Biomedical subjects

F Casares

Publications and source records attributed to F Casares.

At least 19 recordsLinked to original sources

PubMed2001

Presence of morphine and morphine-6-glucuronide in the marine mollusk Mytilus edulis ganglia determined by GC/MS and Q-TOF-MS. Starvation increases opiate alkaloid levels.

Morphine and morphine-6-glucuronide, a morphine metabolite, have been identified and quantified in Mytilus edulis pedal ganglia at a level of 2.67+/-0.44 and 0.98+/-0.14 ng/ganglia, respectively by high performance liquid chromatography coupled to electrochemical detection. These opiate alkaloids were further identified by both gas-chromatography mass spectrometry and nanoflow electrospray ionization double quadrupole orthogonal acceleration Time of Flight mass spectrometry. In animals that were starved, the morphine level rose to 6.38+/-0.88 ng/ganglion and the morphine 6-glucoronide rose to a level of 23.0+/-3.2 ng/ganglion after 30 days. These studies demonstrate that opiate alkaloids are present as naturally occurring signal molecules whose levels respond to stress, i.e., starvation. Opiate alkaloids were not found in the animal's incubation media or food, demonstrating their synthesis occurred in the respective tissue. These new method of opiate alkaloid detection, conclusively proves that morphine and morphine-6-glucuronide are present in animal tissues.

Animals↗

PubMed2001

The presence of morphine in ganglionic tissues of Modiolus deminissus: a highly sensitive method of quantitation for morphine and its derivatives.

Morphine and morphine-6-glucuronide, a morphine metabolite, have been identified and quantified in Modiolus deminissus pedal ganglia at a level of 2.41 and 0.95 ng/ganglia, respectively. These opiate alkaloids are normally found at low concentrations in invertebrate and vertebrate tissues, including neural. Given this problem, we also describe a new opiate extraction protocol as well as a high-performance liquid chromatography purification procedure that can separate and quantify morphine and its derivatives at sub-nanogram concentrations. Furthermore, both morphine and morphine-6-glucuronide were identified in this mollusk's pedal ganglia by mass spectrometry analysis.

Animals↗

PubMed2001

HIV gp120 and morphine alter mu opiate receptor expression in human vascular endothelium.

We find that chronic exposure of human saphenous vein, atria and internal thoracic artery endothelium to the human immunodeficiency virus surface glycoprotein gp120, results in an increase in endothelial mu opioid receptor expression (52%). gp120 acts, in this regard, as a proinflammatory cytokine (e.g. interleukin-1-alpha) by increasing endothelial mu opioid receptor expression. In contrast, morphine decreases mu opioid receptor expression by 90% in a dose dependent fashion. Pretreatment of these tissues with the respective antagonists e.g., naloxone and anti-gp120 blocks the opiate decrease and increase gp120 induced increase in mu expression, respectively. Further, pretreatment of these endothelia with morphine inhibits gp120-stimulated mu transcript expression. Therefore, the immune down-regulating action of morphine may prevent viral replication because this process requires immune activation that can, in part, be provided for by gp120 proinflammatory actions.

Analgesics, Opioid↗

PubMed2000

Lipopolysaccharide increases endogenous morphine levels in rat brain.

The present study was designed to determine whether whole body injection of lipopolysaccharide increases endogenous rat brain morphine levels. High performance liquid chromatography coupled to electrochemical detection and gas-chromatography mass spectrometry reveal that the resting brain morphine level is 7.0+/-3.2ng/g wet weight. In a time dependent manner, intraperitoneal injection of lipopolysaccharide (10 microg), leads to an increase of brain morphine level with a peak reaching after 36h post injection. After 72h following the lipopolysaccharide-injection, morphine levels increased to 66.0+/-5.4ng/g brain wet weight (P<0.001). Furthermore, 96h of fasting also increased the brain morphine level to 44.0+/-3. 6ng/g of brain wet weight. These results suggest that rat brain morphinergic processes respond to organismic challenges after a significant latent period.

Animals↗

PubMed2000

Identification of morphine in the adrenal medullary chromaffin PC-12 cell line.

Morphine was identified in the adrenal medulla chromaffin PC-12 cell line by reversed-phase HPLC, following liquid and solid extraction. The morphine corresponding HPLC fractions (1.746+/-0.615 ng of morphine/million cells) were further analyzed by gas chromatography-mass spectrometry and found to be identical to synthetic morphine. Furthermore, using primers derived from the human neuronal mu 1 opiate receptor, we used RT-PCR to detect expression of mu transcripts from this cell line. The transcript was absent. The study conclusively proves morphine, but not a mu opiate receptor, is constitutively expressed in the adrenal medulla chromaffin PC-12 cell line.

