Biomedical subjects

J Blasi

Publications and source records attributed to J Blasi.

At least 37 records · Page 2Linked to original sources

PubMed1997

Regulated secretion is impaired in AtT-20 endocrine cells stably transfected with botulinum neurotoxin type A light chain.

Botulinum neurotoxin type A (BoNT/A) inhibits neurotransmitter release by specific cleavage of SNAP-25, a synaptosome-associated protein also expressed in the ACTH secretory cell line AtT-20. Expression of light chain BoNT/A (L-BoNT/A) gene transfected into AtT-20 cells resulted in a cleaved form of SNAP-25 indistinguishable from that generated by bona fide BoNT/A. L-BoNT/A-transfected cells showed no difference in replication rate, viability, or phenotype, compared with control AtT-20 cells. In contrast, L-BoNT/A-transfected cells could not be induced to secrete ACTH upon stimulation by 8-bromo-cAMP or KCl. In addition, alpha-latrotoxin induced ACTH release from control cells, but not from L-BoNT/A-transfected cells. These experiments suggest an important role for SNAP-25 in regulated secretion from AtT-20 cells and underline the usefulness of this cell system as a tool for the study of the molecular mechanism of peptide hormone secretion.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Regulation of glucose transport, and glucose transporters expression and trafficking in the heart: studies in cardiac myocytes.

Cardiac muscle is characterized by a high rate of glucose consumption. In the absence of insulin, glucose transport into cardiomyocytes limits the rate of glucose utilization and therefore it is important to understand the regulation of glucose transporters. Cardiac muscle cells express 2 distinct glucose transporters, GLUT4 and GLUT1; although GLUT4 is quantitatively the more important glucose transporter expressed in heart, GLUT1 is also expressed at a substantial level. In isolated rat cardiomyocytes, insulin acutely stimulates glucose transport and translocates both GLUT4 and GLUT1 from an intracellular site to the cell surface. Recent evidence indicates the existence of at least 2 distinct intracellular membrane populations enriched in GLUT4 with a different protein composition. Elucidation of the intracellular location of these 2 GLUT4 vesicle pools in cardiac myocytes, their role in GLUT4 trafficking, and their relation to insulin-induced GLUT4 translocation needs to be addressed.

Animals↗

PubMed1997

Immunocytochemical analysis of the synaptic proteins SNAP-25 and Rab3A in human pituitary adenomas. Overexpression of SNAP-25 in the mammmosomatotroph lineages.

SNAP-25 and Rab3A were originally identified as synaptic proteins involved in neuronal membrane traffic. Recently, both proteins have been detected in several mammalian endocrine cell types and have been proposed as essential components of the exocytotic pathway in neuroendocrine cells. In this study, the expression of SNAP-25 and Rab3A was analysed in biopsied human anterior pituitary tumours (21 cases) by immunocytochemical methods. No differences in Rab3A immunoreactivity were observed between tumour and normal pituitary cells. Strong SNAP-25 immunoreactivity was detected in tumour cells of prolactinomas (n = 3). Several growth hormone (GH)/prolactin (PRL) tumours also displayed intense SNAP-25 immunolabelling (n = 3), whereas the remaining GH-secreting adenomas (n = 4) exhibited moderate to weak SNAP-25 immunoreactivity. In contrast, SNAP-25 near-background immunostaining was observed in tumour cells of adrenocorticotrophic hormone (ACTH)-secreting tumours (n = 4) and non-secreting tumours (n = 7), as well as in normal pituitary cells. Since SNAP-25 and Rab3A have been shown to be involved in exocytotic events in rodent endocrine cells, overexpression of SNAP-25 protein in PRL and GH/PRL tumour cells might be implicated in the mechanism of exocytosis of the neoplastic human mammosomatotroph lineages.

Acromegaly↗

PubMed1997

Characterization of presynaptic proteins involved in synaptic vesicle exocytosis in the nervous system of Torpedo marmorata.

Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a key role in the exocytotic machinery of synaptic vesicles. Their presence, distribution and interactions are reported in central and peripheral nervous systems of the electric fish Torpedo marmorata. These three proteins form a protein complex in all the nervous system regions tested, including the electric lobe and the electric organ which is innervated by pure cholinergic nerve terminals. Immunoblot analysis revealed a double protein pattern of SNAP-25 in the anterior brain and cerebellum, although a single protein band corresponding to SNAP-25 was observed in the electromotor system. Moreover, SNAP-25 showed a differential distribution in the electromotor system. It was present along nerve fibres and terminals that innervated the electric organ but it was not detected in nerve terminals at the electric lobe. Immunoisolation experiments using anti-synaptobrevin antibodies showed a tissue-specific co-existence of SNAP-25 and syntaxin with synaptobrevin in the immunoisolated organelles. In conclusion, the molecular components of the exocytotic machinery are shown to be conserved in Torpedo, although some differences mainly on SNAP-25, suggest a potential diversity in the regulation of neurosecretion.

Animals↗

PubMed1997

Localization of putative receptors for tetanus toxin and botulinum neurotoxin type A in rat central nervous system.

Clostridial neurotoxins (tetanus and botulinum toxins) are potent blockers of neurotransmitter release. These toxins act specifically on the nervous system by interacting with still non-identified protein receptors together with gangliosides. Whereas many biochemical data are available on their binding properties to neuronal membranes in vitro, there is poor morphological evidence of their binding to mammalian central nervous system. In the present study, the binding of tetanus and botulinum neurotoxin type A to rat brain sections is reported. Both toxins bound to nerve terminals with a broad distribution in brain. Tetanus toxin additionally bound to nerve fibres. The staining patterns were clearly shown to be due to the interaction of the heavy chains, which contain the binding moiety, with the tissue. In an attempt to investigate the nature of the acceptors present in the tissue, some sections were pre-incubated with periodic acid. This treatment resulted in the additional binding of botulinum neurotoxin type A to nerve fibres. Since the extended staining of nerve terminals was not modified by this pretreatment, it is suggested that protein receptors of clostridial neurotoxins are located at the nerve terminals, which may be common constituents of the synapses.

Animals↗

PubMed1997

Characterization of two distinct intracellular GLUT4 membrane populations in muscle fiber. Differential protein composition and sensitivity to insulin.

A major objective for the understanding of muscle glucose disposal is the elucidation of the intracellular trafficking pathway of GLUT4 glucose carriers in the muscle fiber. In this report, we provide functional and biochemical characterization of two distinct intracellular GLUT4 vesicle pools obtained from rat skeletal muscle. The two pools showed a differential response to insulin; thus, one showed a marked decrease in GLUT4 levels but the other did not. They also showed a markedly different protein composition as detected by quantitative vesicle immunoisolation analysis. The GLUT4 pool showing no response to insulin contained SCAMP proteins and the vSNARE proteins VAMP2 and cellubrevin, whereas only VAMP2 was found in the insulin-recruitable GLUT4 pool. SDS-PAGE and further silver staining of the immunoprecipitates revealed discrete polypeptide bands associated to the insulin-sensitive pool, and all these polypeptide bands were found in the insulin-insensitive population. Furthermore, some polypeptide bands were exclusive to the insulin-insensitive population. The presence of cellubrevin and SCAMP proteins, endosomal markers, suggest that the insulin-insensitive GLUT4 membrane population belongs to an endosomal compartment. In addition, we favor the view that the insulin-sensitive GLUT4 membrane pool is segregated from the endosomal GLUT4 population and is undergoes exocytosis to the cell surface in response to insulin. Intracellular GLUT4 membranes obtained from skeletal muscle contain cellubrevin, and VAMP2 and GLUT4-vesicles from cardiomyocytes also contain cellubrevin. This suggests that vSNARE proteins are key constituents of GLUT4 vesicles. The presence of the tSNARE protein SNAP25 in skeletal muscle membranes and SNAP25 and syntaxin 1A and syntaxin 1B in cardiomyocyte plasma membranes further suggest a role of the SNAREs in GLUT4 trafficking in muscle.

