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Biomedical subjects

J Villanueva

Publications and source records attributed to J Villanueva.

At least 19 recordsLinked to original sources
PubMed2006

Perforin polymorphism A91V and susceptibility to B-precursor childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group.

Perforin plays a key role in the cytotoxicity of natural killer and cytotoxic T cells. Genetic mutations in the perforin gene (PRF1) give rise to approximately 30% cases of familial hemophagocytic lymphohistiocytosis. A frequent polymorphism, A91V (C to T transition at position 272), may impair processing of perforin protein to the active form, and has been suggested to increase susceptibility to childhood acute lymphoblastic leukemia (ALL). To investigate the role of A91V in ALL, we genotyped 2272 children with de novo ALL registered on the Pediatric Oncology Group ALL Classification study P9900 and 655 normal controls. Allele frequencies in the controls showed a very low frequency of the variant allele in blacks, 0.7% compared to 4% in white controls. In light of this, analysis was restricted to a comparison of white cases and controls only. Overall genotype frequencies were similar in white ALL cases and normal white controls (P=0.58), indicating that in contrast to the previous report, A91V polymorphism is not associated with increased risk of childhood ALL. PRF1 A91V frequency was significantly increased in children with BCR-ABL positive ALL (24 vs 8.5%; P=0.0048); however, this observation includes a relatively small number of cases and needs further exploration.

Base Sequence↗
PubMed2003

Non-linear effects in the determination of paleotemperature U37(k') alkenone ratios by chemical ionization mass spectrometry.

The performance of gas chromatography coupled to mass spectrometry in the positive chemical ionization mode using ammonia as reagent gas (GC-PCI-MS) in the analysis of C37 alkenones for paleotemperature estimation has been re-evaluated. In some conditions, the discrepancies observed in the measurement of the U37(k') index with this technique as compared with GC equipped with flame ionization detection (GC-FID) cannot be explained by differences in sensitivity between the tri- and diunsaturated alkenones. Thus, at low (currently <0.3) or high (currently >0.4) U37(k') values the GC-PCI-MS determinations may be observed to be lower or higher, respectively, than those measured with GC-FID. As shown by analysis of a series of synthetic C37 alkenone standards these discrepant results can be explained by non linear effects in the GC-PCI-MS response factors. Second-order polynomial functions provide equations that describe better the signal to amount of analyte ratios. Users of GC-PCI-MS should calibrate their instruments with standards of known C37 alkenone composition in order to minimize non-linear effects.

Gas Chromatography-Mass Spectrometry↗
PubMed2002

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) up-regulates death receptor 5 (DR5) mediated by NFkappaB activation in epithelial derived cell lines.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L) activates nuclear factor kappaB (NFkappaB). This activation is regulated by the recruitment of an adaptor protein Fas associating death domain (FADD) to TRAIL death receptors, death receptor 4 (DR4, TRAIL-R1) and death receptor 5 (DR5 TRAIL-R2). This leads to recruitment of caspase 8 and receptor interacting protein (RIP) to the receptor complex. Upon recruitment of caspase 8 and RIP, NFkappaB inducing kinase (NIK) becomes activated causing NFkappaB activation. The role of TRAIL induced NFkappaB activation in epithelial cells is unknown. Herein we demonstrate that TRAIL increases expression of DR5 in human embryonic kidney (HEK) 293, MCF-7 and MDA MB 231 epithelial cell lines while DR4 expression remains unchanged. Blockage of NFkappaB activation either by expression of dominant negative IkappaB or treatment with proteasome inhibitor lactacystin eliminates TRAIL induced DR5 expression. Expression of FADD dominant negative in HEK 293 cells that prevents the recruitment of caspase 8 and RIP to TRAIL death receptors also eliminates this increase. By over expression of the p65 subunit of NFkappaB that increases NFkappaB transcriptional activity, DR5 expression was increased compared to vector alone expressing cells. By blocking TRAIL induced NFkappaB activation, the sensitivity of cells to undergo TRAIL induced apoptosis was significantly decreased. Conversely, the amount of TRAIL induced apoptosis was increased in HEK 293 cells over expressing p65 subunit of NFkappaB. Finally blockage of NFkappaB activation eliminates the synergistic apoptotic response of TRAIL and etoposide. Thus, TRAIL mediated NFkappaB activation increases DR5 expression thereby amplifying the apoptotic response of TRAIL in epithelial derived cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Monitoring the expression and purification of recombinant proteins by MALDI-TOF mass spectrometry.

