Biomedical subjects

Alfredo Martínez

Publications and source records attributed to Alfredo Martínez.

At least 19 recordsLinked to original sources

PubMed2007

The nitric oxide donor LA 419 decreases ischemic brain damage.

Stroke represents a major clinical problem with limited available therapeutic treatments. Nitric oxide (NO) and the enzymes that produce it are involved in the pathogenesis of this disease. Here we investigated whether the novel NO donor LA 419 was able to ameliorate the consequences of stroke in an experimental model of global ischemia. We observed a sharp increase in the amounts of inducible NO synthase (iNOS) and nitrotyrosine in the cerebral cortex of experimental rats and a moderate increase of neuronal NO synthase (nNOS), as demonstrated by immunohistochemistry, Western blotting, and enzymatic activity assays. Treatment of these animals with LA 419 completely prevented ischemia-induced upregulation of nitrergic markers. Magnetic resonance imaging of the experimental brains showed a marked decrease in apparent diffusion coefficient (ADC) following ischemia-reperfusion, which was significantly corrected by pre-treatment with LA 419. These results clearly show that LA 419 is an efficient modulator of NO-related pathophysiological events and could eventually be used for the treatment of patients with cerebrovascular pathologies.

Animals↗

PubMed2006

Bayesian estimation of Tritrichomonas foetus diagnostic test sensitivity and specificity in range beef bulls.

Accuracy of culture for diagnosis of Tritrichomonas foetus was investigated in 2832 naturally exposed range beef bulls from 124 herds. Preputial fluid samples were inoculated into the culture medium, incubated at 37 degrees C, and daily examined. Diagnostic test was evaluated using Bayesian techniques to estimate sensitivity and specificity without a gold standard. Median posterior test sensitivity was 72.04% (95% probability interval: 58.07-86.38%) and specificity was 95.37% (95% probability interval: 94.07-96.65%). Low diagnostic test accuracy may have resulted from host and/or diagnostic test procedure related factors. Under natural range conditions, more accurate methods for T. foetus diagnostic and repeated preputial samplings of bulls may be necessary on trichomonosis control programs.

Animals↗

PubMed2006

Molecular evolution of proadrenomedullin N-terminal 20 peptide (PAMP): evidence for gene co-option.

Posttranslational processing of proadrenomedullin generates two biologically active peptides, adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Sequence comparison of homologous proadrenomedullin genes in vertebrate evolution shows a high degree of stability in the reading frame for AM, whereas PAMP sequence changes rapidly. Here we investigate the functional significance of PAMP phylogenetic variation studying two of PAMP's better characterized physiological activities, angiogenic potential and antimicrobial capability, with synthetic peptides carrying the predicted sequence for human, mouse, chicken, and fish PAMP. All tested peptides induced angiogenesis when compared with untreated controls, but chicken and fish PAMP, which lack terminal amidation, were apparently less angiogenic than their human and mouse homologs. Confirming the role of amidation in angiogenesis, Gly-extended and free acid variants of human PAMP produced responses similar to the natural nonamidated peptides. In contrast, antimicrobial activity was restricted to human PAMP, indicating that this function may have been acquired at a late time during the evolution of PAMP. Interestingly, free acid human PAMP retained antimicrobial activity whereas the Gly-extended form did not. This fact may reflect the need for maintaining a tightly defined structural conformation in the pore-forming mechanism proposed for these antimicrobial agents. The evolution of PAMP provides an example of an angiogenic peptide that developed antimicrobial capabilities without losing its original function.

1-Naphthylamine↗

PubMed2006

Adrenomedullin (AM) and adrenomedullin binding protein (AM-BP) in the bovine mammary gland and milk: Effects of stage of lactation and experimental intramammary E. coli infection.

Adrenomedullin (AM) has been characterized as an endogenous tissue survival factor and modulator of many inflammatory processes. Because of the increased susceptibility of the mammary gland to infection during the time surrounding parturition in the cow, we investigated how milk and tissue content of AM and its binding protein (AM-BP) might be affected by the stage of lactation and the udder health status. Milk and mammary biopsy samples were obtained from Holstein cows 21 days prior to and at various times after calving to represent the dry period and early and mid-stages of lactation. Additional cows received an intramammary challenge with Escherichia coli for immunohistochemical characterization of AM and AM-BP. Milk AM concentrations were relatively constant across the stages of lactation while AM-BP increased two-fold (P<0.04) between early and mid-lactation. Milk AM (P<0.04) and AM-BP (P<0.03) increased as somatic cell counts (SCCs) increased within a given stage of lactation. Tissue content of both (AM and AM-BP) were significantly affected by stage of lactation, lowest in the dry period and progressively increasing to peak at mid-lactation as well as increasing in association with higher levels of SCCs. Following E. coli challenge, AM increased in epithelial cells surrounding mammary alveoli presenting high levels of SCCs. The data suggest that AM and AM-BP are cooperatively regulated in the mammary gland during lactation; changes in localized tissue AM and AM-BP content reflect a dynamic regulation of these tissue factors in the bovine mammary gland consistent with their protective effects within inflamed tissue.