Animals↗

PubMed1997

BDNF and TrkB co-localize in CA1 neurons resistant to transient forebrain ischemia in the adult gerbil.

Delayed cell death of projection cells in the CA1 area of the hippocampus is produced in the adult gerbil following 5 minutes (min) of transient forebrain ischemia. Parvalbumin-immunoreactive local-circuit neurons are resistant to the ischemic insult. Brain-Derived Neurotrophic Factor (BDNF) immunoreactivity is localized in all neurons of the CA1 area in control gerbils. However, TrkB immunoreactivity is observed in a minority of BDNF-immunoreactive neurons in the CA1 area. The number of BDNF-immunoreactive cells in CA1 is dramatically reduced in ischemic gerbils as early as 24 h after ischemia, but the number of TrkB-immunoreactive cells in the CA1 area is maintained following ischemia. Moreover, about 90% of BDNF-immunoreactive cells and about 85% of TrkB-immunoreactive cells in ischemic gerbils co-localize the calcium-binding protein parvalbumin. Finally, BDNF and TrkB are coexpressed in about 95% of CA1 neurons surviving the ischemic insult. These results indicate that a subpopulation of CA1 hippocampal neurons coexpressing TrkB, parvalbumin and BDNF is resistant to transient forebrain ischemia in the gerbil. These results also suggest that a subpopulation of CA1 hippocampal neurons in the gerbil hippocampus is endowed with a putative BDNF/TrkB autocrine regulatory loop that may be involved in both cell survival and synaptic remodeling of the damaged gerbil hippocampus following transient forebrain ischemia.

Animals↗

PubMed1996

Fatty acylation of synaptotagmin in PC12 cells and synaptosomes.

Synaptotagmin I is localized to synaptic vesicles where it functions in the calcium-triggered release of neurotransmitters. Here we demonstrate that synaptotagmin I covalently incorporated [3H]palmitate after metabolic labelling of PC-12 cells and rat brain synaptosomes. Labeling was localized to a tryptic fragment that contains a cluster of cysteine residues adjacent to the molecule's single transmembrane anchor. Neutral hydroxylamine released the [3H]palmitate from this fragment and increased its electrophoretic mobility, demonstrating that acylation occurs at the membrane-proximal cysteine cluster. In addition, hydroxylamine-induced mobility shifts were also apparent for synaptotagmins II and III, suggesting that posttranslational palmitoylation via thioester bonds may be a general modification of all synaptotagmins.

Acylation↗

PubMed1996

Differential distribution of syntaxin isoforms 1A and 1B in the rat central nervous system.

Syntaxin 1 binds to several proteins of the synaptic terminal and is a central component in the pathway of protein-protein interactions that underlies docking and fusion of synaptic vesicles. Molecular studies revealed the occurrence of two isoforms, syntaxin 1A and syntaxin 1B, which coexpress in neural tissues. However, they display differential expression patterns in endocrine cell types. We generated isoform-specific antibodies that were used in Western blotting and immunocytochemical studies. First, we confirmed the sole presence of syntaxin 1A in endocrine pituitary cells. Second, we found distinctive immunolabelling patterns of each isoform in the rat olfactory system, hippocampus, striatum, thalamus and spinal cord. In addition, the principal white matter commissures displayed distinct immunoreactivity for each isoform. This report shows, for the first time, major differences between the distributions of syntaxin 1A and syntaxin 1B isoforms in the rat central nervous system.

Animals↗

PubMed1996

Expression of synaptosomal-associated protein SNAP-25 in endocrine anterior pituitary cells.

