Biomedical subjects

I Martinez

Publications and source records attributed to I Martinez.

At least 37 records · Page 2Linked to original sources

PubMed1997

Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.

A very simple, fast, universally applicable and reproducible method to extract high quality megabase genomic DNA from different organisms is described. We applied the same method to extract high quality complex genomic DNA from different tissues (wheat, barley, potato, beans, pear and almond leaves as well as fungi, insects and shrimps' fresh tissue) without any modification. The method does not require expensive and environmentally hazardous reagents and equipment. It can be performed even in low technology laboratories. The amount of tissue required by this method is approximately 50-100 mg. The quantity and the quality of the DNA extracted by this method is high enough to perform hundreds of PCR-based reactions and also to be used in other DNA manipulation techniques such as restriction digestion, Southern blot and cloning.

Animals↗

PubMed1997

Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods.

Five grams of seafood products were inoculated with one to 500 viable or 10(9) heat-killed cells of Listeria monocytogenes. The presence of the pathogen was detected by the polymerase chain reaction (PCR) with primers specific for fragments of the listeriolysin O (hly) gene (two sets) and for the invasion-associated protein (iap) gene (one set). For DNA preparation, boiling, either alone or in combination with lysozyme and proteinase K treatment, was not always sufficient to lyse L. monocytogenes, while treatment with Triton X-100 produced consistently good DNA suitable for amplification. To avoid false-negative and false-positive results, 48 h incubations were necessary and a subculturing step after an initial 24 h incubation greatly improved the results. The primers that amplified regions of the listeriolysin O gene gave clearer and stronger products than primers for the invasion-associated protein gene. Using this method we were able to detect one to five L. monocytogenes cells in 5 g of product in a total of 55 h.

Animals↗

PubMed1997

Cloning and characterization of a cDNA encoding a maize seedling phytase.

During germination, maize seedlings express a phytase able to hydrolyse the large amount of phytin stored in the dry seed. Previous studies allowed purification and characterization of this enzyme as a homodimer of 38 kDa subunits [Laboure, Gagnon and Lescure, Biochem. J. (1993) 295, 413-419]. In the present work, an antibody against the purified maize phytase has been used to screen a maize seedling cDNA expression library. Several positive clones containing an insert of about 1400 bp were isolated. The nucleotide sequence of the insert of one of these clones has been established. This cDNA, called phy S11, was 1335 bp long and contained an open reading frame of 387 amino acids. The sequence of N-terminal residues (23 amino acids) of the purified phytase has been established. These residues are found at positions 19-41 of the amino acid sequence encoded by phy S11. This confirms that this cDNA codes for the maize phytase. The deduced amino acid sequence appears to be very different from those of published Aspergillus niger phytases; however, an homologous region of 33 amino acids was detected. This region of the fungal sequence contains the RHGxRxP consensus motif found in various high molecular mass acid phosphatases and believed to be the acceptor site for phosphate. Expression of the phy S11 cDNA in Escherichia coli allowed the production of the phytase subunit and its assembly to give a protein of the same size as the native phytase. The time course of phy S11 mRNA accumulation during germination showed that no transcript was present in dry seeds. The mRNA accumulated during the first day of germination, to reach a maximum after 2 days (radicle protrusion), and then decreased in young seedlings. Genomic Southern blot analyses suggest the existence of at least two genes and genetic mapping reveals two loci separated by 1 cM on chromosome 3 of maize. The cloning of this first cDNA coding for a plant phytase, will allow the isolation of the corresponding genes and the study of their regulation during germination.

6-Phytase↗

PubMed1997

The Breakdown of Olivine to Perovskite and Magnesiowustite

San Carlos olivine crystals under laboratory conditions of 26 gigapascals and 973 to 1473 kelvin (conditions typical of subducted slabs at a depth of 720 kilometers) for periods of a few minutes to 19 hours transformed to the phase assemblage of perovskite and magnesiowustite in two stages: (i) the oxygen sublattice transformed into a cubic close-packed lattice, forming a metastable spinelloid, and (ii) at higher temperatures or longer run durations, this spinelloid broke down to perovskite and magnesiowustite by redistributing silicon and magnesium while maintaining the general oxygen framework. The breakdown was characterized by a blocking temperature of 1000 kelvin, below which olivine remained metastable, and by rapid kinetics once the reaction was activated.

