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M Blázquez

Publications and source records attributed to M Blázquez.

At least 19 recordsLinked to original sources
PubMed2001

Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic.

Testosterone and oestradiol can modulate GABA synthesis in sexually regressed goldfish. Here we investigated their effects on the mRNA expression of two isoforms of the GABA synthesizing enzyme glutamate decarboxylase (GAD(65) and GAD(67), EC 4.1.1.15). Full-length GAD clones were isolated from a goldfish cDNA library and sequenced. Goldfish GAD(65) encodes a polypeptide of 583 amino acid residues, which is 77% identical to human GAD(65). Goldfish GAD(67) encodes a polypeptide of 587 amino acid residues and is 82% identical to human GAD(67). Goldfish GAD(65) and GAD(67) are 63% identical. Sexually regressed male and female goldfish were implanted with solid silastic pellets containing testosterone, oestradiol or no steroid. Semiquantitative PCR analysis showed that oestradiol significantly increased GAD(65) mRNA expression in female hypothalamus and telencephalon, while testosterone resulted in a significant increase only in telencephalon. GAD(67) mRNA levels were not affected by steroids in females. In contrast, both steroids induced significant decreases of GAD(65) and GAD(67) mRNA levels in male hypothalamus, but had no effect on GAD mRNA expression in male telencephalon. Our results indicate that modulation of GAD mRNA expression is a possible mechanism for steroid action on GABA synthesis, which may have opposite effects in males and females.

Amino Acid Sequence↗
PubMed2001

Association of prohormone convertase 3 with membrane lipid rafts.

Prohormone convertase 3 (PC3) is a neuroendocrine-specific member of the subtilisin-kexin family, involved in the intracellular processing and maturation of prohormones and proneuropeptides. PC3 is synthesised as a proprotein that undergoes two different cleavages resulting in the mature PC3 and the enzymatically active PC3DeltaC. In vitro translated proPC3 and proPC3DeltaC bind to trans-Golgi network (TGN)/granule-enriched membranes from the AtT20 neuroendocrine cell line in a pH-dependent manner suggesting both a dominant role for the pro-region in membrane association and that the C-terminal region is not essential. However, while PC3 bound to membranes the majority of PC3DeltaC did not, suggesting that either the pro-region or the C-terminal region of PC3 is required for membrane association. Removal of peripheral membrane proteins did not affect the binding properties of any of the in vitro translated proteins. Chromaffin granule membranes (CGMs) were used to study the binding characteristics of endogenous PC3 and its active C-terminal truncated counterpart (PC3DeltaC). Incubation of CGMs with Triton X-100 did not completely solubilise either of these forms of PC3. Moreover, both PC3 and PC3DeltaC remained associated with detergent-resistant membrane microdomains, termed lipid rafts, purified from CGMs. The data raise the possibility that PC3 and PC3DeltaC are sorted to the regulated secretory pathway via their association with membrane lipid rafts.

Aspartic Acid Endopeptidases↗
PubMed2000

Involvement of the membrane lipid bilayer in sorting prohormone convertase 2 into the regulated secretory pathway.

Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45-84 of proPC2 (proPC2(45-84)) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC2(45-84). Rather, proPC2(45-84) was shown to bind to protein-free liposomes in a pH- and Ca(2+)-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.

Animals↗
PubMed2000

Basic mechanisms of secretion: sorting into the regulated secretory pathway.

Targeting proteins to their correct cellular location is crucial for their biological function. In neuroendocrine cells, proteins can be secreted by either the constitutive or the regulated secretory pathways but the mechanism(s) whereby proteins are sorted into either pathway is unclear. In this review we discuss the possibility that sorting is either an active process occurring at the level of the trans-Golgi network, or that sorting occurs passively in the immature granules, The possible involvement of protein-lipid interactions in the sorting process is also raised.

Animals↗
PubMed1999

Multiplicity of glutamic acid decarboxylases (GAD) in vertebrates: molecular phylogeny and evidence for a new GAD paralog.

The evolution of chordate glutamic acid decarboxylase (GAD; EC 4.1.1.15), a key enzyme in the central nervous system synthesizing the neurotransmitter gamma-amino-butyric acid (GABA) from glutamate, was studied. Prior to this study, molecular data of GAD had been restricted to mammals, which express two distinct forms, GAD65 and GAD67. These are the products of separate genes and probably are derived from a common ancestral GAD following gene duplication at some point during vertebrate evolution. To enable a comprehensive phylogenetic analysis, molecular information of GAD forms in other vertebrate classes was essential. By reverse transcriptase-polymerase chain reaction (RT-PCR), partial nucleotide sequences of GAD were cloned from brains of zebra finch (Taeniopygia guttata), turtle (Trachemys scripta), goldfish (Carassius auratus), zebrafish (Danio rerio), and armoured grenadier (Coryphaenoides (Nematonurus) armatus, a deep-sea fish), and from the cerebral ganglion plus neural gland of Ciona intestinalis, a protochordate. Whereas GAD65 and GAD67 homologs were expressed in birds, reptiles, and fish, only a single GAD cDNA with equal similarities to both vertebrate GAD forms was found in the protochordate. This indicates that the duplication of the vertebrate GAD gene occurred between 400 and 560 million years ago. For both GAD65 and GAD67, the generated phylogenetic tree followed the general tree topology for the major vertebrate classes. In turtle, an alternative spliced form of GAD65, putatively encoding a truncated, nonactive GAD, was found. Furthermore, a third GAD form, which is equally divergent from both GAD65 and GAD67, is expressed in C. (N.) armatus. This third form might have originated from an ancient genome duplication specific to modern ray-finned fishes.

