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Biomedical subjects

L Vallar

Publications and source records attributed to L Vallar.

At least 37 records · Page 2Linked to original sources
PubMed1992

Purification of biologically active human plasma transthyretin by dye-affinity chromatography: studies on dye leakage and possibility of heat treatment for virus inactivation.

The application of a purification procedure for the industrial preparation from human plasma of a therapeutic protein may be hindered by several safety concerns. The dye leaching from Remazol Yellow GGL-Sepharose used for the affinity chromatography of human plasma transthyretin was quantitatively studied by a sensitive competitive enzyme immunoassay. The possibility of including a heat treatment step for virus inactivation in the purification process while preserving the biochemical and functional characteristics of the protein is also reported.

Azo Compounds↗
PubMed1992

Characterization of small-molecular-mass guanine-nucleotide-binding regulatory proteins in insulin-secreting cells and PC12 cells.

The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine-secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.

Adenosine Diphosphate Ribose↗
PubMed1992

Dye-affinity purification of transthyretin from an unexploited by-product of human plasma chromatographic fractionation.

Dye-affinity chromatography of human plasma transthyretin on Remazol Yellow GGL-Sepharose from an unexploited by-product of chromatographic fractionation of plasma was optimized for large-scale preparation of a therapeutic product. With this system, transthyretin is only weakly bound to the gel. The residence time on gel and the transthyretin level in the by-product were observed to have no influence on the binding capacity of gel, and the optimum amount of transthyretin to be applied to the gel was found to be 1 g/l of gel. The adsorbent can be used more than ten times. The procedure resulted in the isolation, with a 30% yield with respect to plasma, of an 80% pure protein, which retained its thyroxine-binding capacity. Although the purity is acceptable for substitutive therapy, it can be improved further with a second chromatography on Cibacron Blue-Sepharose.

Adsorption↗
PubMed1992

A sensitive enzyme immunoassay for the detection of a synthetic affinity ligand, the reactive yellow 13 dye.

Dye-affinity chromatography is widely and increasingly used for the isolation of various proteins. In particular, the purification of transthyretin can be efficiently achieved by affinity chromatography on immobilized Reactive Yellow 13. Measurement of trace-amounts of dye leaching from affinity columns is important because of possible toxicity or side effects linked with the presence of dye in therapeutic transthyretin preparations. A competitive enzyme immunoassay was developed to monitor yellow-dye column leaching. Biotinylated rabbit anti-Reactive Yellow 13 antibodies (immunoglobulin G fraction) were used as principal reagent. The assay is specific sensitive to 1 ng/ml of Reactive Yellow 13, has a good reproducibility and allows the accurate detection of the dye in the presence of transthyretin.

Affinity Labels↗
PubMed1992

G-protein oncogenes in acromegaly.

G-proteins belong to a family of proteins which share the common properties of GTP binding and hydrolysis. Heterotrimeric G-proteins are composed of alpha-, beta- and gamma-subunits. The alpha-subunit which differs from one G-protein to another contains the GDP/GTP binding site and has intrinsic GTPase activity. The receptor occupancy causes displacement of bound GDP by GTP, dissociation of free beta gamma-dimer and alpha-GTP complex, interaction of the activated alpha-GTP complex with intracellular effectors, such as enzymes and ion channels. The turn off of the reaction is due to the GTPase activity which causes the hydrolysis of GTP to GDP. G-proteins are essential for transferring hormonal signals from cell surface receptors to intracellular effectors. Since G-proteins generate intracellular effectors involved in cell growth, G-protein genes have the propensity to be converted into oncogenes. In fact, mutations in the alpha-subunit of Gs (the G-protein involved in the activation of adenylyl cyclase) have been demonstrated in 40% of human GH secreting pituitary adenomas. Single amino acid substitutions replacing Arg 201 with either Cys or His or Gln 227 with either Arg or Leu cause constitutive activation of adenylyl cyclase by inhibiting GTPase (gsp oncogene). The same mutations were identified in about 10% of thyroid adenomas and in the McCune-Albright syndrome.

Acromegaly↗
PubMed1991

Mutations in the alpha subunit of the stimulatory regulatory protein of adenylyl cyclase (Gs) in human GH-secreting pituitary adenomas. Biochemical, clinical, and morphological aspects.