Journal Article↗

PubMed1997

The importance of dietary calcium and phosphorous in the secondary hyperparathyroidism of patients with early renal failure.

Secondary hyperparathyroidism (HPT) was evaluated in 157 patients with chronic renal failure (CRF). It was noted that HPT developed early in CRF at a time when plasma calcium and phosphorous were within normal limits. As creatinine clearance decreased below 80 mL/m, there was a significant decrease in plasma calcitriol and a slow and progressive significant increment in plasma parathyroid hormone (PTH). The effect of dietary intake of calcium and phosphorous was evaluated in these patients with early renal failure (ERF). They were divided into two groups. Group A was placed on a protein- and phosphorous-restricted diet (10 days) followed by a daily phosphorous-load diet (10 days). Group B had similar sequential diets plus a calcium supplement throughout the study. Dietary protein and phosphorous restriction resulted in an amelioration of the HPT only in the group of patients receiving calcium supplementation. The phosphate-load diet resulted in worsening of HPT in both groups. In summary, a deficit of calcitriol occurs early in CRF, which in turn leads to a significant increase in PTH. Phosphorous restriction, together with calcium supplementation, ameliorated the HPT of patients with ERF.

Adolescent↗

PubMed1997

Vaccine evaluation studies of replication-defective SIVsmB7.

Non-infectious virus-like particles of SIVsmB7 that expresses env and gag gene products but are defective in pol and vpx/vpr were assessed for their ability to induce protective immunity against infection with pathogenic SIVsmE660 in rhesus macaques. Animals were immunized in three groups: group A was primed with cell-associated SIVsmB7 and boosted with cell-free SIVsmB7; group B was primed with cell-free SIVsmB7 and boosted with cell-free SIVsmB7 conjugated to iron oxide microbeads; group C was primed with cell-free SIVsmB7 mixed with Titer Max adjuvant and boosted with cell-free SIVsmB7 mixed with SAF-M adjuvant followed by secondary boosting with cell-free SIVsmB7 conjugated to microbeads. Animals were challenged intravenously with 20 animal infectious doses of SIVsmE660 grown in rhesus peripheral blood mononuclear cells 3 weeks after final boosting. All animals became infected as evidenced by quantitative virus cultivation. Sera from immunized animals contained low-titer antibodies by ELISA and low or undetectable neutralizing antibodies on the day of challenge but strong anamnestic antibody responses were observed following challenge. Interestingly, 2 of 3 animals in group A showed evidence of transient viremia and more stable CD4 counts following challenge as compared to the other immunized animals and to non-immunized controls. Thus, immunization with cell-associated SIVsmB7 did not provide sterilizing immunity against challenge with a highly pathogenic SIV strain but might have caused virus clearance later in infection.

Animals↗

PubMed1996

Nitric oxide down-regulates endocytosis in rat liver endothelial cells.

Hepatic sinusoidal endothelial cells (SEC) were challenged with inducers, blockers or donor of nitric oxide (NO) production in vitro to test the effect of this reactive nitrogen agent on endocytosis in SEC. NO was measured as its stable form nitrite (NO2-) in culture media and trace amounts of radioiodinated ligands were tested for endocytosis and binding after 6 and 24 h of incubation. Among several proinflammatory reagents tested, IL-1 beta induced most strongly NO synthesis after 24 h in culture. Although endocytosis was significantly enhanced in SEC that had been exposed to IL-1 beta for 6 h, prolonged exposure (24 h) to this proinflammatory cytokine, which triggered increased production of NO by the cells, yielded a decreased endocytic activity. The presence of aminoguanidine, an inhibitor of NO synthase, gave significant up-regulation of endocytosis compared with control cells. To fully verify the role of NO as an endocytosis modulator, SEC were preincubated with sodium nitroprusside, an exogenous NO donor. Again, increased concentration of NO2- in the medium was associated with decreased endocytosis of SEC. Binding studies at 4 degrees C revealed that the down-regulation of endocytosis in SEC after increased exposure to NO was due to a decreased number or affinity of receptors on the cell surface.