Alternative Splicing↗
PubMed1998

Fish as models for the neuroendocrine regulation of reproduction and growth.

Models are essential for the full understanding of neuroendocrine control processes. In this regard fish offer a rich source of biological material. They have diverse growth and reproductive strategies, inhabiting most of the Earth's aquatic ecological niches. Fish possess many of the common vertebrate features but also offer several unique aspects to allow the biologist easy access to the study of hypothalamic and pituitary function. Several key examples of how teleosts, or the bony fish, can offer insight into fundamental mechanisms of vertebrate sex differentiation, growth and reproduction are reviewed.

Animals↗
PubMed1998

Gamma-aminobutyric acid up-regulates the expression of a novel secretogranin-II messenger ribonucleic acid in the goldfish pituitary.

An RNA-arbitrarily primed PCR differential display strategy was used to identify candidate genes in the pituitary that are up-regulated by endogenously activated gamma-aminobutyric acid (GABA) systems that may also be involved in the control of reproduction. Goldfish were injected with the GABA metabolism inhibitor gamma-vinyl-GABA (GVG), known for its high efficiency to specifically increase endogenous brain and pituitary GABA levels in this species, resulting in higher levels of circulating gonadotropin-II (GTH-II). Several transcripts related to hormone secretion, signal transduction pathways, and messenger RNA (mRNA) editing were shown to be up-regulated after GVG injection. Among these transcripts we characterized an mRNA coding for the secretory vesicle protein secretogranin-II (SgII), a member of the chromogranin family, which is the precursor of a novel 34 amino acid neuropeptide, goldfish secretoneurin (SN). A semiquantitative PCR developed to measure pituitary SgII mRNA levels showed a 5-fold increase in GVG treated fish vs. control fish. Moreover, GVG treatment specifically increased SgII mRNA levels in gonadotrophs, concomitant with a decrease in GTH-II cell content. In addition, i.p. injection of synthetic goldfish SN increased GTH-II release in goldfish pretreated with the dopamine antagonist domperidone. Activation of GABAergic neurons has two effects, enhancing in vivo GTH-II release and up-regulating SgII mRNA specifically in goldfish gonadotrophs. Together with our SN bioactivity data, this suggests the existence in the pituitary of an autocrine or paracrine mechanism linked to the regulated secretory pathway in the gonadotrophs.

Amino Acid Sequence↗
PubMed1997

Electrochemical evidence on the molten globule conformation of cytochrome c.

To explore a new approach for characterizing the molten globule conformation, cyclic voltammetric studies of salt induced transitions at acidic pH of cyt c have been carried out. The use of modified electrodes has made the observation of direct electrochemistry in native cyt c possible. However, most of these electrodes do not show reversible responses at acidic pH, due to the fact that, for this system, a deprotonated electrode surface is needed. In these studies, we have used a 6-mercaptopurine and cysteine-modified gold electrodes which are effective for direct rapid electron transfer to cyt c, even in acid solutions. The change in the absorption bands of cyt c are used to monitor the conformational states and, hence, to compare the voltammetric results. Under the experimental conditions where the A state of cyt c is obtained, a reversible voltammetric signal is observed. The midpoint peak potentials are found to be very close to the formal potential of native cyt c. Results are discussed in terms of a cooperative two-state transition between the acid unfolded and the globular acidic states of cyt c. This finding establishes, for the first time, the similarity of both the native and the molten globule-like conformations in terms of its redox properties.

Animals↗
PubMed1996

Oxidation of sulfhydryl groups of ribonuclease inhibitor in epithelial cells is sufficient for its intracellular degradation.

Ribonuclease inhibitor (RI) is a cytoplasmic protein (50 kDa) that inhibits a variety of pancreatic type RNases. The porcine inhibitor contains 30 cysteine residues, all of which occur in the reduced state. It is well known that in vitro modification of the thiol groups inactivates the protein and greatly increases its susceptibility to proteolysis. Here we show that oxidation of thiol groups in RI can also occur within the cell. Induction of an oxidative insult in cultured LLC-PK1 cells, either with a general oxidant, H2O2, or with a thiol-specific oxidant, diamide, led to the loss of RI activity. By using specific antibodies it was demonstrated that the decrease correlated with a decline in the amount of RI protein in the cells. Furthermore, analysis of RI mRNA levels and half-life of the protein excluded inhibition of the synthesis of RI as the cause of its depletion. The results indicate that oxidation of thiol groups in RI is sufficient to cause its rapid inactivation and disappearance from the cell. Most likely this results from intracellular degradation of the protein.

Animals↗