tests that were detected by the defibrillator. CONCLUSIONS: These results suggest that electromagnetic interference by GSM mobile phones are not a probable cause of implantable defibrillators dysfunction.

Defibrillators, Implantable↗

PubMed1997

Use of interferon-alpha in laryngeal papillomatosis: eight years of the Cuban national programme.

Laryngeal papillomatosis is one of the first diseases where interferon (IFN) was found to be effective. In 1983, a programme for the treatment of all such cases started in Cuba. Up to December 1991, 125 patients (92 children, 33 adults) have been treated: 102 with leucocyte IFN-alpha, 12 with recombinant IFN-alpha-2b, and 11 have received both preparations. Case management consisted of surgical removal of the lesions followed by an IFN schedule starting with 10(5) IU/kg of weight in children or 6 x 10(6) IU in adults, i.m. daily. The dose was progressively reduced, as long as no relapses occurred. At the end of the one-year schedule the doses were reduced to 5 x 10(4) IU/kg in children or 3 x 10(6) IU in adults, weekly. If there was a relapse, it was removed surgically and the patient returned to a higher dose level. Most cases (89; 71 per cent) have not relapsed after the treatment; 60 of them have been followed for more than three years. In those with relapses, the frequency of recurrence decreased in all but four patients. The treatment seemed to be more effective if initiated less than three months after the disease onset. The tracheostomy could be removed in five out of seven patients who needed it before the IFN treatment and was necessary in only three new cases during IFN treatment. In two of these, decannulation was possible later on. In a total of 14 patients relapses persisted after several cycles of IFN treatment. They were considered resistant to such treatment. No severe side effects were reported. The most frequent ones were fever, drowsiness, increased bronchial secretion, chills and headache. The establishment of this programme has maintained the disease under control in Cuba.

Adolescent↗

PubMed1997

Identification of a Zn(2+)-sensitive component of Ehrlich cell plasma membrane redox system by CHAPS-agarose-polyacrylamide electrophoresis and in situ staining of activity.

A procedure based on CHAPS-agarose-polyacrylamide electrophoresis and in situ staining of activity was used to detect a Zn(2+)-sensitive component of Ehrlich cell plasma membrane redox system. The procedure is so powerful that it allows to use crude plasma membrane fractions and can be easily adapted for use in an electrophoretic approach to the purification of this protein.

Animals↗

PubMed1996

Effects of protein kinase C and phosphoprotein phosphatase modulators on Ehrlich cell plasma membrane redox system activity.

Diacyl glycerols and phorbol esters, which activate protein kinases C, stimulated Ehrlich ascites tumor cell ferricyanide reductase activity. On the contrary, selective inhibition of active protein kinases C with bis-indolyl maleimide did not change the rate of ferricyanide reduction by Ehrlich cells. Selective inhibitors of phosphoprotein phosphatases, okadaic acid and cyclosporin A, also stimulated plasma membrane redox system. Taking all these data together, protein kinases or phosphoprotein phosphatases seemed to be involved in the multiple and complex regulation of Ehrlich cell plasma membrane redox system.

Animals↗

PubMed1996

Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells.

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis-Menten kinetics for the substrates, with apparent K(m) values of 4.3 X 10(-5) M and 6.7 X 10(-5) M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thio-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

Animals↗

PubMed1996

Three-dimensional analysis of the arrangement of compact chromatin in the nucleus of G0 rat lymphocytes.

The arrangement of compact chromatin of G0 lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was serially cut and sections were contrasted with procedures preferential for DNA. Electron microscopy images were digitized, processed, and displayed using a commercial software package, complemented by a system for three-dimensional reconstruction and analysis developed by us on an IBM-compatible microcomputer provided with an image acquisition board. The reconstructions showed a continuous layer of compact chromatin in contact with the nuclear envelope that prevents the automatic recognition of individual chromatin clumps. The ensemble of the arrangement of compact chromatin was found to be very similar in different lymphocytes. After morphological filtering procedures, the initial mass was divided into individual bodies of compact chromatin, which were tagged. Most of these bodies contact the nuclear envelope. The number of bodies as well as the number of contacts with the envelope are similar and correspond to a haploid number of chromosomes. The largest body is always the one containing nucleolus-associated chromatin. When the cell has two nucleoli, the nucleolus-associated chromatin bodies contact the envelope in diametrically opposed areas. This feature was also described in rat liver cells. It is concluded that: (a) the individualized compact chromatin bodies do not correspond to an entire chromosome or to a pair of chromosomes; (b) the arrangement of compact chromatin is not identical in each G0 lymphocyte, but there are patterns that are repeated with limited changes; and (c) there are common features that appear in different cell types of individuals of the same species.