Very recently a subset of human GH-secreting pituitary adenomas carrying a somatic mutation in the alpha subunit of the stimulatory regulatory protein of adenylyl cyclase (Gs) was identified. In all these tumors (Group 2; about 30% of all the GH secreting tumors studied) the alpha s cDNAs contained mutations; in 8 tumors mutations replaced Arginine 201 with either Cystein or Histidine while in the remaining tumors Glutamine 227 was replaced by either Arginine or Leucine. No mutations were observed in the remaining adenomas (Group 1). The two mutations caused a constitutive activation of adenylyl cyclase and a turn on of cAMP synthesis by inhibiting GTPase activity. The transformed phenotype was reflected in adenomatous cells with high rate of cAMP production and in vitro GH secretion. No difference in age, sex, clinical features, duration of the disease and cure rate were observed between the patients without (Group 1) or with alpha s mutation (Group 2), while higher serum GH levels and smaller tumor size were present in Group 2 patients. Moreover, hypersecretory activity in Group 2 tumors was also apparent at electron microscopy; cells of Group 2 tumors were densely granulated and showed prominent rough endoplasmic reticulum and Golgi complex. With respect to Group 1, Group 2 patients were less responsive to GH-releasing hormone (GHRH), while they were more sensitive to somastostatin. The former finding is in agreement with the hypothesis that the oncogenic proteins mimic the effects of extracellular growth factors, so removing the requirement for GHRH; the latter might explain the low rate of tumor growth as due to the counteracting role of endogenous inhibitory factors.

Adenoma↗
PubMed1990

Two G protein oncogenes in human endocrine tumors.

Somatic mutations in a subset of growth hormone (GH)-secreting pituitary tumors convert the gene for the alpha polypeptide chain (alpha s) of Gs into a putative oncogene, termed gsp. These mutations, which activate alpha s by inhibiting its guanosine triphosphatase (GTPase) activity, are found in codons for either of two amino acids, each of which is completely conserved in all known G protein alpha chains. The likelihood that similar mutations would activate other G proteins prompted a survey of human tumors for mutations that replace either of these two amino acids in other G protein alpha chain genes. The first gene so far tested, which encodes the alpha chain of Gi2, showed mutations that replaced arginine-179 with either cysteine or histidine in 3 of 11 tumors of the adrenal cortex and 3 of 10 endocrine tumors of the ovary. The mutant alpha i2 gene is a putative oncogene, referred to as gip2. In addition, gsp mutations were found in 18 of 42 GH-secreting pituitary tumors and in an autonomously functioning thyroid adenoma. These findings suggest that human tumors may harbor oncogenic mutations in various G protein alpha chain genes.

Amino Acid Sequence↗
PubMed1990

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells.

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.

Adenosine Triphosphate↗
PubMed1990

Rapid and reversible secretion changes during uncoupling of rat insulin-producing cells.

To determine whether insulin secretion is affected by a blockage of gap junctions between B cells, we have studied the secretion of rat pancreatic islets of Langerhans, primary dispersed islet cells, and cells of the RINm5F line, during short-term exposure to heptanol. Within minutes, this alkanol blocked gap junctions between the B cells of intact islets and abolished their normal secretory response to glucose. These two changes were rapidly and fully reversible after return of the islets to control medium. We further found that heptanol had no significant effect on the glucose-stimulated secretion of single B cells but inhibited that of B cell pairs. In the clone of RINm5F cells, whose junctional coupling and D-glyceraldehyde-induced stimulation of insulin release by aggregated cells were also inhibited by heptanol, this alkanol did not perturb intracellular pH and Ca2+ and the most distal steps of the secretion pathway. In summary, a gap junction blocker affected the secretion of insulin-producing cells by a mechanism which is dependent on cell contact and is not associated with detectable pleiotropic perturbations of the cell secretory machinery. The data provide evidence for the involvement of junctional coupling in the control of insulin secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Clinical, biochemical, and morphological correlates in patients bearing growth hormone-secreting pituitary tumors with or without constitutively active adenylyl cyclase.

