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M Arribas

Publications and source records attributed to M Arribas.

18 recordsLinked to original sources
PubMed2001

Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance.

Although insulin regulates metabolism in both brown and white adipocytes, the role of these tissues in energy storage and utilization is quite different. Recombination technology using the Cre-loxP approach allows inactivation of the insulin receptor in a tissue-specific manner. Mice lacking insulin receptors in brown adipocytes show an age-dependent loss of interscapular brown fat but increased expression of uncoupling protein-1 and -2. In parallel, these mice develop an insulin-secretion defect resulting in a progressive glucose intolerance, without insulin resistance. This model provides direct evidence for not only a role for the insulin receptors in brown fat adipogenesis, the data also suggest a novel role of brown adipose tissue in the regulation of insulin secretion and glucose homeostasis.

Adipose Tissue, Brown↗
PubMed2000

Weibel-Palade body membrane proteins exhibit differential trafficking after exocytosis in endothelial cells.

Weibel-Palade bodies, the secretory granules of endothelial cells, possess two different membrane proteins. However, P-selectin is seen only in Weibel-Palade bodies in HUVECs, whereas CD63 is also seen in late endosomes/lysosomes. Since P-selectin is targeted to lysosomes in heterologous expression studies, we have determined whether a lysosomal targeting signal also operates within HUVECs. We have also examined the trafficking of CD63 to its two different intracellular locations. By following antibodies bound at the plasma membrane during stimulation, we have discovered that while half of the P-selectin recycles to the WPBs, 50% is rapidly delivered to a lamp-1-positive compartment. Thus, the lysosomal targeting signal of this protein also operates in HUVECs. CD63 is found constitutively at the cell surface of HUVECs and most of it is delivered to the late endosomes/lysosomes after internalisation. However, stimulation causes both a rise in the CD63 plasma membrane level and in the amount that recycles to the WPBs. Our data strongly suggest that the CD63 that originates in the WPB preferentially recycles to the granule rather than being delivered to the late endosome/lysosome, and that there are, therefore, two separate pools of this protein within HUVECs. Our findings indicate that although P-selectin and CD63 are both targeted to the same compartments from the PM, the kinetics and the ratio of their targeting to Weibel-Palade bodies versus lysosomes are very different.

Antibodies↗
PubMed1999

Secretagogue-triggered transfer of membrane proteins from neuroendocrine secretory granules to synaptic-like microvesicles.

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.

Animals↗
PubMed1999

Interhemispheric supratentorial intracranial pressure gradients in head-injured patients: are they clinically important?

OBJECT: It is generally accepted that the intracranial compartment behaves as a unicameral space in which intracranial pressure (ICP) is uniformly distributed. However, this concept has been challenged many times. Although there is general agreement on the existence of craniospinal and suprainfratentorial gradients, the existence of interhemispheric gradients is still a matter of debate. The object of this study was to reexamine the issue of interhemispheric supratentorial ICP gradients in patients with head injuries and the clinical significance of these gradients in their management. METHODS: The authors present the results of a prospective study conducted in 50 head-injured patients to determine the clinical significance of supratentorial ICP gradients. In each case a concurrent bilateral frontal intraparenchymatous device was implanted within the 6-hour window after computerized tomography (CT) scanning. According to CT criteria, each patient was categorized into one of three different groups: 1) diffuse lesions, in which no unilaterally measured volumes greater than 25 ml were present and the midline shift was 3 mm or less; 2) Focal A, in which added hemispheric volumes were greater than 25 ml and midline shift was 3 mm or less; and 3) Focal B, in which all patients with a midline shift greater than 3 mm were included. From the results of the entire group the authors were able to distinguish four different patterns of supratentorial ICP. In Pattern I, the intracranial compartment behaved as a true unicameral space with similar mean ICPs and pulse amplitudes in both hemispheres; in Pattern II, different mean ICPs and amplitudes were observed although ICP increases or decreases were congruent; and in Pattern III, patients with different mean ICPs, different ICP amplitudes, and no congruent increases or decreases of ICP were included. All (15 cases) but one patient with a diffuse lesion presented with ICP Pattern I. Fifteen patients with focal lesions showed a Type II pattern, whereas only one patient presented with a Type III pattern. In 10 patients, of whom all but one presented with a focal lesion, transient gradients that disappeared in less than 4 hours were also observed. CONCLUSIONS: In many patients with focal lesions, clinically important interhemispheric ICP gradients exist. In this subset, transient gradients that disappear with time are frequently observed and may indicate an increase in the size of the lesion. The clinical relevance of such gradients is discussed and guidelines for adequately monitoring ICP are suggested to optimize head injury management and to avoid suboptimal or even harmful care in patients with mass lesions.