A growing body of evidence indicates that the fundamental molecular mechanism of exocytosis in the secretory pathway may be structurally similar in all eukaryotic cells. The synaptosomal-associated protein of 25 kDa (SNAP-25) is a plasma membrane protein involved in regulated exocytosis in neurons. In order to compare exocytotic components in neurons and endocrine cells, we have analyzed the expression of SNAP-25 in the rat anterior pituitary. Western blotting analysis documented the presence of SNAP-25 in anterior pituitary homogenates and cultured anterior pituitary cells. In addition to SNAP-25, other neuronal proteins involved in exocytosis (syntaxin, VAMP/synaptobrevin and Rab3A) were also detected in the anterior pituitary. The specific expression of SNAP-25 mRNA in anterior pituitary cells was also corroborated by Northern analysis. SNAP-25 immunoreactivity was located at the plasma membrane of endocrine anterior pituitary cells. Characteristically, patches of fine punctate deposits exhibited intense SNAP-25 immunoreactivity. Double-labeling immunocytochemistry revealed that SNAP-25 was mainly associated with gonadotroph cell populations. Furthermore, we demonstrate that in the anterior pituitary, SNAP-25 is selectively cleaved by clostridial neurotoxins. In conclusion, our results establish the presence of SNAP-25 in secretory anterior pituitary cells and suggest a potential role of this protein in the secretion of adenohypophysial hormone.

Animals↗

PubMed1995

Tetanus toxin inhibits spontaneous quantal release and cleaves VAMP/synaptobrevin.

Tetanus toxin decreased the frequency of spontaneous events at the electric organ of Torpedo marmorata. This reduction was up to 70% in poisoned electric organ. According to distribution analysis of miniature end plate currents, only a subpopulation of events which have small amplitudes were recorded after poisoning. Furthermore, isolated cholinergic nerve terminals showed a decrease in VAMP/synaptobrevin when poisoned with tetanus toxin under similar conditions. The relationship between the two effects of the toxin, i.e. inhibition of vesicle exocytosis and peptidase activity on synaptobrevin, is discussed.

Acetylcholine↗

PubMed1995

Poisoning by botulinum neurotoxin A does not inhibit formation or disassembly of the synaptosomal fusion complex.

We investigated the effect of poisoning rat brain synaptosomes with botulinum neurotoxin A on the NSF-mediated disassembly of a complex consisting of syntaxin, SNAP-25 and synaptobrevin (fusion complex). Botulinum neurotoxin A specifically removes 9 amino acids from the C-terminus of SNAP-25 and efficiently blocks KCl-evoked glutamate release from synaptosomes. We report that truncated SNAP-25 is incorporated into the fusion complex of poisoned synaptosomes. The presence of truncated SNAP-25 does not interfere with the NSF-induced disassembly of the fusion complex. Also, the release of truncated SNAP-25 from the fusion complex is similar to that of the native SNAP-25. Since neither the formation of the complex nor its disassembly seems to be affected by the SNAP-25 fragment, this fragment is likely to block exocytosis by disrupting events between disassembly of the synaptosomal fusion complex and membrane fusion itself.

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↗

PubMed1995

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

Animals↗

PubMed1994

Transmission at the squid giant synapse was blocked by tetanus toxin by affecting synaptobrevin, a vesicle-bound protein.

1. The effect of whole tetanus toxin (TeTX) and of its light chain (TeTX L-chain) on transmitter release was determined by presynaptic pressure-injection in the squid giant synapse. 2. The results indicate that whole TeTX does not modify transmission while the L-chain blocks transmission within 20-30 min. This block does not involve changes in the sodium or potassium conductances responsible for spike generation or the voltage-dependent presynaptic calcium current responsible for transmitter release. 3. Western blotting of protein fractions from the squid optic lobe demonstrated the presence of a protein which reacted with specific antibodies against mammalian synaptobrevin, a vesicular protein. In addition, this protein was enzymatically cleaved by the L-chain component of the toxin in a similar fashion to its mammalian counterpart. 4. These results demonstrate that TeTX L-chain toxin acts directly on a squid synaptobrevin and prevents synaptic release probably by interfering with the docking-fusion synaptic vesicles at the active zone.