Somatic mutations in the alpha-chain (alpha s) of the stimulatory regulatory protein of adenylyl cyclase (Gs) causing constitutive activation of the enzyme have been identified in a subset of human GH-secreting pituitary adenomas. This study reports on the differences between acromegalic patients bearing tumors without (group 1; n = 51) or with (group 2; n = 29) this alteration. No difference in age, sex, clinical features, duration of the disease, or cure rate was observed between the two groups. By contrast, group 2 patients had higher basal GH levels than group 1. Moreover, a significant difference in sellar morphology was found; group 2 patients more frequently showed sellas of normal size (grade I) than group 1. Hypersecretory activity of group 2 tumors was also apparent at electron microscopy; contrary to those of group 1, cells of group 2 tumors were densely granulated and showed prominent rough endoplasmic reticulum and Golgi complex. With respect to group 1, group 2 patients were less responsive to GH-releasing hormone, while they were more sensitive to somatostatin- and dopamine-induced GH inhibition. These results suggest that patients with constitutively active adenylyl cyclase have hyperactive tumors; the sensitivity of these tumors to inhibitory agents (somatostatin and dopamine), possibly counteracting the expression of activating mutations, might explain the low rate of tumor growth.

Acromegaly↗
PubMed1990

GTPase-inhibiting mutations activate the alpha-chain of Gs in human tumours.

Several trophic hormones transmit their signals by coupling to G-proteins. A subset of human growth hormone (GH)-secreting pituitary tumours, characterized by elevated GH secretion, cyclic AMP levels, and adenylyl cyclase activity, carries mutations in the gene that encodes the alpha-chain of Gs. By substitution of a single amino acid (Arg-201 or Gln-227), these mutations inhibit the intrinsic GTPase activity of alpha s, thus stabilizing the protein in its active conformation. The discovery of mutant alpha s proteins in human tumours suggests that the alpha s gene can be converted into an oncogene, called gsp, in cells that proliferate in response to cyclic AMP. Most likely, oncogenes can result from similar mutations in other G-protein alpha-chain genes.

GTP Phosphohydrolases↗
PubMed1989

GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours.

A subset of growth hormone-secreting human pituitary tumours carries somatic mutations that inhibit GTPase activity of a G protein alpha chain, alpha(s). The resulting activation of adenylyl cyclase bypasses the cells' normal requirement for trophic hormone. Amino acids substituted in the putative gsp oncogene identify a domain of G protein alpha-chains required for intrinsic ability to hydrolyse GTP. This domain may serve as a built-in counter-part of the separate GTPase-activating proteins required for GTP hydrolysis by small GTP-binding proteins such as p21ras.

Adenylyl Cyclases↗
PubMed1989

Mechanisms of signal transduction at the dopamine D2 receptor.

D2 dopamine receptor activation induces inhibition of adenylate cyclase, with a rapid decrease of cAMP levels, and an ensuing blockade of IP3-dependent release of Ca2+ from intracellular stores. K+ channels are concomitantly activated and Ca2+ channels are possibly also inhibited. The increased K+ conductance causes hyperpolarization, which may be responsible for the abolition of Ca2+ action potentials and [Ca2+]i fluctuations occurring both at rest and after activation of receptors coupled to PIP2 hydrolysis. Lucia Vallar and Jacopo Meldolesi analyse this spectrum of intracellular signals which might be sufficient to sustain inhibition of secretion in pituitary lactotroph cells and possibly the other effects of D2 receptors in other cell systems.

Animals↗
PubMed1988

Inhibition of inositol phosphate production is a late, Ca2+-dependent effect of D2 dopaminergic receptor activation in rat lactotroph cells.

The effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Regulation of inositol 1,4,5-trisphosphate metabolism in insulin-secreting RINm5F cells.

Factors underlying the transience of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] accumulation following muscarinic stimulation of RINm5F cells were examined. Transience was not due to a protein kinase C-mediated stimulation of Ins(1,4,5)P3 dephosphorylation, since pretreatment of cells with tetradecanoyl-phorbol acetate (TPA) did not alter the rate of this conversion. However, preincubation with TPA did inhibit carbamoylcholine-stimulated Ins(1,4,5)P3 formation. In permeabilized cells, the conversion of Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was slightly enhanced in the presence of TPA or cyclic AMP, but much more markedly by raising the Ca2+ concentration from 10(-7) M to 10(-6) or 10(-5) M. In intact cells the most rapid rate of accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 occurred in the first 2 s following stimulation, whereas the levels of inositol 1,4-bisphosphate were not increased until after 5 s. This suggests that Ins(1,4,5)P3 kinase is chiefly responsible for the early disposal of Ins(1,4,5)P3 following cellular stimulation. The results are consistent with the proposal that the transient accumulation of Ins(1,4,5)P3 is due both to its enhanced metabolism via the Ca2+-calmodulin-sensitive Ins(1,4,5)P3 kinase, as well as a down-regulation of phosphatidylinositol 4,5-bisphosphate hydrolysis.