1Samples coming from biologic sources usually contain several contaminants that interfere seriously with Mass Spectrometry (MS) measurements. In this paper we report the application of MALDI-TOF MS to monitor recombinant protein expression and purification. The technique is based on the use of a C18 resin to clean and concentrate proteins in batch. The utility of this method is demonstrated for samples coming from different bacterial cultures expressing secreted and intracellular proteins ranging from 4 to 53 kDa. MALDI-TOF MS of peptide and proteins can be accomplished directly from complex bacterial cultures or from any purification step in a few minutes using the conventional stainless steel sample targets, allowing for a nearly instantaneous monitoring of the nature and integrity of recombinant expression products.

Journal Article↗
PubMed2001

Low rate of acute lung allograft rejection after the use of daclizumab, an interleukin 2 receptor antibody.

BACKGROUND: The incidence and the severity of acute lung allograft rejection has been linked to the development of bronchiolitis obliterans syndrome. Therefore, we investigated the effects of daclizumab, a humanized monoclonal antibody directed against the alpha subunit of the interleukin 2 receptor, in reducing acute rejection after transplantation. METHODS: We retrospectively evaluated 27 patients who received daclizumab as induction immunosuppression and compared them with a historical control group of 34 patients. Both groups received similar immunosuppressive regimens involving tacrolimus, prednisone, and either azathioprine or mycophenolate mofetil. All patients received cytomegalovirus and aspergillus prophylaxis. RESULTS: Twenty-one patients in the control group and 22 patients in the daclizumab group were available for analysis at 6 months after lung transplantation. Ten (48%) patients in the control group had at least grade 2 acute rejection compared with four (18%) in the daclizumab group (P<0.04). The incidence of infection was similar in both groups. One patient in each group developed posttransplant lymphoproliferative disease. CONCLUSION: Therapy with daclizumab resulted in a significant decrease in the incidence of grade 2 or greater acute rejection after lung transplantation compared with historical controls. There seems to be no increase in the incidence of adverse effects in the patients treated with daclizumab.

Acute Disease↗
PubMed2001

Timing of cyclin D1 expression within G1 phase is controlled by Rho.

The expression of cyclin D1 in mid-G1 phase is associated with sustained ERK activity, and we show here that Rho is required for the sustained ERK signal. However, we also report that Rho inhibits an alternative Rac/Cdc42-dependent pathway, which results in a strikingly early G1-phase expression of cyclin D1. Thus, cyclin D1 is induced in mid-G1 phase because a Rho switch couples its expression to sustained ERK activity rather than Rac and Cdc42. Our results show that Rho is crucial for maintaining the correct timing of cyclin D1 expression in G1 phase and describe a new role for cytoskeletal integrity in the regulation of cell cycle progression.

3T3 Cells↗
PubMed2000

Horseradish peroxidase binding to intestinal brush-border membranes of Cyprinus carpio. Identification of a putative receptor.

Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush-border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K(d) = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin-type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3-kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization.

Animals↗
PubMed2000

Hydrogen exchange monitored by MALDI-TOF mass spectrometry for rapid characterization of the stability and conformation of proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back-exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially compare the activation domain of human procarboxypeptidase A2 versus three site-directed mutants of different conformational stability. In this work, we show that by using MALDI-TOF MS to monitor hydrogen exchange in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minimal amounts of sample.

Carboxypeptidases↗
PubMed2000

Extracorporeal photopheresis for the treatment of lung allograft rejection.

OBJECTIVES: Acute and chronic rejection continue to limit the survival of lung transplant recipients. Extracorporeal photopheresis has evolved as a possible therapy for patients with acute nd chronic lung allograft rejection. METHODS: We retrospectively reviewed 14 patients diagnosed with BOS who underwent therapy with extracorporeal photopheresis. RESULTS: Three patients were classified as BOS 0'b', five as BOS 1, three as BOS 2, and, three as BOS 3 at the time of diagnosis. Of the patients with BOS 0'b' or BOS 1 seven remain alive and one died of lung cancer. Two have progressed to BOS 2. Of the patients with BOS 2 or 3, four have died of BOS, one died of lung cancer, and one was re-transplanted. In three patients with BOS and concurrent acute rejection, therapy with extracorporeal photopheresis led to the resolution of the acute rejection episode. Two of the 14 patients developed line related sepsis. CONCLUSION: Extracorporeal photopheresis appears to be a promising therapy for patients with early BOS. It may also have a role in the treatment of acute lung allograft rejection.

Acute Disease↗
PubMed1998

Characterization of the wound-induced metallocarboxypeptidase inhibitor from potato. cDNA sequence, induction of gene expression, subcellular immunolocalization and potential roles of the C-terminal propeptide.