Adrenomedullin↗

PubMed2006

Adrenomedullin: a new and promising target for drug discovery.

Adrenomedullin (AM) is a 52 amino acid peptide that plays a critical role in several diseases such as hypertension, cancer, diabetes, cardiovascular and renal disorders, among others. Interestingly, AM behaves as a protective agent against some pathologies, yet is a stimulating factor for other disorders. Thus, AM can be considered as a new and promising target for the design of non-peptidic modulators that could be useful for the treatment of those pathologies, by regulating AM levels or the activity of AM. A full decade on from its discovery, much more is known about AM molecular biology and pharmacology, but this knowledge still needs to be applied to the development of clinically useful drugs.

Adrenomedullin↗

PubMed2006

Adrenomedullin is a cross-talk molecule that regulates tumor and mast cell function during human carcinogenesis.

We have previously demonstrated that adrenomedullin (AM) plays a critical role as an autocrine/paracrine tumor cell survival factor. We now present evidence that AM is an important regulator of mast cell (MC) function and that this modulation is potentially involved in tumor promotion. AM induced histamine or beta-hexosaminidase release from rat and human MCs through a receptor-independent pathway. AM was chemotactic for human MCs and stimulated mRNA expression of vascular endothelial growth factor, monocyte chemoattractant protein-1, and basic fibroblast growth factor in this cell type. Differentiated but not undifferentiated human MCs responded to hypoxic insult with elevated AM mRNA/protein expression. Using confocal microscopy, we identified AM-producing MCs in tumor infiltrates of human breast and lung cancer patients. In mixed culture assays the AM-producing human MC line HMC-1 augmented both anchorage-dependent and -independent growth of human lung cancer A549 cells, an effect that was suppressed by MC-targeted siRNA AM knockdown. Finally, HMC-1 cells induced in vivo angiogenesis as assessed by directed in vivo angiogenesis assay analysis; neutralizing anti-AM monoclonal antibody blocked this ability. Our collective data suggest a new role for AM as a cross-talk molecule that integrates tumor and MC communication, underlying a unique promotion mechanism of human cancers.

Adrenomedullin↗

PubMed2005

Synthesis, biological evaluation, and three-dimensional quantitative structure-activity relationship study of small-molecule positive modulators of adrenomedullin.

Adrenomedullin (AM) is a peptide hormone implicated in blood pressure regulation and in the pathophysiology of several diseases such as hypertension, cancer, diabetes, and renal disorders, becoming an interesting new target for the development of drugs. In a recent high-throughput screening study, a positive modulator with a bistriazole structure has been identified.(1) In this work, a new series of structurally related compounds has been synthesized by reaction of phenoxyacetic acid with the corresponding dihydrazide, followed by treatment of the formed bisoxadiazoles with benzylamine. The affinity toward AM of the lead compound, and a structurally related family obtained from the small-molecule NCI library together with the synthesized series, has been determined. A three-dimensional quantitative structure-activity relationship (3D-QSAR) study and conformational and molecular dynamics simulations have shown that the presence of a free NH and a phenyl group is essential for the interaction of these compounds with AM.

Adrenomedullin↗

PubMed2005

Gastrin-releasing peptide (GRP) induces angiogenesis and the specific GRP blocker 77427 inhibits tumor growth in vitro and in vivo.