Animals↗

PubMed1994

Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin.

Tetanus toxin (TeTx) and the various forms of botulinal neurotoxins (BoNT/A to BoNT/G) potently inhibit neurotransmission by means of their L chains which selectively proteolyze synaptic proteins such as synaptobrevin (TeTx, BoNT/B, BoNT/F), SNAP-25 (BoNT/A), and syntaxin (BoNT/C1). Here we show that BoNT/D cleaves rat synaptobrevin 1 and 2 in toxified synaptosomes and in isolated vesicles. In contrast, synaptobrevin 1, as generated by in vitro translation, is only a poor substrate for BoNT/D, whereas this species is cleaved by BoNT/F with similar potency. Cleavage by BoNT/D occurs at the peptide bond Lys59-Leu60 which is adjacent to the BoNT/F cleavage site (Gln58-Lys59) and again differs from the site hydrolyzed by TeTx and BoNT/B (Gln76-Phe77). Cellubrevin, a recently discovered isoform expressed outside the nervous system, is efficiently cleaved by all three toxins examined. For further characterization of the substrate requirements of BoNT/D, we tested amino- and carboxyl-terminal deletion mutants of synaptobrevin 2 as well as synthetic peptides. Shorter peptides containing up to 15 amino acids on either side of the cleavage site were not cleaved, and a peptide extending from Arg47 to Thr116 was a poor substrate for all three toxins tested. However, cleavability was restored when the peptide is further extended at the NH2 terminus (Thr27-Thr116) demonstrating that NH2 terminally located sequences of synaptobrevin which are distal from the respective cleavage sites are required for proteolysis. To further examine the isoform specificity, several mutants of rat synaptobrevin 2 were generated in which individual amino acids were replaced with those found in rat synaptobrevin 1. We show that a Met46 to Ile46 substitution drastically diminishes cleavability by BoNT/D and that the presence of Val76 instead of Gln76 dictates the reduced cleavability of synaptobrevin isoforms by TeTx.

Amino Acid Sequence↗

PubMed1994

Proteolysis of SNAP-25 by types E and A botulinal neurotoxins.

Clostridial neurotoxins, tetanus toxin (TeTx) and the seven related but serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G), are potent inhibitors of synaptic vesicle exocytosis in nerve endings. Recently it was reported that the light chains of clostridial neurotoxins act as zinc-dependent metalloproteases which specifically cleave synaptic target proteins such as synaptobrevin/VAMPs, HPC-1/syntaxin (BoNT/C1), and SNAP-25 (BoNT/A). We show here that BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli. BoNT/E cleaves the Arg180-Ile181 bond. This site is different from that of BoNT/A, which cleaves SNAP-25 between the amino acid residues Gln197 and Arg198. These findings further support the view that clostridial neurotoxins have evolved from an ancestral protease recognizing the exocytotic fusion machinery of synaptic vesicles whereby individual toxins target different members of the membrane fusion complex.

Amino Acid Sequence↗

PubMed1994

Inhibition of neurotransmitter release by clostridial neurotoxins correlates with specific proteolysis of synaptosomal proteins.

Rat brain synaptosomes were used to study the effect of several clostridial neurotoxins on the neurotransmitter release. In this system the blockade of transmitter release correlated with the proteolytic activity of the toxins. Blockade of glutamate release was linked to selective proteolysis of one of the following synaptic proteins: synaptobrevin (BoNT/D, BoNT/F); SNAP-25 (BoNT/A, BoNT/E), or HPC-1/syntaxin (BoNT/C1). All the toxins used had an inhibitory effect on synaptosomes with the exception of BoNT/F. BoNT/F cleaved synaptobrevin in permeabilized synaptosomes but failed to produce the same effect on intact synaptosomes.

Animals↗