Adolescent↗
PubMed1997

Synaptojanin 2, a novel synaptojanin isoform with a distinct targeting domain and expression pattern.

Synaptojanin (synaptojanin 1) is a recently identified inositol 5'-phosphatase, which is highly enriched in nerve terminals and is implicated in synaptic vesicle recycling. It is composed of three domains: an amino-terminal SacI homology region, a central inositol 5'-phosphatase homology region, and a carboxyl-terminal proline-rich region. We have now identified and characterized a novel form of synaptojanin, synaptojanin 2, which has a broader tissue distribution. Synaptojanin 2 cDNA from rat brain library encodes a protein of 1,248 amino acids with a predicted Mr of 138,268. The two synaptojanin isoforms share 57.2 and 53.8% amino acid identity in their SacI and phosphatase domains, respectively. In marked contrast, their carboxyl-terminal proline-rich regions bear little homology. Expression of synaptojanin 2 in COS7 cells produced a 140-kDa protein with inositol 5'-phosphatase actvity. Protein binding assays demonstrated that among the major src homology 3-proteins known to bind to the proline-rich region of synaptojanin 1, Grb2, amphiphysin, and members of SH3p4/8/13 protein family, only Grb2 bound to that of synaptojanin 2. Furthermore, subcellular fractionation studies in transfected Chinese hamster ovary cells revealed that synaptojanin 2 was predominantly associated with the particulate fraction while synaptojanin 1 was mainly localized in the soluble fraction. This observation suggests that the proline-rich regions of synaptojanins 1 and 2 are implicated in different protein-protein interactions and direct the two isoforms to different subcellular compartments. Our results demonstrate the presence of a family of synaptojanin-type inositol 5'-phosphatases with different tissue and subcellular distributions, which may be involved in distinct membrane trafficking and signal transduction pathways in mammalian cells.

Amino Acid Sequence↗
PubMed1997

The stimulatory effect of rabphilin 3a on regulated exocytosis from insulin-secreting cells does not require an association-dissociation cycle with membranes mediated by Rab 3.

Rabphilin 3a is a Rab 3-GTP binding protein concentrated on secretory vesicles of neurons and endocrine cells. There is evidence that rabphilin 3a undergoes cycles of association-dissociation with membranes and that recruitment of rabphilin 3a to secretory vesicles is mediated by Rab 3a, suggesting that rabphilin 3a is a downstream effector of this Rab. In this study we have investigated whether a membrane-anchored form of rabphilin 3a mimics the action of rabphilin 3a on secretion and bypasses the need for Rab 3 function. Overexpression of both wild-type rabphilin 3a and of a transmembrane anchored form of rabphilin 3a stimulated (about 2-fold) evoked secretion of coexpressed human proinsulin from clonal HIT-T15 cells. A similar transmembrane-anchored protein which lacked the Rab 3 binding region stimulated secretion even more effectively. Unexpectedly, a rabphilin 3a deletion mutant missing the Rab 3 binding domain was also stimulatory on secretion, although a further deletion of rabphilin to exclude the first of the two proline-rich regions abolished its stimulatory effect. The first of these two mutants was primarily particulate, while the second mutant was primarily soluble, suggesting that the first proline-rich region of rabphilin 3a plays a role in targeting rabphilin to its site of action. We conclude that the action of rabphilin 3a can be independent of Rab 3 if other mechanisms produce a sufficient concentration of the protein in proximity of exocytotic sites. These results provide new evidence for a fundamental similarity in the mechanisms by which Ras and Rab GTPase produce their distinct physiological effects.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Botulinum toxin type A inhibits Ca(2+)-dependent transport of acetylcholine in reconstituted giant liposomes made from presynaptic membranes from cholinergic nerve terminals.