Angiogenesis is becoming a major target for antitumor therapies, and identifying new angiogenic factors and their specific inhibitors may provide new avenues for tumor management. Here we identify gastrin-releasing peptide (GRP) as a new angiogenic molecule that is secreted by tumors and acts directly upon GRP receptors in the endothelial cells. Addition of GRP increases endothelial cell migration and cord formation in vitro, and induces angiogenesis in an in vivo assay. We have recently identified a small molecule GRP blocker, compound 77427. This inhibitor significantly reduced endothelial cell cord formation in vitro and angiogenesis in vivo. Conversely, when applied to VEGF-induced angiogenesis, the small molecule did not have any effect, demonstrating its specificity. Furthermore, this GRP blocker was able to reduce lung tumor cell growth in vitro as demonstrated by MTT and clonogenic assays. When applied to a xenograft model with lung cancer cells, compound 77427 reduced tumor volume to undetectable sizes, although when the treatment was suspended, tumors began to grow again at normal rates. Our collective observations indicate that GRP is a new angiogenic peptide and that its inhibition offers an attractive tool to reduce tumor burden.

Base Sequence↗

PubMed2005

A new family of angiogenic factors.

Angiogenesis is the production of new blood vessels from pre-existing ones. This process is tightly regulated by a series of pro- and anti-angiogenic molecules in normal physiology and when this equilibrium is broken serious consequences may arise. Solid tumors are characterized by a fast growth that eventually pushes cells away from their natural source of oxygen and nutrients from the capillaries. To survive in this hypoxic environment, tumor cells secrete a variety of pro-angiogenic molecules that would elicit proliferation of new blood vessels, thus re-establishing oxygen and nutrient supply. Blockade of angiogenesis may provide a rational approach to managing tumor growth and novel strategies are being developed. The identification of new targets is of paramount importance in the search for a clinically proficient anti-angiogenic therapy. The adrenomedullin family of peptides and gastrin-releasing peptide (GRP) are newly identified pro-angiogenic molecules, secreted by the tumors, whose inhibition results in a considerable reduction of angiogenesis and of tumor growth in animal models. The recent identification of small molecules that reduce the angiogenic effect of these peptides opens new avenues for the development of new anti-tumorigenic drugs.

Adrenomedullin↗

PubMed2005

Inhibitors of the arachidonic acid pathway and peroxisome proliferator-activated receptor ligands have superadditive effects on lung cancer growth inhibition.

Arachidonic acid (AA) metabolizing enzymes and peroxisome proliferator-activated receptors (PPARs) have been shown to regulate the growth of epithelial cells. We have previously reported that exposure to the 5-lipoxygenase activating protein-directed inhibitor MK886 but not the cyclooxygenase inhibitor, indomethacin, reduced growth, increased apoptosis, and up-regulated PPARalpha and gamma expression in breast cancer cell lines. In the present study, we explore approaches to maximizing the proapoptotic effects of PPARgamma on lung cancer cell lines. Non-small-cell cancer cell line A549 revealed dose-dependent PPARgamma reporter activity after treatment with MK886. The addition of indomethacin in combination with MK886 further increases reporter activity. We also show increased growth inhibition and up-regulation of apoptosis after exposure to MK886 alone, or in combination with indomethacin and the PPAR ligand, 15-deoxy-Delta12,14-prostaglandin J2 compared with single drug exposures on the adenocarcinoma cell line A549 and small-cell cancer cell lines H345, N417, and H510. Real-time PCR analyses showed increased PPAR mRNA and retinoid X receptor (RXR)alpha mRNA expression after exposure to MK886 and indomethacin in a time-dependent fashion. The results suggest that the principal proapoptotic effect of these drugs may be mediated through the known antiproliferative effects of the PPARgamma-RXR interaction. We therefore explored a three-drug approach to attempt to maximize this effect. The combination of low-dose MK886, ciglitazone, and 13-cis-retinoic acid interacted at least in a superadditive fashion to inhibit the growth of lung cancer cell lines A549 and H1299, suggesting that targeting PPARgamma and AA action is a promising approach to lung cancer growth with a favorable therapeutic index.

Acetophenones↗

PubMed2005

Characterization of adrenomedullin in birds.

Adrenomedullin (AM) is a multifunctional evolutionarily highly conserved peptide. Although its genomic and amino acid (aa) sequences are known in several mammalian species and in fish, the structure of the AM gene remains unknown in intermediate phyla, including birds. Here, we report the structure and aa sequence of the chicken (c) AM ortholog. The cAM gene is located at the short arm of chromosome 5, which shows high synteny with the short arm of human (h) chromosome 11, where hAM is located. Key sequences in the third intron have been conserved which allow for an alternative splicing mechanism, similar to the one found in mammals. The preprohormone contains two peptides with high homology to human proadrenomedullin N-terminal 20 peptide (PAMP) and hAM. We found through real-time PCR and immunocytochemistry cAM mRNA and peptide expression in a variety of chicken tissues, which parallel patterns observed for mammals, with the exception that cAM levels are almost non-detectable in brain. Similarly to mammals, cAM expression is upregulated under hypoxic conditions and following dexamethasone treatment. These data demonstrate a high degree of homology between the cAM gene and its mammalian ortholog and evolutionary conservation of the regulatory mechanisms controlling its expression.