Adrenal Gland Neoplasms↗

PubMed2000

Ascaris suum, an intestinal parasite, produces morphine.

The parasitic worm Ascaris suum contains the opiate alkaloid morphine as determined by HPLC coupled to electrochemical detection and by gas chromatography/mass spectrometry. The level of this material is 1168 +/- 278 ng/g worm wet weight. Furthermore, Ascaris maintained for 5 days contained a significant amount of morphine, as did their medium, demonstrating their ability to synthesize the opiate alkaloid. To determine whether the morphine was active, we exposed human monocytes to the material, and they immediately released nitric oxide in a naloxone-reversible manner. The anatomic distribution of morphine immunoreactivity reveals that the material is in the subcuticle layers and in the animals' nerve chords. Furthermore, as determined by RT-PCR, Ascaris does not express the transcript of the neuronal mu receptor. Failure to demonstrate the expression of this opioid receptor, as well as the morphine-like tissue localization in Ascaris, suggests that the endogenous morphine is intended for secretion into the microenvironment.

Animals↗

PubMed2000

Endogenous morphine.

It is now well accepted that endogenous morphine is present in animals, both in invertebrates and vertebrates. It is a key signaling molecule that plays an important role in downregulating physiological responses, such as those in the immune system, including immune elements in the CNS. It has been demonstrated that a specific mu-opiate-receptor subtype, mu3, mediates these downregulatory effects through release of NO. This article examines morphine as an endogenous signaling molecule, in terms of its role in neural and immune regulation.

Animals↗

PubMed2000

homothorax and iroquois-C genes are required for the establishment of territories within the developing eye disc.

In Drosophila the eye-antennal disc gives rise to most adult structures of the fly's head. Yet the molecular basis for its regionalization during development is poorly understood. Here we show that homothorax is required early during development for normal eye development and is necessary for the formation of the ventral head capsule. In the ventral region of the disc only, homothorax and wingless are involved in a positive feedback loop necessary to restrict eye formation. homothorax is able to prevent the initiation and progression of the morphogenetic furrow without inducing wingless, which points to homothorax as a key negative regulator of eye development. In addition, we show that the iroquois-complex genes are required for dorsal head development antagonizing the function of homothorax in this region of the disc.

Alleles↗

PubMed2000

A dual role for homothorax in inhibiting wing blade development and specifying proximal wing identities in Drosophila.

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal (P-D) axis: the wing blade, the wing hinge and the mesonotum. Development of the wing blade initiates along part of the dorsal/ventral (D/V) compartment boundary and requires input from both the Notch and wingless (wg) signal transduction pathways. In the wing blade, wg activates the gene vestigial (vg), which is required for the wing blade to grow. wg is also required for hinge development, but wg does not activate vg in the hinge, raising the question of what target genes are activated by wg to generate hinge structures. Here we show that wg activates the gene homothorax (hth) in the hinge and that hth is necessary for hinge development. Further, we demonstrate that hth also limits where along the D/V compartment boundary wing blade development can initiate, thus helping to define the size and position of the wing blade within the disc epithelium. We also show that the gene teashirt (tsh), which is coexpressed with hth throughout most of wing disc development, collaborates with hth to repress vg and block wing blade development. Our results suggest that tsh and hth block wing blade development by repressing some of the activities of the Notch pathway at the D/V compartment boundary.

Animals↗

PubMed1999

Endogenous morphine levels increase in molluscan neural and immune tissues after physical trauma.

The aim of this study was to demonstrate by biochemical and immunocytochemical methods the presence of endogenous morphine in nervous and immune tissues of the freshwater snail, Planorbarius corneus. High performance liquid chromatography (HPLC) coupled to electrochemical detection performed on tissues from control snails, revealed that the CNS contains 6.20+/-2.0 pmol/g of the alkaloid, the foot tissue contains a much lower level, 0.30+/-0.03 pmol/g, whilst morphine is not detected in the hemolymph and hepatopancreas. In specimens that were traumatized, we detected a significant rise of the CNS morphine level 24 h later (43.7+/-5.2 pmol/g) and an initial decrease after 48 h (19.3+/-4.6 pmol/g). At the same times, we found the appearance of the opiate in the hemolymph (0.38+/-0.04 pmol/ml and 0.12+/-0.03 pmol/ml) but not in the hepatopancreas. Using indirect immunocytochemistry, a morphine-like molecule was localized to a number of neurons and a type of glial cell in the CNS, to some immunocytes in the hemolymph and to amoebocytes in the foot, as well as to fibers in the aorta wall. Simultaneously to the rise of morphine biochemical level following trauma, morphine-like immunoreactivity (MIR) increased in both intensity and the number of structures responding positively, i.e., neurons and fiber terminals. In another mollusc, the mussel Mytilus galloprovincialis, the same pattern of enhanced MIR was found after trauma. Taken together, the data suggest the presence of a morphinergic signaling in invertebrate neural and immune processes resembling those of classical messenger systems and an involvement in trauma response.