Giant liposomes were made from a mixture of asolectin phospholipid vesicles and presynaptic plasma membranes isolated from Torpedo cholinergic nerve endings. Acetylcholine filled giant liposomes were able to release neurotransmitter upon stimulation by the Ca2+ ionophore A23187 and Ca2+. Botulinum neurotoxin type A inhibited this Ca(2+)-dependent acetylcholine transport. Additionally, Botulinum toxin type A decreased membrane fluidity of liposomes. These results suggest that Botulinum toxin can interact directly with components of the presynaptic plasma membrane and inhibit acetylcholine translocation. Furthermore, since the reconstituted liposomes do not have synaptic vesicle components, the observed effects may account for the action of Botulinum toxin on the non-quantal release of acetylcholine from motor nerve terminals.

Acetylcholine↗
PubMed1995

Characterization of a rabbit serum raised against a botulinum toxin type A binding protein from presynaptic plasma membranes from Torpedo electric organ.

Botulinum neurotoxin type A blocks acetylcholine release from the peripheral nervous system. We have previously described a putative botulinum neurotoxin type A receptor of presynaptic plasma membranes from Torpedo. The electric organ of Torpedo, which is largely enriched in cholinergic nerve endings, is homologous to the neuromuscular junction, allowing us to isolate large scale of presynaptic components. In order to characterize this protein we have raised a polyclonal antibody (a-P140) against this receptor. The antiserum a-P140 recognizes a 140,000 mol. wt band in non-reducing conditions and an 80,000 band in reducing conditions. The immunohistochemistry assay reveals the P140 protein on the ventral face of the electrocytes where the nerve terminals are localized. Moreover, a-P140 antiserum recognizes the P140-BoNT/A complex after binding and cross-linking experiments. In addition, we have immunoprecipitated an in vitro translated product which is closely coincident in mol. wt to the 80,000 band of the receptor.

Animals↗
PubMed1995

Tetanus toxin mechanism of action in Torpedo electromotor system: a study on different steps in the intoxication process.

The mechanism of action of tetanus toxin was characterized in the electromotor system of Torpedo marmorata either at peripheral and central nervous system. The consecutive steps of the intoxication pathway were observed: (i) [125I]tetanus toxin specifically bound to neuronal plasma membranes isolated both from electric organ and electric lobe of Torpedo, exhibiting one and two binding sites respectively; (ii) [125I]tetanus toxin was internalized into nerve terminals and retrogradely transported to the electric lobe after its injection in the electric organ; (iii) finally, intracellular effect of tetanus toxin was studied either at electric organ and electric lobe membrane fractions. In both preparations tetanus toxin cleaved synaptobrevin, as detected by immunoblotting methods. In conclusion, our findings exhibit the presence of two different populations of acceptors for tetanus toxin in central and peripheral nervous system and show that synaptobrevin cleavage may account for intracellular toxicity in Torpedo.

Animals↗
PubMed1993

High resolution labeling of cholinergic nerve terminals using a specific fully active biotinylated botulinum neurotoxin type A.

We report here on the synthesis and characterization of a fully active biotinylated derivative of the botulinum neurotoxin type A. Different ratios of biotin: botulinum toxin were tested to optimize derivatizing conditions and a ratio of 35:1 was selected for further experiments. The average number of biotin groups per toxin molecule was estimated to be 7.8, occurring at both heavy and light chains, and almost all externally located and easily accessible to recognition by streptavidin. The modified toxin retained its toxicity and its ability to interact with biological membranes. Apart from its suitability for detection in Western blots and in microtiter well plates, biotinylated botulinum toxin proved to be adequate for morphological labeling studies at both light and electron microscopy. Peroxidase histochemistry in cryostat sections of intoxicated rat hemidiaphragm muscles showed a distinct labeling of end-plates. Electron microscopy studies were performed on the electric organ of Torpedo marmorata using colloidal gold-conjugated streptavidin for detection. After intoxication of electric organ fragments with the modified toxin, gold labels were found associated with the presynaptic plasma membrane of nerve terminals and with the membrane of synaptic vesicles. Moreover, the distribution of biotinylated botulinum toxin binding sites over the membrane of synaptosomes isolated from the electric organ of Torpedo and their relationship with intramembrane particles were analyzed using the replica-staining label-fracture technique. It was found that the toxin is never associated with intramembrane particles.

