Biomedical subjects

C Pedraza

Publications and source records attributed to C Pedraza.

13 recordsLinked to original sources

PubMed2001

Laminin-8 (alpha4beta1gamma1) is synthesized by lymphoid cells, promotes lymphocyte migration and costimulates T cell proliferation.

Laminins are a growing family of large heterotrimeric proteins with cell adhesive and signalling functions. They are major components of basement membranes and are found in many organs, including the vasculature and other compartments of bone marrow, thymus, lymph nodes and spleen. However, expression, recognition and use of laminin isoforms by lymphoid cells are poorly understood. In the present study, lymphoid T cells (Jurkat) were found to synthesize laminin alpha4, beta1 and gamma1 mRNAs and polypeptides and to assemble the chains into laminin-8. Lymphoblastoid B (NAD-20) cells, lymphoid NK (NKL) cells and blood lymphocytes also contained laminin-8 and, after cell permeabilization, practically all blood lymphocytes reacted with mAbs to laminin beta1 and gamma1 chains. Following stimulation, blood lymphocytes secreted laminin-8, and this laminin isoform, but not laminin-10/11(alpha5beta1gamma1/alpha5beta2gamma1), promoted chemokine-induced migration of the cells. In an activation-dependent manner, purified blood CD4 T cells adhered to immobilized laminin-8 and laminin-10/11 by using alpha6beta1 integrin, but minimally to laminin-1 (alpha1beta1gamma1). Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via the same integrin. Thus, lymphoid cells are able to synthesize and secrete complete laminin molecules. In addition, synthesis of laminin-8 and recognition of laminin-8 and -10/11 by lymphocytes indicate relevance of these laminin isoforms in lymphocyte physiology.

Antibodies, Monoclonal↗

PubMed2001

Attitudes toward animal research among psychology students in Spain.

Animal research plays a central role in psychology, and its use, prevalence and quality depends on the attitudes of students who enter psychology in Spain. Attitudes among psychology students about the use of laboratory animals are not known, so the aim of this work was to analyze the attitudes of Spanish psychology students toward animal research. An attitude questionnaire of 15 items was given to 661 undergraduate students of the School of Psychology at the University of Málaga, Spain. Several results were found: (a) 65.7% of the respondents strongly agreed or agreed with animal research. General support for animal research was significantly higher by men than by women. (b) Support for animal research was higher for senior students, suggesting that the psychology curriculum or self-selection to remain in the program might influence students' attitudes. (c) Attitudes toward animal research were similar among students independent of the type of animal being used for research in biological or psychological studies. (d) 58% considered that laboratory animals never or almost never are inappropriately handled. (c) 55.6% indicated that research in psychology on animals could be generalized to humans. Overall, these results suggest that among Spanish psychology students animals research is considered important for the advancement of the science. Likewise, a majority of students displayed positive attitudes toward animal research.

Adult↗

PubMed2000

Monocytic cells synthesize, adhere to, and migrate on laminin-8 (alpha 4 beta 1 gamma 1).

Laminins, a growing family of large heterotrimeric proteins with cell adhesive and signaling properties, are major components of vascular and other basement membranes. Expression, recognition, and use of laminin isoforms by leukocytes are poorly understood. In monoblastic THP-1 cells, transcripts for laminin gamma(1)-, beta(1)-, and alpha(4)-chains were detected by RT-PCR. Following immunoaffinity purification on a laminin beta(1) Ab-Sepharose column, laminin beta(1)- (220 kDa), gamma(1)- (200 kDa), and alpha(4)- (180/200 kDa) chains were detected by Western blotting in THP-1 cells and in two other monoblastic cell lines, U-937 and Mono Mac 6. After cell permeabilization, a mAb to laminin gamma(1)-chain reacted with practically all blood monocytes by immunofluorescence flow cytometry, and laminin-8 (alpha(4)beta(1)gamma(1)) could be isolated also from these cells. Monoblastic JOSK-I cells adhered constitutively to immobilized recombinant laminin-8, less than to laminin-10/11 (alpha(5)beta(1)gamma(1)/alpha(5)beta(2)gamma(1)) but to a higher level than to laminin-1 (alpha(1)beta(1)gamma(1)). Compared with the other laminin isoforms, adhesion to laminin-8 was preferentially mediated by alpha(6)beta(1) and beta(2) integrins. Laminin-8 and, to a lower extent, laminin-1 promoted spontaneous and chemokine-induced migration of blood monocytes, whereas laminin-10/11 was inhibitory. Altogether, the results indicate that leukocytes, as other cell types, are able to synthesize complete laminin molecules. Expression, recognition, and use of laminin-8 by leukocytes suggest a major role of this laminin isoform in leukocyte physiology.