Animals↗

PubMed1999

Regulation of Hox target genes by a DNA bound Homothorax/Hox/Extradenticle complex.

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (EXD). For EXD to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (HTH). Here we show that a conserved N-terminal domain of HTH directly binds to EXD in vitro, and is sufficient to induce the nuclear localization of EXD in vivo. However, mutating a key DNA binding residue in the HTH homeodomain abolishes many of its in vivo functions. HTH binds to DNA as part of a HTH/Hox/EXD trimeric complex, and we show that this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of HTH we provide evidence that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of HTH, Hox, and EXD proteins, bound to DNA.

Amino Acid Sequence↗

PubMed1998

Control of antennal versus leg development in Drosophila.

During the evolution of insects from a millipede-like ancestor, the Hox genes are thought to have promoted the diversification of originally identical body structures. In Drosophila melanogaster, antennae and legs are homologous structures that differ from each other as a result of the Hox gene Antennapedia (Antp), which promotes leg identities by repressing unknown antennal-determining genes. Here we present four lines of evidence that identify extradenticle (exd) and homothorax (hth) as antennal-determining genes. First, removing the function of exd or hth, which is required for the nuclear localization of Exd protein, transforms the antenna into leg; such transformations occur without activation of Antp. Second, hth is expressed and Exd is nuclear in most antennal cells, whereas both are restricted to proximal cells of the leg. Third, Antp is a repressor of hth. Fourth, ectopic expression of Meis1, a murine hth homologue, can trigger antennal development elsewhere in the fly. Taken together, these data indicate that hth is an antennal selector gene, and that Antp promotes leg development by repressing hth and consequently nuclear Exd.

Animals↗

PubMed1997

Nuclear translocation of extradenticle requires homothorax, which encodes an extradenticle-related homeodomain protein.

We show that homothorax (hth) is required for the Hox genes to pattern the body of the fruit fly, Drosophila melanogaster. hth is necessary for the nuclear localization of an essential HOX cofactor, Extradenticle (EXD), and encodes a homeodomain protein that shares extensive identity with the product of Meis1, a murine proto-oncogene. MEIS1 is able to rescue hth mutant phenotypes and can induce the cytoplasmic-to-nuclear translocation of EXD in cell culture and Drosophila embryos. Thus, Meis1 is a murine homolog of hth. MEIS1/HTH also specifically binds to EXD with high affinity in vitro. These data suggest a novel and evolutionarily conserved mechanism for regulating HOX activity in which a direct protein-protein interaction between EXD and HTH results in EXD's nuclear translocation.

Animal Structures↗

PubMed1997

Interactions of Drosophila Ultrabithorax regulatory regions with native and foreign promoters.

The Ultrabithorax (Ubx) gene of the Drosophila bithorax complex is required to specify parasegments 5 and 6. Two P-element "enhancer traps" have been recovered within the locus that contain the bacterial lacZ gene under the control of the P-element promoter. The P insertion that is closer to the Ubx promoter expresses lacZ in a pattern similar to that of the normal Ubx gene, but also in parasegment 4 during embryonic development. Two deletions have been recovered that remove the normal Ubx promoter plus several kilobases on either side, but retain the lacZ reporter gene. The lacZ patterns from the deletion derivatives closely match the normal pattern of Ubx expression in late embryos and imaginal discs. The lacZ genes in the deletion derivatives are also negatively regulated by Ubx and activated in trans by Contrabithorax mutations, again like the normal Ubx gene. Thus, the deleted regions, including several kilobases around the Ubx promoter, are not required for long range interactions with Ubx regulatory regions. The deletion derivatives also stimulate transvection, a pairing-dependent interaction with the Ubx promoter on the homologous chromosome.

Animals↗

PubMed1996

Functional similarity in appendage specification by the Ultrabithorax and abdominal-A Drosophila HOX genes.

In Drosophila, the Ultrabithorax, abdominal-A and Abdominal-B HOX genes of the bithorax complex determine the identity of part of the thorax and the whole abdomen. Either the absence of these genes or their ectopic expression transform segments into the identity of different ones along the antero-posterior axis. Here we show that misexpression of Ultrabithorax, abdominal-A and, to some extent, Abdominal-B genes cause similar transformations in some of the fruitfly appendages: antennal tissue into leg tissue and wing tissue into haltere tissue. abdominal-A can fully, and Abdominal-B partially, substitute for Ultrabithorax in haltere development. By contrast, when ectopically expressed, the three genes specify different segments in regions of the main body axis like notum or abdomen. Insects may have originally used the HOX genes primarily to specify this main body axis. By contrast, the homeotic requirement to form appendages is, in some cases, non-specific.