Animals↗
PubMed1993

Calcium-dependent and -independent acetylcholine release from electric organ synaptosomes by pardaxin: evidence of a biphasic action of an excitatory neurotoxin.

The effect of pardaxin, a new excitatory neurotoxin, on neurotransmitter release was tested using purely cholinergic synaptosomes of Torpedo marmorata electric organ. Pardaxin elicited the release of acetylcholine with a biphasic dose dependency. At low concentrations (up to 3 x 10(-7) M), the release was calcium-dependent and synaptosomal structure was well preserved as revealed by electron microscopy and measurements of occluded lactate dehydrogenase activity. At concentrations from 3 x 10(-7) M to 10(-5) M, the pardaxin-induced release of acetylcholine was independent of extracellular calcium, and occluded synaptosomal lactate dehydrogenase activity was lowered, indicating a synaptosomal membrane perturbation. Electron microscopy of 10(-6) M pardaxin-treated synaptosomes revealed nerve terminals depleted of synaptic vesicles and containing cisternae. At higher toxin concentrations (> or = 10(-5) M), there were striking effects on synaptosomal morphology and occluded lactate dehydrogenase activity, suggesting a membrane lytic effect. We conclude that, at low concentrations, this neurotoxin is a promising tool to investigate calcium-dependent mechanisms of neurotransmitter release in the nervous system.

Acetylcholine↗
PubMed1993

[Epidemiological data on chronic myeloid leukemia in Asturias, 1972-1986].

PURPOSE: To evaluate the incidence of CML in Asturias during the period 1972-1986, studying the variations in relation to age, sex and geographical zone. MATERIAL AND METHODS: Information relating to the date of diagnosis, name, age, sex and home address of the 108 cases (65 men and 43 women) diagnosed CML during the period 1972-1986 according to conventional criteria was collected from the hospital records of the region. For the calculations, SADEI demographic data were taken as a reference, comparing the incidence between sexes and geographical zones by means of the CHI2. RESULTS: A rate of incidence of 0.64/100,000 inhabitants/year was obtained, this being 0.8 in men and 0.5 in women (p < 0.05). The rate of incidence increased progressively with age until the 45-59 age group when it was maximum. There were no differences between the three main centres of population Gijón (0.53), Oviedo (0.55) and Avilés (0.69) nor the latter with the rest of the zones in Asturias; nor was three either any difference between coastal towns (0.56) and those of the interior (0.71) (p = NS). CONCLUSIONS: Limited incidence of CML in Asturias without significant differences in its geographical distribution within the region. The incidence, which was higher among males, increased progressively with age until reaching maximum importance in the 45-59 age group.

Adolescent↗
PubMed1992

Binding of botulinum neurotoxin to pure cholinergic nerve terminals isolated from the electric organ of Torpedo.