Carbachol↗
PubMed1988

Alpha 1-adrenergic stimulation of in vitro growth hormone release and cytosolic free Ca2+ in rat somatotrophs.

The effects of alpha 1-adrenergic agents on GH release and intracellular free Ca2+ concentration ([Ca2+]i) were investigated in purified rat somatotroph preparations. Phenylephrine (PHE) stimulated in vitro GH release; the maximal effect (2.5-fold stimulation) occurred at 1 microM PHE. The effect was completely blocked by the alpha-adrenergic antagonist phentolamine and partially counteracted by the beta-antagonist propranolol. Experiments with the fluorescent Ca2+ probe fura 2 show that PHE causes [Ca2+]i to rise from 178 +/- 31 nM (mean +/- SE; n = 25) to 370 +/- 55 nM (n = 9). This effect was complete within 20 sec and was maintained for at least 5-10 min. The rise was rapidly interrupted by administration of 1 microM phentolamine. The beta-receptor agonist isoproterenol caused a small [Ca2+]i rise due to action on alpha 1-adrenoreceptors. The PHE-induced [Ca2+]i rise showed two components: an initial peak due to Ca2+ mobilization from intracellular stores and a subsequent rise due to Ca2+ influx from the extracellular space. Somatostatin (SRIF) lowered both resting [Ca2+]i and Ca2+ influx stimulated by PHE. Pertussis toxin pretreatment did not modify PHE-induced [Ca2+]i changes, while it completely prevented the effect of SRIF on both resting and triggered [Ca2+]i, thus suggesting that a GTP-binding protein sensitive to the toxin is involved in the transduction of SRIF action. The increase in cAMP induced by cholera toxin pretreatment modified neither PHE nor SRIF action on [Ca2+]i. In conclusion, in rat somatotrophs Ca2+ mobilization and influx are stimulated by alpha 1-adrenergic agents, and this triggered [Ca2+]i rise results in a stimulation of GH release. In these cells SRIF is able to reduce both resting [Ca2+]i levels and [Ca2+]i increases induced by alpha 1-adrenergic activation.

Adenosine Diphosphate Ribose↗
PubMed1987

Dopamine inhibits cytosolic Ca2+ increases in rat lactotroph cells. Evidence of a dual mechanism of action.

Single rat lactotroph cells were studied after loading with the cytosolic free Ca2+ concentration ([Ca2+]i) indicator fura-2 either 1 or 3 days after cell dispersion. Under unstimulated conditions, two groups of lactotrophs were observed, the first (predominant at day 1) with large [Ca2+]i fluctuations (peaks up to 300 nM) probably due to spontaneous action potentials and the second (predominant at 3 days) with stable [Ca2+]i (values variable between 65 and 200 nM). The effect of dopamine on the resting [Ca2+]i was different in the two groups. Even at high dopamine concentrations, no change occurred in the second group; whereas in the first, disappearance of fluctuations and marked decrease of [Ca2+]i were observed. These effects of dopamine appear to be due to hyperpolarization that was demonstrated by the use of a specific fluorescent indicator, bis(oxonol). Two types of triggered [Ca2+]i transients were studied in detail: those due to redistribution of Ca2+ from the intracellular stores (induced by thyrotropin-releasing hormone) and those due to Ca2+ influx through voltage-gated Ca2+ channels (induced by high [K+]). Dopamine (1 microM) markedly inhibited both these transients by the action of D2 receptors (blocked by 1-sulpiride and domperidone). All effects of dopamine were prevented by treatment of the cells with pertussis toxin, indicating the involvement of one (or more) GTP-binding protein(s). Another consequence of D2 receptor activation is the inhibition of adenylate cyclase. Treatments (cholera toxin, forskolin), known to raise cAMP levels, were found to dissociate the effects of dopamine on [Ca2+]i inasmuch as they markedly relieved the inhibition of the redistributive transients by thyrotropin-releasing hormone but left hyperpolarization and inhibition of K+ transients unaffected. The spectrum of intracellular signals elicited by the activation of D2 receptors is therefore complex and includes at least two mechanisms that involve [Ca2+]i, one related and the other independent of the decrease of cAMP levels.

Adenylate Cyclase Toxin↗