Animals↗

PubMed1996

Hyperstimulation of leukocytes by plasma from cardiopulmonary bypass patients is diminished by alpha-MSH pretreatment.

Cardiopulmonary bypass (CPB) results in a diffuse inflammatory response characterized in part by hyperstimulation of leukocytes. We have previously shown that this hyperstimulation appears to be due, in part, to an increase in the release of biological response modifiers (BRMs) such as cytokines. In the present study, we evaluated the ability of a naturally occurring immunocyte inhibitory substance, alpha-melanocyte-stimulating hormone (alpha-MSH), to prevent the hyperstimulation caused by CPB. Monocytes and granulocytes were pretreated with alpha-MSH (10(-6) M) before exposing the cells to plasma obtained from patients who had undergone CPB, as CPB plasma would stimulate native monocytes and granulocytes in a manner similar to that observed in CPB patients. Pretreatment of these cells with alpha-MSH significantly diminished the hyperstimulation induced by CPB plasma in a concentration-dependent manner. In contrast, when the cells were first or simultaneously exposed to CPB plasma and then to alpha-MSH, alpha-MSH had no effect. Furthermore, use of the specific neutral endopeptidase inhibitor, phosphoramidon, significantly increased the efficacy of alpha-MSH in inhibiting CPB-induced immunocyte activation. The data demonstrate that pretreatment of monocyte/macrophages and granulocytes with alpha-MSH effectively inhibits the immune hyperstimulation induced by CPB-plasma exposure. In addition, the data strongly suggest that preexposure to other naturally occurring immune inhibitory substances may diminish the hyperstimulation associated with CPB. The study also further confirms that this hyperstimulation may, in part, be due to BRMs released from immunocytes.

Cardiopulmonary Bypass↗

PubMed1995

Presence of the mu3 opiate receptor in endothelial cells. Coupling to nitric oxide production and vasodilation.

Initial confinement of opiate receptors to the nervous system has recently been broadened to several other cell types. Based on the well established hypotensive effect of morphine, we hypothesized that endothelial cells may represent a target for this opiate substance. Endothelial cells (human arterial and rat microvascular) contain a high affinity, saturable opiate binding site presumed to mediate the morphine effects that is stereoselectively and characteristically antagonized by naloxone. This opiate alkaloid-specific binding site is insensitive to opioid peptides. It is, therefore, considered to be the same subtype of opiate receptor (designated mu3) used in the mediation of morphine in other cell types exhibiting the same binding profile. Experiments with endothelial cultures and the aortic ring of rats cultured in vitro demonstrate that morphine exerts direct modulatory control over the activities of endothelial cells, which leads to vasodilation. It induces the production of nitric oxide, a process that is sensitive to naloxone antagonism and nitric oxide synthase inhibition. In contrast with that of opiates, the administration of opioid peptides does not induce nitric oxide production by endothelial cells. In conclusion, the data presented above reveal a novel site of morphine action, endothelial cells, where a mu3 receptor is coupled to nitric oxide release and vasodilation.

Animals↗

PubMed1995

Changes in the blood-thymus barrier of adult rats after estradiol-treatment.

The accessibility of the thymus parenchyma for relatively large Mw (+/- 150 Kd) proteins has been studied by the intravenous injection of monoclonal antibodies (mAbs) specific either for all T cells (His-17) or MHC class II molecules (His-19) in control and estradiol benzoate (EB)-treated adult Wistar rats. In controls, the transcapsular route rather than cortical capillaries seems to be involved in the entry of molecules into the thymus. By contrast, a specific staining for either T cells (His-17) or MHC class II molecules (His-19 positive cells) disappears almost completely from the thymic cortex of EB-treated rats except in the immediate subcapsular epithelial cell layer. In these rats, T cells and epithelial cells intimately associated to blood vessels from both inner cortex and corticomedullary border showed additional staining with the respective mAbs confirmed by electron microscopy. The disappearance of the transcapsular route together with the increased vascular permeability of cortical blood vessels would be related to the reinforcement of the subcapsular epithelial cell layer and to direct effects of EB on vascular endothelia, respectively. These results are discussed in relationship to the cell migration into and out of adult thymus, as suggested by the changes in intrathymic T cell subsets evaluated by flow cytometry.

Animals↗