Torpedo electric organ has been used to study the binding of botulinum neurotoxin type A to pure cholinergic synaptosomes and presynaptic plasma membrane. 125I-labeled botulinum neurotoxin type A exhibits specific binding to cholinergic fractions. Two binding sites have been determined according to data analysis: a high affinity binding site (synaptosomes: Kd = 0.11 +/- 0.03 nM, Bmax = 50 +/- 10 fmol.mg prot-1; presynaptic plasma membrane: Kd = 0.2 +/- 0.05 nM, Bmax = 150 +/- 15 fmol.mg prot-1) and a low affinity binding site (synaptosomes: Kd approximately 26 nM, Bmax approximately 7.5 pmol.mg prot-1; presynaptic plasma membrane: Kd approximately 30 nM, Bmax approximately 52 pmol.mg prot-1). The binding of 125I-botulinum neurotoxin type A is decreased by previous treatment of synaptosomes by neuraminidase and trypsin, and by a preincubation with bovine brain gangliosides or antiserum raised against Torpedo presynaptic plasma membrane. When presynaptic plasma membranes are blotted to nitrocellulose sheet, either 125I-botulinum neurotoxin or botulinum toxin-gold complexes bind to a M(r) approximately 140,000 protein. Botulinum toxin-gold complexes have also been used to study the toxin internalization process into Torpedo synaptosomes. The images fit the three step sequence model in the pathway of botulinum neurotoxin poisoning.

Acetylcholine↗
PubMed1992

[Features of chronic myeloid leukemia at diagnosis. Study of a series of 134 cases].

PURPOSE: To analyse the clinico-biological features of 134 patients with chronic myelogenous leukaemia (CML) at presentation. MATERIAL AND METHODS: The series is comprised of 134 patients from the Asturias Central Hospital and other hospital of the region, diagnosed of CML with conventional criteria between 1970 and 1989. A retrospective study was carried out revising the clinical records and the clinico-biological data at diagnosis. Cytogenetic studies were available in 62 cases. The statistical analysis was based upon descriptive statistics and comparison of means and proportions by the chi square and Student's tests. Univariate study was also performed for several variables. RESULTS: The mean age of the group was 50 years, ranging between 2 and 81. The M/F ratio was 76/58. The commonest symptoms at onset were those secondary to hypermetabolism and splenomegaly, 8% of the patients being asymptomatic. Splenomegaly was present in 73.8% of the patients and hepatomegaly in 37.6%. The median white cell count was 132 x 10(9)/L. Absolute basophilia and eosinophilia were seen in 83% and 78% of the cases, respectively. Anaemia was found in 47.4% of the patients, usually mild, and 39% of them had nucleated red cells in peripheral blood. The median platelet count was 400 x 10(9)/L. Thrombocytosis was found in 48% of the cases, while 11% had thrombocytopenia. The mean number of blast cells in the bone marrow was 1.72%. The histopathologic study of the bone marrow revealed decreased red cells in 94.5% of the patients and decreased megakaryocytes in 29.5%; these last were increased in 50% of the patients. Increased reticulin fibres were found in 38.5% of the bone marrow samples. In addition to the Ph' chromosome, which was present in 51 patients, chromosomal abnormalities were seen in 15.6% of the cases in the chronic phase and in 69.2% in the terminal stages of the disease. Positive correlation could be established between the white cell count and the size of spleen (p < 0.001) and liver (p < 0.05), and there was a negative correlation between white blood cell count and haemoglobin rate and platelet count (p < 0.05 for both). CONCLUSIONS: (1) The analysis of this series shows that the CML cases in this region have similar characteristics to those in other western world communities (2). The mean age of this group is somewhat higher than in other series, which should be re-evaluated after discarding the Ph'-negative cases. (3) There seems to be positive correlation between leucocyte count and spleen and liver enlargement, and negative correlation between leucocyte count and haemoglobin and platelet count.

Adolescent↗
PubMed1981

[Neonatal septic arthritis. Our experience in 14 cases (author's transl)].

A survey was carried out from october 1973 to december 1979 of fourteen newborn who presented septic arthritis secondary to sepsis, developed during their admission at our neonatology unit. Authors point out the high incidence of Klebsiella as the organism isolated in 48% of the positive cultures and the good response to general and local treatments. A discussion is done on the sequels and their long-term evolution.

Arthritis, Infectious↗
PubMed1979

[Meckel syndrome (author's transl)].

Authors study four cases, two siblings in two different families, with several anomalies allowing to consider them as examples of the Meckel-Grüber syndrome. A review of the recent literature about this entity is made, as well as a comparative study of the anomalie present in the published cases.

Abnormalities, Multiple↗