Adrenomedullin↗

PubMed2005

Differentially expressed nucleolar TGF-beta1 target (DENTT) shows tissue-specific nuclear and cytoplasmic localization and increases TGF-beta1-responsive transcription in primates.

Differentially Expressed Nucleolar TGF-beta1 Target (DENTT) is a new member of the TSPY/TSPY-like/SET/NAP-1 (TTSN) superfamily whose mRNA is induced by TGF-beta1 in TGF-beta1-responsive human lung cancer cells. Monkey DENTT mRNA contains a 2085-bp open reading frame that encodes a predicted polypeptide of 695 amino acids with five nuclear localization signals, two coiled-coil regions, and a domain that shows significant identity to a region that defines the TTSN superfamily. RT-PCR amplification and Western blot analyses showed DENTT mRNA and protein in adult monkey tissues, including the adrenal gland, cerebral cortex, and ovary. Immunohistochemical staining showed that numerous neurons were intensely immunoreactive for DENTT, as were anterior pituitary secretory cells, thyroid follicular cells, and smooth muscle cells of arteries and lung bronchial walls. DENTT expression was also prominent in monkey bronchiolar-alveolar adenomas and cell lines. While the addition of TGF-beta1 or retinoic acid to monkey normal lung bronchial 12MBr6 cells and human lung cancer NCI-H727 cells increased DENTT protein production, TGF-beta1 together with retinoic acid resulted in a more sustained increase in DENTT production than with TGF-beta1 or retinoic acid alone. Transient transfection studies showed that ectopic DENTT expression significantly increased TGF-beta1-responsive 3TP-Lux and CAGA12-Lux reporter transcription in 12MBr6 and NCI-H727 cells with TGF-beta1 addition, while ectopic DENTT expression had no significant effect on the transcription of a retinoic acid-responsive element reporter in the presence of retinoic acid or TGF-beta1. These findings suggest new possibilities for DENTT as a TGF-beta1-regulated, but not a retinoic acid-regulated member of the TTSN superfamily in primate physiology.

Adrenal Glands↗

PubMed2005

Adrenomedullin: a new target for the design of small molecule modulators with promising pharmacological activities.

Adrenomedullin (AM) is a 52-amino acid peptide with a pluripotential activity. AM is expressed in many tissues throughout the body, and plays a critical role in several diseases such as cancer, diabetes, cardiovascular and renal disorders, among others. While AM is a protective agent against cardiovascular disorders, it behaves as a stimulating factor in other pathologies such as cancer and diabetes. Therefore, AM is a new and promising target for the development of molecules which, through their ability to regulate AM levels, could be used in the treatment of these pathologies.

Adrenomedullin↗

PubMed2005

The contributions of Santiago Ramón y Cajal to cancer research - 100 years on.

In 1906, Santiago Ramón y Cajal received the Nobel Prize for his detailed description of the nervous system. But Cajal (pronounced 'Cahal') worked in many fields and his contributions to cancer research have been overlooked, mainly because his observations were published in Spanish. As we near Cajal's Nobel Prize centennial celebration, we review his histological slides, original drawings and publications that are related to cancer. There is an impressive body of evidence that shows Cajal's interest in tumour biology.

Anatomy↗

PubMed2005

Pigment epithelium-derived factor is a substrate for matrix metalloproteinase type 2 and type 9: implications for downregulation in hypoxia.