Animals↗

PubMed1996

Immunocytochemical localization of allergenic proteins in Parietaria judaica L. (Urticaceae) pollen grains.

Inhalation of pollen grains and fungal spores leads to allergenic rhinitis and type I asthma mediated by IgE. The etiology of these conditions is due to the presence of substances that can induce anaphylactic processes in susceptible subjects. A combination of transmission electron microscopy with immunocytochemical methods was used to localize allergenic proteins in Parietaria judaica L. (Urticaceae) pollen grains. Germination of pollen grains was induced in vitro for 10 and 30 min to activate proteins in the intine and in material exuded from the pollen grains that are related to pollen-stigma recognition; these activated proteins would be the ones that react with the antibodies. The activation time, labeling intensity and specificity of the antigens depended on whether IgG (rabbit) or IgE (allergenic patient) was used. Abundant, non-specific labeling was observed with rabbit IgG. The labeling observed with human IgE was located more specifically in the intine (oncus) at random points near the apertures. The immunolocalization of allergenic proteins in the intine indicates the presence of proteins related to activation of the pollen grains. This fact confirms the function attributed to the intine in the processes of pollen tube formation and fertilization, and also the possible mechanism that is activated in the pollen grains when these reach the mucosa of sensitive subjects. In the case of P. judaica, the existence of a thickening of the intine below the apertures (oncus) and the release of its contents in an explosive fashion seem to be the main causes of their strong allergenic activity.

Allergens↗

PubMed1995

Glutamine transport by vesicles isolated from tumour-cell mitochondrial inner membrane.

Mitochondrial-inner-membrane vesicles, isolated from Ehrlich ascites carcinoma cells by titration with detergents, accumulated L-glutamine by a very efficient transport system. The vesicles lack any phosphate-activated glutaminase activity, allowing measurement of transport rates without interference by L-glutamine metabolism. The time course of the transport was linear for the first 60 s, reaching a steady state after 120 min. L-Glutamine transport showed co-operativity, with a Hill coefficient of 2.2; the kinetic parameters S0.5 and Vmax had values of 5 mM and 26 nmol/30 s per mg of protein respectively. The pH-dependence curve showed a bell shape, with a pH optimum about 8.0. The uptake of L-glutamine was not affected by the presence of a 50-fold molar excess of D-glutamine, L-cysteine, L-histidine, L-alanine, L-serine and L-leucine, whereas L-glutamate behaved as a poor inhibitor. The structural analogue L-glutamate gamma-hydroxamate (5mM) inhibited the net uptake by 68%; interestingly, other analogues (6-diazo-5-oxo-L-norleucine, acivicin and L-glutamate gamma-hydrazide) were ineffective. The impermeant thiol reagent p-chloromercuriphenylsulphonic acid (0.5mM) completely abolished the mitochondrial L-glutamine uptake; in contrast, other thiol reagents (mersalyl and N-ethylmaleimide) did not significantly affect the transport. These data confirm the existence of a specific transport system with high capacity for L-glutamine in the mitochondrial inner membrane, a step preceding the highly operative glutaminolysis in tumour cells.

Amino Acids↗

PubMed1995

Tumor glutaminase purification.

Two alternative purification schemes to obtain the glutaminase from Ehrlich tumor cells in a highly purified form have been developed. One experimental approach is based on conventional and high-performance liquid chromatography fractionation techniques, yielding a 37-fold higher purification than has been previously reported. The method comprises: isolation of mitochondria, solubilization with Triton X-100, ion-exchange and hydroxyapatite chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography. A second purification schedule has been optimized employing native polyacrylamide gel electrophoresis, in situ activity staining, and electroelution of the protein band. This approach resulted in a simple and rapid isolation of a 10-fold higher purified glutaminase than before, minimizing also the potential for proteolytic inactivation of the enzyme. The apparent molecular weight of the protein in native form was determined by gel filtration and sucrose density gradient ultracentrifugation. Polyclonal antibodies raised against Ehrlich glutaminase were immunopurified against the pig kidney enzyme. Immunoblot analyses employing these antibodies as well as anti-rat kidney glutaminase antibodies revealed the same pattern of bands seen with the purified enzyme.

Animals↗