Animals↗

PubMed1996

Partial reconstitution of a replication-competent retrovirus in helper cells with partial overlaps between vector and helper cell genomes.

Contamination of retroviral vector preparations with replication-competent retroviruses is a major safety concern in human gene therapy. These can arise by recombination between retroviral vectors and packaging cell sequences. Recently, we constructed new packaging lines, derived from spleen necrosis virus (SNV) that do not contain overlapping regions of homology between these two components (DSH134G and DSH29B cells). These cell lines were tested for the presence of recombination products and replication-competent viruses in comparison to a similar packaging line (DSN) that contains a partial overlap between vector and viral protein coding regions. No recombination products were detected in DSH cells. However, we found that recombination had occurred in DSN cells, partially reconstituting a provirus-like structure that was capable of being spread, although inefficiently, through an infected cell culture. Our data indicate that even small regions of sequence homology eventually allow homologous recombination between vector and helper cell genomes.

Animals↗

PubMed1996

Hepatic sinusoidal endothelium heterogeneity with respect to mannose receptor activity is interleukin-1 dependent.

Using fluorescein isothiocyanate-conjugated ovalbumin (OVA-FITC), 125I-mannan, or 125I-invertase as specific ligands for the mannose receptor, we have quantified its activity in mouse and rat hepatic sinusoidal endothelium (HSE), under both basal conditions and after lipopolysaccharide (LPS) or human recombinant interleukin-1beta (IL-1beta) stimulations. Mouse treatment for 4 hours with 5 microg/kg IL-1beta significantly increased OVA-FITC uptake by HSE. Ligand uptake exhibited a sublobular compartmentalization: In control mice as well as in IL-1beta-stimulated mice, the ligand distributed preferentially in the periportal and septal areas; no OVA-FITC was observed in the perivenous sinusoids. In vitro exposure of mouse HSE to 100 pg/mL LPS or 1 ng/mL IL-1beta for 6 hours significantly (P < .01) increased OVA-FITC uptake. Blocking IL-1 receptors in HSE by addition of 100 ng/mL IL-1 receptor antagonist (IL-1Ra) before stimulation with LPS or IL-1beta abrogated the increase in mannose receptor-mediated uptake. In vitro endocytosis assays showed that rat HSE uptake of 125I-mannan or 125I-invertase progressively increased with both exposure time and concentration of added IL-1beta. Upregulation of mannose receptor-mediated uptake in response to IL-1beta or LPS was also blocked by previous addition of IL-1Ra to rat HSE. Flow cytometric analysis showed a significant HSE heterogeneity in mannose receptor-mediated endocytosis in response to IL-1beta treatment: type I endothelial cells (EC-I, defined by their small size and high cytoplasmic density) significantly (P < .01) increased OVA-FITC uptake compared with type II endothelial cells (EC-II, defined by their large size and low cytoplasmic density). In addition, the subset of EC-I contained three times more IL-1beta-binding cells than the EC-II subset. Because EC-I and EC-II are preferentially located in the periportal and perivenous segments of hepatic sinusoids, respectively, these results suggest that IL-1beta, apart from upregulating mannose receptor activity, contributes to the sublobular compartmentalization of this endothelial cell function.

Animals↗

PubMed1996

A deficit of calcitriol synthesis may not be the initial factor in the pathogenesis of secondary hyperparathyroidism.

Secondary hyperparathyroidism (HPT) develops early in chronic renal failure (CRF) at a time when plasma calcitriol levels are normal. At this time, PTH are higher than normal controls and serum phosphorous levels are lower. A decrement in total serum Ca is noted, after an oral phosphate load, only in patients with ERF. These data suggest that factors, other than a decrease in calcitriol synthesis, may be involved in the pathogenesis of HPT. A hypothesis is forwarded suggesting that an alteration in the newly cloned calcium sensor receptor may be the earliest abnormality in the HPT, preceding a decrease in plasma calcitriol levels.

Adolescent↗

PubMed1996

Mutational analysis of the envelope protein of spleen necrosis virus.