PURPOSE: Pigment epithelium-derived factor (PEDF), a protein secreted by the retinal pigment epithelium (RPE), acts on retinal survival and angiogenesis. Because hypoxia and VEGF regulate matrix metalloproteinases (MMPs), their effects on PEDF proteolysis were explored. METHODS: Mouse models for retinopathy of prematurity (ROP) were used. Cultured monkey RPE cells were exposed to low oxygen and chemical hypoxia mimetics. PEDF and VEGF mRNA levels in RPE were determined by RT-PCR. MMPs were assessed by zymography, DQ-gelatin degradation solution assays, and MMP immunostaining. PEDF proteolysis was assayed in solution and followed by SDS-PAGE and immunostaining. MMP induction by VEGF was performed in baby hamster kidney (BHK) cells. Retinal R28 cell survival, ex vivo chick embryonic aortic vessel sprouting, and directed in vivo angiogenesis assays were performed. RESULTS: Levels of PEDF in RPE/choroid significantly decreased in the ROP model. Hypoxia decreased PEDF levels in the media conditioned by RPE cells, with no significant change in PEDF mRNA. Conversely, PEDF proteolysis, gelatinolytic activities of approximately 57-kDa and approximately 86-kDa zymogens, and MMP-2 immunoreactivities increased with hypoxia. Addition of VEGF to BHK cells caused a time and dose-related upregulation of approximately 57-kDa zymogens and of DQ-gelatinolytic and PEDF-degrading activity. The PEDF-degrading activity and approximately 57-kDa zymogens in the BHK media shared MMP protease inhibition patterns and MMP-2 immunoreactivities with those in the vitreous. Limited proteolysis with MMP-2 and -9 degraded PEDF in a Ca(+2)-dependent fashion. MMP-mediated proteolysis of PEDF abolished the retinal survival and antiangiogenic activities of the PEDF protein. CONCLUSIONS: Hypoxia and VEGF can downregulate PEDF through proteolytic degradation. PEDF is a novel substrate for MMP-2 and -9. These results reveal a novel posttranslational mechanism for downregulating PEDF, and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios, in angiogenesis and/or in neuronal death.

Animals↗

PubMed2005

Identification of adrenomedullin in avian type II pneumocytes: increased expression after exposure to air pollutants.

Adrenomedullin (AM) is a potent vasodilator peptide present in the lung of mammals where it is expressed mainly in the columnar epithelium and alveolar macrophages. AM increases the secretion of phosphatidylcholine by type II pneumocytes, which suggests a role as an autocrine modulator of surfactant secretion. In this study we show the expression of an AM-like protein in the lung of the pigeon, Columba livia. Using an antibody against its human ortholog, AM-like immunoreactivity was found to be associated with membranous structures of the multivesicular bodies of type II pneumocytes. We also studied the differential expression of AM-like peptide in the lung of pigeons exposed to polluted city air vs cleaner countryside conditions and found that AM-like expression was higher in city animals. Similar results were obtained in an experimental study in which pigeons were exposed to increasing concentrations of a single pollutant, ozone. Taken together, our findings support the implication of AM in the response of type II pneumocytes to air pollutants.

Adrenomedullin↗

PubMed2004

Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator.

MMPs (matrix metalloproteinases) play a major role in the pathogenesis of hypertension by altering the extracellular matrix during cardiovascular remodelling. In the present study we show that MMP-2, but not MMP-9, cleaves the vasodilator peptide AM (adrenomedullin). Addition of the AM-binding protein, complement factor H, prevents this cleavage, providing a hitherto unknown mechanism of action for this binding protein. We identified the signature cleavage fragments and found some of them in human urine, suggesting that MMP-2 processing of AM may occur in vivo. Synthetic AM fragments regulated blood pressure in rats. The larger peptides are vasodilators, as is intact AM, whereas intermediate fragments did not affect blood pressure. In contrast, AM(11-22) elicited vasoconstriction. Studies of AM receptor activation in Rat2 cells confirm that the larger AM cleavage peptides activated this receptor, whereas AM(11-22) did not. The present study defines a new mechanism through which MMP-2 may regulate blood pressure by simultaneously eliminating a vasodilator and generating a vasoconstrictor.

Adrenomedullin↗

PubMed2004

Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, and its inhibition results in reduction of tumor growth.

We have found through ex vivo and in vivo angiogenesis models that the adrenomedullin gene-related peptide, proadrenomedullin NH2-terminal 20 peptide (PAMP), exhibits a potent angiogenic potential at femtomolar concentrations, whereas classic angiogenic factors such as vascular endothelial growth factor and adrenomedullin mediate a comparable effect at nanomolar concentrations. We found that human microvascular endothelial cells express PAMP receptors and respond to exogenous addition of PAMP by increasing migration and cord formation. Exposure of endothelial cells to PAMP increases gene expression of other angiogenic factors such as adrenomedullin, vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor C. In addition, the peptide fragment PAMP(12-20) inhibits tumor cell-induced angiogenesis in vivo and reduces tumor growth in xenograft models. Together, our data demonstrate PAMP to be an extremely potent angiogenic factor and implicate this peptide as an attractive molecular target for angiogenesis-based antitumor therapy.

Adrenomedullin↗