CD18 Antigens↗

PubMed2000

Erythromegakaryocytic cells synthesize laminin-8 (alpha4beta1gamma1).

Blood platelets contain laminin-8 (alpha4beta1gamma1), a recently described laminin isoform, but the origin of platelet laminin is at present unknown. Laminin of platelets could be synthesized by megakaryocytes or, alternatively, endocytosed from plasma or other sources. In the present study, the synthesis and presence of laminin-8 in erythromegakaryocytic HEL and DAMI cells were explored. In HEL cells, transcripts for alpha4, beta1, and gamma1 laminin chains were readily detected by RT-PCR. Immunofluorescence flow cytometry demonstrated reactivity of mAbs to laminin beta1 and gamma1 chains with permeabilized cells. Metabolic labeling of HEL cells using [(35)S]methionine and [(35)S]cysteine followed by immunoprecipitation with monoclonal antibodies to beta1 and gamma1 chains revealed bands of approximately 220 and 200 kDa. In the HEL cell lysate, polypeptides of 220 and 200 kDa were recognized by monoclonal antibodies to laminin beta1 and gamma1 chains, respectively, whereas immunoaffinity-purified rabbit antibodies to laminin alpha4 chain gave inconclusive results. However, following immunoaffinity purification on a laminin beta1 antibody-Sepharose column, a 200-kDa band was readily detected by the antibodies to laminin alpha4 chain. Similar results were obtained with DAMI cells. The size of laminin chains of HEL/DAMI cells was similar, though not identical, to the one of platelets, and the alpha4 chain was noncovalently associated to disulfide-bonded beta1gamma1 heterodimer, as in platelets. We conclude that erythromegakaryocytic cells synthesize laminin-8.

Animals↗

PubMed1999

Blood platelets contain and secrete laminin-8 (alpha4beta1gamma1) and adhere to laminin-8 via alpha6beta1 integrin.

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.

Amino Acid Sequence↗

PubMed1999

Uracil/tegafur plus oral calcium folinate in advanced breast cancer.

Uracil and tegafur (in a molar ratio of 4:1 [UFT]) has proven activity against breast cancer and is delivered in an easy-to-administer oral formulation. Orzel, which combines UFT with the oral biomodulator, calcium folinate, may provide even greater antitumor efficacy against breast cancer. Here, we describe the preliminary results of this phase II trial investigating the feasibility of 250 mg/m2/day of UFT plus 45 mg/day of oral calcium folinate administered to highly pretreated patients with advanced breast cancer. The results indicate a highly tolerable regimen and an overall response rate of 27.8% in a group of poor-prognosis patients. These findings warrant continued investigation.

Administration, Oral↗

PubMed1998

Tiapride-induced catalepsy is potentiated by gamma-hydroxybutyric acid administration.

1. The effect of administration of gammahydroxybutyrate (GHB) and tiapride, either alone or in combination, on catalepsy behavior was examined in male mice. 2. Catalepsy was measured by bar and grid tests. Two successive evaluations were carried out 30 and 60 min after injections. 3. Tiapride (175 and 200 mg/kg) and gammahydroxybutyrate (200 mg/kg) provoked an increase of catalepsy scores, exhibiting different time courses. GHB produced a marked but short lasting catalepsy with a peak of action at 30 min, while tiapride produced a catalepsy state with a peak of action at 60 min. 4. Tiapride-induced catalepsy was potentiated by gammahydroxybutyrate administration at 30 min (bar test) and 60 min (bar and grid tests). 5. These results underlie the view that GHB interacts with central dopamine D2 transmission.