Spleen necrosis virus (SNV) is an amphotropic type C retrovirus originally isolated from a duck. The envelope protein is related to that of type D retroviruses, and SNV appears to use the same receptor as do simian retroviruses. However, little is known about envelope-receptor interactions of SNV. We constructed a series of envelope mutants to characterize the SU peptide of SNV. Point mutations were introduced throughout SU in regions that are conserved among all retroviruses belonging to the same receptor interference group. The biological and biochemical properties of these mutants were analyzed. All mutants were transported efficiently to the cell surface. Almost all mutations in the amino-terminal one-third caused a conformational change of the envelope and a significant drop in infectivity and abolished the ability to confer superinfection interference. Similar observations were made with only two of seven mutants with mutations in the middle of SU. Four mutations in this region had little or no effect on biological activity. One mutant envelope protein (Asp to Arg at position 192) was processed normally but showed little infectivity and had no ability to confer superinfection interference. A detailed mutational analysis suggested that this amino acid forms a hydrogen bond to its cellular receptor. Mutations within the carboxy-terminal part of SU had very little or no effect on biological function. Aberrantly processed envelope proteins were proteolytically cleaved at a new point upstream of and differing in sequence from the conserved retroviral SU/TM cleavage site. Surprisingly, these mutants still retained some infectivity (0.01 to 1% of that of the wild type). Our data indicate that the envelope of SNV behaves in a manner very different from that of the envelopes of other studied retroviruses.

Amino Acid Sequence↗

PubMed1996

Analytical and clinical evaluation of a procedure for measuring CA 15-3 in the IMx analyser.

CA 15-3 is the most widely used tumour marker in the follow-up of patients with breast cancer. Its postoperative blood level is closely related to the response to the treatment applied and to the evolution of the disease. In this study, we assessed both the clinical and analytical features of a new microparticle enzyme immunoassay for CA 15-3 quantification in the Abbott IMx system. In the precision study, the coefficients of variation within runs, between runs and between laboratories were 3.5, 5.1 and 6.5% or less, respectively. The analytical sensitivity of the assay was 0.11 U/ml. Linear regression analyses of the IMx CA 15-3 assay with the Centocor and CIS CA 15-3 RIAs gave correlation coefficients of 0.88 and 0.95, respectively. The upper limit of the reference range, obtained from serum of healthy women, was 27.3 U/ml, while the ROC curve analysis for patients with active breast cancer compared to patients with no evidence of the disease gave an optimum cutoff of 33 U/ml (sensitivity 0.76 and specificity 0.87). The overall agreement of the three methods was over 90%.

Breast Neoplasms↗

PubMed1996

Cerebral infarction and antiphospholipid syndrome in children.

OBJECTIVE: To investigate the prevalence of antiphospholipid antibodies (aPL) in children with acute cerebral infarction. METHODS: The study was carried out in 10 consecutive patients during the first days of neurological symptoms. aPL detected as lupus anticoagulant or anticardiolipin antibodies (aCL) and natural anticoagulant proteins C, S, and antithrombin III were determined in all patients. RESULTS: Seven patients had acute cerebral infarctions associated with aCL; 4 of these patients had high serum aCL concentrations in 2 different determinations (antiphospholipid syndrome). Two patients had temporary protein C deficiency. In one patient with negative aCL, protein C, S, and antithrombin III determinations were not carried out. No patient had evidence of connective tissue disease or family history of hypercoagulable state. After followup of 15.7 months, no patient had recurrent infarction while taking aspirin. CONCLUSION: Our study demonstrates high prevalence of aCL in children who suffer acute cerebral infarction and our results suggest aspirin may be effective therapy to prevent recurrences.

Adolescent↗

PubMed1995

Differential cytokine-mediated modulation of endocytosis in rat liver endothelial cells.

We have studied the effects of exogenous tumor necrosis factor-alpha, interleukin-1 beta, and lipopolysaccharide-induced interleukin-1 beta on receptor-mediated endocytosis in primary cultures of liver sinusoidal endothelial cells. Tumor necrosis factor-alpha and interleukin-1 beta enhanced endocytosis via the scavenger and mannose receptors in a dose-time dependent manner, while the function of the collagen receptor remained unaffected. The modulatory effects of the cytokines were blocked by adding specific inhibitors for both cytokines. Results from studies on binding (4 degrees C) and internalization and degradation (37 degrees C) of ligands indicate that the increased scavenger capacity of liver sinusoidal endothelial cells resulting from exposure to the inflammatory mediators was due to increased rate of intracellular transport of endocytosed ligands to lysosomes, rather than to increased binding to receptors.