A partial cDNA clone for the potato wound-inducible metallocarboxypeptidase inhibitor (PCI) was isolated from a cDNA library constructed from mRNA of abscisic acid (ABA)-treated potato leaves. The full 5' region of the cDNA was obtained through a RACE-PCR protocol. PCI mRNA encodes a precursor polypeptide which comprises a 29 residue N-terminal signal peptide, a 27 residue N-terminal pro-region, the 39 residue mature PCI protein, and a 7 residue C-terminal extension. Northern blot analysis demonstrates that the PCI gene is transcriptionally activated by wounding, and wound signaling can be induced by ABA and jasmonic acid. Subcellular localization of the protein was investigated by immunocytochemistry and electron microscopy, showing that PCI accumulates within the vacuole. A partial PCI precursor form, comprising the mature protein and the C-terminal extension, has been expressed in Escherichia coli and characterized. Its inability to inhibit carboxypeptidases, and stability to carboxypeptidase digestion, suggest that the C-terminal pro-domain may have, besides a probable vacuolar sorting function, a role in modulation of the inhibitory activity of PCI.

Abscisic Acid↗
PubMed1998

CD30 expression identifies a functional alloreactive human T-lymphocyte subset.

BACKGROUND: CD30 is a member of the tumor necrosis factor/nerve growth factor receptor family and has been proposed as a marker of specific cytokine-producing subsets in humans. Previous studies have examined the expression of CD30 on established T helper type 1 and T helper type 2 cell clones and the function of CD30+ cells after mitogenic stimulation. In this study, we examined the development and function of CD30+ T cells generated in response to alloantigen. METHODS: Primary one-way mixed lymphocyte reactions were established, and the expression of CD30 on T lymphocytes was determined by immunofluorescence and flow cytometry. Fluorescence-activated cell sorting was utilized to define the cytokine profile of alloactivated CD30+ cells after restimulation with anti-CD3 monoclonal antibodies or alloantigen. The effect of cyclosporine on the development of CD30+ cells, and on cytokines produced by CD30+ T lymphocytes, in response to alloantigen was determined. RESULTS: CD30+ T lymphocytes could be detected on day 2 of mixed lymphocyte reactions and continued to increase in number and proportion through day 6. Both CD4 and CD8 T cells expressed CD30 after primary alloantigenic stimulation. CD30+ T cells are a subset of alloactivated T cells and are the major source of interferon-gamma and interleukin-5 produced in response to alloantigen. Cyclosporine partially, but not completely, inhibits the development of CD30+ cells, and has a greater effect on interferon-gamma production than on interleukin-5 production. CONCLUSIONS: CD30+ T lymphocytes may constitute an important immunoregulatory subset in human allograft rejection.

CD4-Positive T-Lymphocytes↗
PubMed1998

Membrane and tissue distribution of folate binding protein in pig.

Folate binding protein may participate in folate homeostasis by regulating monoglutamyl folate transport across relevant cell membranes. We compared the activity, immunoreactivity, and transcripts of folate binding protein in pig liver, kidney, and jejunal mucosa and their relevant cell membranes. Binding of [3H]folic acid was sixfold greater to pig liver plasma membranes than to kidney brush-border membranes, whereas there was no binding to jejunal brush-border membranes. The IgG fraction of rabbit antibody detected pig recombinant folate binding protein at 30 kDa and stained pig liver plasma membranes and kidney brush-border membranes but did not react with jejunal brush-border membranes. Folate binding protein transcripts were present in threefold greater abundance in pig liver than in kidney. Species comparisons showed folate binding protein transcripts in rat and human kidney but not in liver. Thus folate binding protein participates in folate homeostasis by regulating uptake by renal tubular membranes and uniquely by pig liver plasma membranes, but it is not involved in jejunal folate absorption.

Animals↗
PubMed1998

[Central lipoma of the mandible. A new case].

Lipomas are benign neoplasms affecting many organs of the body with adipose tissue. As a bone central lesion they represent less than 1% of all lipomas. In the literature lipomas are reported in different bones: calcaneum, ribs, fibula, phalanges, ulna, frontal and parietal bone. In the jaws 8 cases have been reported, most of them representing radiographic findings the clinician diagnosing cyst or tumor, specially odontogenic tumor. No preference has been reported for any gender, age or race but the condition appears after the fourth decade. We report a case of central lipoma in the mandible with the clinical, radiological and histopathological findings and discuss its origin.

Adipose Tissue↗