Anesthetics, Intravenous↗

PubMed1996

Midkine as a novel target gene for the Wilms' tumor suppressor gene (WT1).

Midkine (MK) is a heparin-binding growth factor which is strongly expressed during the midgestation period of mouse embryogenesis. Wilms' tumor is an embryonal kidney malignancy in infants, and WT1 has been identified as its tumor suppressor gene. The high expression level of MK in all Wilms' tumor specimens so far examined and the presence of two WT1 elements (5'-GCGGGGGCG-3') in the human MK promoter region led us to examine the possible role of the WT1 gene product in the regulation of MK gene expression. A gel shift assay verified the complex formation between the WT1 gene product and WT1 consensus sequence of MK gene. DNase1 footprint analysis also demonstrated that the downstream WT1 element was protected from DNase1 cleavage by the addition of the WT1 protein. The human MK promoter fused with the chloramphenicol acetyltransferase gene (phMK2.3kCAT) was co-transfected with an effector plasmid containing the WT1 gene into several cell lines. Transient transfection assays showed suppression of the MK promoter by WT1 co-transfection in recipient cells; deletion of the WT1 binding site abolished the suppression. The evidence reported in this study indicates that MK gene is a newly identified WT1 target gene.

3T3 Cells↗

PubMed1995

The fourth armadillo repeat of plakoglobin (gamma-catenin) is required for its high affinity binding to the cytoplasmic domains of E-cadherin and desmosomal cadherin Dsg2, and the tumor suppressor APC protein.

Plakoglobin is a member of a protein family with a repeated amino acid motif, the armadillo repeat, and is a cytoplasmic protein found in both adherens junctions and desmosomes. Plakoglobin has been shown to form distinct complexes with cadherins or desmosomal cadherins. Also, plakoglobin has been shown to complex with APC, the tumor suppressor gene product. Recently we isolated a cDNA clone encoding plakoglobin lacking the fourth armadillo repeat of the original 13-repeat protein [Ozawa et al. (1995) J. Biochem. 118, 836-840]. In this study, we established an in vitro assay system to study the molecular interaction of plakoglobin with cadherins, the APC gene product, and alpha-catenin. Establishment of the system and cloning of an alternate form of plakoglobin cDNA allowed us to examine the biological activity of plakoglobin lacking the fourth armadillo repeat. Experiments with the bacterially expressed 12-repeat plakoglobin revealed that the protein binds to E-cadherin, desmoglein (Dsg2), and APC with lower affinity than the 13-repeat form does. Consistent with the observation that the affinity of alpha-catenin for these two alternate forms was similar, we found amino acid residues 104 to 145 of plakoglobin, the residues present in both isoforms, are sufficient for its binding to alpha-catenin.

Adenomatous Polyposis Coli Protein↗

PubMed1995

A retinoic acid-responsive element in human midkine gene.

Midkine is a growth/differentiation factor which is found as a product of a retinoic acid-responsive gene. The 2.3-kb upstream sequence of the human MK gene has cis acting elements which confer retinoic acid-induced expression of fused chloramphenicol acetyl-transferase (CAT) gene in F9 embryonal carcinoma cells. In the 5'-region of the human gene, a sequence resembling the DR5-type retinoic acid-responsive element (AGGTCA-related direct repeats separated by 5 nucleotides) was present in a small block of highly homologous 5'-sequences shared by the human and mouse genes. Deletion of this direct repeat reduced retinoic acid-induced CAT gene expression. The core element in the human gene differs from the consensus sequence of retinoic acid-responsive element in two nucleotides and from the retinoic acid responsive element of the mouse MK gene in one nucleotide.

Animals↗

PubMed1994

Mapping and characterization of a retinoic acid-responsive enhancer of midkine, a novel heparin-binding growth/differentiation factor with neurotrophic activity.