Animals↗

PubMed1995

Improved retroviral packaging lines derived from spleen necrosis virus.

Using highly efficient gene expression vectors, we constructed new retroviral packaging lines derived from spleen necrosis virus. Core proteins are expressed from the murine leukemia virus promoter and enhancer followed by the tripartite leader sequence of an adenovirus. Using different plasmids for envelope expression, we found that the efficiency of vector transduction is dependent on the level of gag-pol expression. The level of envelope expression did not have a measurable impact on vector virus titers. The new helper cell lines do not contain any sequences homologous to vector genomes. They transduce standard retrovirus vectors with titers up to 10(6) colony forming units per milliliter of supernatant tissue culture medium. No replication-competent virus was observed.

Animals↗

PubMed1995

Mapping of receptor binding domains in the envelope protein of spleen necrosis virus.

Spleen necrosis virus (SNV) is an amphotropic retrovirus originally isolated from a duck. Although of avian origin, it also replicates on some mammalian cells. SNV-derived retroviral vectors work with high efficiency and have a high potential for various gene transfer applications. However, little is known about the envelope-receptor interactions of this virus. We constructed a series of recombinant envelope proteins to characterize the SU peptide of SNV. We found that, in contrast to the envelope proteins of other retroviruses, truncated envelope proteins of SNV are transported to the cell surface. Surprisingly, particles displaying truncated envelope proteins can still infect cells, although at reduced efficiencies. Furthermore, these proteins can confer partial superinfection interference. Our data suggest that peptides throughout SU are involved in envelope-receptor interactions. To more precisely determine the localization of the main receptor binding domain, point mutations were introduced at certain regions of the SNV SU which are highly conserved among retroviruses belonging to the same receptor interference group. We identified one point mutation in the middle of SU (position 192) which drastically reduced infectivity and strongly reduced the ability to confer superinfection interference. The level of expression was not abolished, and translocation to the cell membrane of the mutant envelope occurred efficiently. This indicates that amino acid 192 may be directly involved in receptor binding.

Amino Acid Sequence↗

PubMed1995

Fluconazole treatment of candida peritonitis with delayed removal of the peritoneal dialysis catheter.

Candida peritonitis was treated with fluconazole in ten continuous ambulatory peritoneal dialysis (CAPD) patients without immediate removal of the peritoneal catheter. Shortly prior to diagnosis, six patients (60%) had received broad-spectrum antibiotics. Gram stain of peritoneal fluid detected yeast in 70% of cases. In eight patients the peritoneal dialysis catheter was removed within one week of diagnosis because of clinical deterioration. In the majority of cases (90%), candida peritonitis resolved only after catheter removal in spite of ongoing fluconazole therapy. Fluconazole was well tolerated by all patients.

Antifungal Agents↗

PubMed1995

Highly efficient eukaryotic gene expression vectors for peptide secretion.

Recently, we constructed a series of highly efficient universal eukaryotic gene expression vectors (Sheay et al., BioTechniques 15:856-862, 1993). Such vectors contain a viral promoter and enhancer followed by the adenovirus tripartite leader sequence, a multiple cloning site for the insertion of the gene of interest and a polyadenylation sequence. To enable expression of peptides to be secreted into the tissue culture medium or to be incorporated into the cell membrane, several modifications have been introduced into such vectors: stop codons in all three reading frames were inserted at the end of the multiple cloning site and a DNA sequence coding for a signal peptide for transport through the endoplasmatic reticulum (ER) was introduced downstream of the adenovirus tripartite leader sequence followed by two unique restriction enzyme recognition sites. A protocol is described that allows fast and easy cloning of peptide-coding regions, i.e., PCR products, for expression and secretion. The transport of a genetically engineered chimeric transmembrane protein connected to this ER leader sequence was as efficient as that of the original protein from which the ER sequence has been derived. These universal vectors can also be used for the easy construction of any chimeric transmembrane or secretion proteins.

Amino Acid Sequence↗