MK is a gene that is activated by retinoic acid in embryonal carcinoma (EC) cells and is expressed temporarily during the mid-gestation period of mouse embryogenesis. Midkine, the product of the gene is a novel heparin-binding growth/differentiation factor with neurite outgrowth and neurotrophic activities. The regulatory DNA element in the retinoic acid-induced expression of the MK gene has been investigated. The 1.9 kb 5'-flanking region of the MK gene can mediate retinoic acid-responsive gene expression in F9 and HM-1 EC cells. Analysis of this region by deletion mutagenesis in F9 EC cells shows that there is a retinoic acid-responsive enhancer (designated as REM1) around 900 bp upstream from the transcription start site. This enhancer is composed of two sequence elements, which are located between -1006 and -895 and between -901 and -794. The core element of the upstream region (-971 to -955), whose deletion abolished the retinoic acid responsiveness, contained a sequence highly homologous to a binding site for retinoic acid receptors. Binding of a retinoic acid receptor heterodimer to this core element was verified by gel shift assay. Thus, retinoic acid and the receptor complex can directly induce the expression of a growth/differentiation factor gene.

Animals↗

PubMedDate not supplied

Effects of L-741,741, a selective dopamine receptor antagonist, on anxiety tested in the elevated plus-maze in mice.

Dopamine systems are involved in modulation of anxiety. Although the action of D1/5, D2 and D3 receptor antagonists on anxiety has been documented in different animal models, little is known about the influence of D4 dopamine antagonists. The aim of this study was to examine the effects of L-741,741 (0.75, 1.5 and 3 mg/kg i.p.), a selective D4 dopamine receptor antagonist, on anxiety tested in the elevated plus-maze. Forty-eight albino adult male mice of the OF.1 strain were used. Animals were randomly allocated to one control group receiving physiological saline and dymethyl-sulphoxide (DMSO) (10%) and three experimental groups receiving L-741,741 injections. Tests were performed 30 min after injections for a duration of 5 min. A number of classical parameters were collected: i) open arm duration; ii) closed arm duration; iii) central platform duration; iv) open arm frequency; v) closed arm frequency; vi) central platform frequency; and vii) number of rearings. L-741,741 did not produce any significant behavioral changes under present test conditions, suggesting that the dopamine D4 receptor might not be involved in the modulation of anxiety-related behaviors in the plus-maze.

Animals↗

PubMedDate not supplied

[Update on gamma-hydroxybutyric acid].

AIMS: The article offers an updated review of the main pharmacological aspects of gamma-hydroxybutyric acid (GHB), as well as its clinical and behavioural effects. DEVELOPMENT: A number of pharmacological, neurochemical and electrophysiological studies have clearly shown that endogenous GHB plays a role as a neurotransmitter and/or neuromodulator in the central nervous system (CNS). GHB displays specific synthesis, release and reuptake mechanisms, as well as particular binding sites that suggest the existence of a central GHBergic system. This substance, popularly known as 'liquid ecstasy', is also a potentially abusable drug; if administered for prolonged periods of time it can lead to dependence and withdrawal symptoms after the patient stops taking it. Its chief behavioural actions include sedation/sleepiness, induction of absence seizures, catalepsy or reduced aggression, among others. Some of these effects appear to be related to an interaction that has been reported to exist between the GHBergic system and the dopaminergic and GABAergic receptors in the CNS. From the clinical point of view, its use has been approved in some countries to treat the narcoleptic syndrome, and it has also been considered for possible use in the treatment of alcohol or opiate abuse. Finally, recent studies conducted with laboratory animals suggest the existence of a possible neurotoxic effect following prolonged administration in abusable dosages. CONCLUSIONS: GHB is an extraordinarily interesting compound. It acts as a neurotransmitter/neuromodulator in the CNS. It is also an abusable recreational drug and may also be used to treat a number of different pathological conditions, the most important of which is narcolepsy. The possible development of neurotoxicity following prolonged administration, however, imposes considerable limitations on its usefulness in clinical contexts.

Animals↗