Biomedical subjects
M Costa
Publications and source records attributed to M Costa.
The regulation of flowering time by daylength in Arabidopsis.
We are studying several Arabidopsis mutants that show altered regulation of flowering time in response to daylength. One of the mutations we are studying, constans, delays flowering under long days but has no effect under short days. Analysis of the expression of the CONSTANS gene, and modification of its expression in transgenic plants, suggests that this gene promotes flowering in response to long days and that the delay in flowering that occurs in wild-type plants under short days is at least in part due to regulation of CONSTANS gene transcription. We describe genetic approaches that we are taking to identify genes that act in the same genetic pathway as CONSTANS, and in particular the relationship between CONSTANS and two other genes that we are studying. These are LATE ELONGATED HYPOCOTYL, for which we have a dominant mutant allele that causes late flowering, and EARLY SHORT DAYS 4, whose inactivation causes early flowering. In addition to their effects on flowering time, the over-expression of CONSTANS and the inactivation of EARLY SHORT DAYS 4 cause the Arabidopsis shoot to become determinate and therefore to terminate development prematurely. This phenotype is discussed in light of other genes that have previously been shown to be required to maintain indeterminate development of the shoot.
[Splanchnic artery aneurysms. Personal experience. A case of celiac and superior mesenteric artery aneurysm and a case of splenic artery aneurysms].
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Contingent negative variation and cognitive performance in hypotension.
The difference in attention and cognitive performance between 26 hypotensive (systolic blood pressure < 100 mmHg and diastolic blood pressure < 60 mmHg) and 22 normotensive female university students was assessed. Attention was examined with contingent negative variation (CNV) recorded using light and tone as S1 and S2. Cognitive performance was assessed by free recall of a list of words and two German tests of cognitive speed performance and sustained attention: Zahlen-Verbindungs-Test and d2. The hypotensive participants demonstrated a lower increase in negativity on the CNV. Moreover, in the free recall test, hypotensive individuals remembered fewer words, in comparison with normotensive subjects. Scores for hypotensive individuals on the Zahlen-Verbindungs-Test and d2 were also lower. No difference was found in reaction times to imperative stimuli (S2).
The role of histamine H1, H2 and H3 receptors on enteric ascending synaptic transmission in the guinea pig ileum.
The role of histamine H1-, H2- and H3-receptors was studied on neural transmission in ascending excitatory pathways of the guinea pig ileum. A two-compartment (oral and anal compartments) bath was used: ascending neural pathways were activated by electrical stimulation in the anal compartment and the resulting contraction of the circular muscle in the oral compartment was recorded. Drugs were applied in the anal compartment and each agonist was evaluated in the presence of the antagonists of the other two receptors. In the presence of cimetidine (10 microM) and thioperamide (1 microM), histamine (0.03-3 microM) depressed the nerve-mediated contractions (5-70% inhibition, P <.05-.01). The inhibitory effect of histamine was antagonized by mepyramine. At the higher concentrations (10 and 30 microM), histamine elicited contractions of the circular muscle in the oral compartment, and these were abolished by mepyramine (1 microM) and tetrodotoxin (0.6 microM). The H2 agonists dimaprit (30 and 100 microM) and amphamine (0.1-300 microM) produced small contractions of the circular muscle in the oral compartment. These contractile responses were abolished by tetrodotoxin (0.6 microM) and cimetidine (10 microM). The H3 agonist R-alpha-methylhistamine (0.001-1 microM) inhibited (2-58%, P <.05) the nerve-mediated contractions. This inhibitory effect was antagonized by the H3 antagonist thioperamide. These results indicate that 1) histamine, acting at H1 receptors, at lower concentrations depresses synaptic transmission, although at higher concentrations activates the enteric excitatory ascending pathway; 2) activation of H2 receptors by H2 agonists stimulates the enteric excitatory ascending pathways and 3) activation of H3 receptors inhibits synaptic transmission.
A group II self-splicing intron from the brown alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation.
We have investigated the reactivity of three of the seven group II introns encoded by the mitochondrial genome of the brown alga Pylaiella littoralis. While the first intron in the protein-coding cox1 gene could not be induced to self-splice under any of the conditions tested, the first two introns in the gene encoding the large ribosomal subunit are reactive in vitro and splice primarily by the standard group II two-step transesterification pathway. Intron 2 proved to be of exceptional interest, because in contrast to all group II molecules known so far, its optimal magnesium concentration is less than 10 mM and it still carries out accurate splicing at concentrations as low as 0.1 mM magnesium. Analysis of reaction products under optimal conditions showed no evidence of hydrolysis at the 5' splice site and up to 90% of precursor molecules could be converted into excised lariat intron, which migrated as a single band on non-denaturing polyacrylamide gels. Absorbance versus temperature profiles generated from the lariat form of intron 2 reveal the existence of an early melting component, the amplitude of which does not depend on the way the molecules were purified, i.e. with or without a denaturation step. This highly cooperative transition, whose position along the temperature axis changes with the concentration of magnesium, is proposed to consist of the unfolding of the tertiary structure of the molecule. We conclude that group II introns, which are the largest known ribozymes, can form conformationally homogeneous populations of molecules suitable for physical-chemical studies of higher-order structure.
Orally projecting interneurones in the guinea-pig small intestine.
1. Orally projecting, cholinergic interneurones are important in mediating ascending excitatory reflexes in the small intestine. We have shown that there is just one major class of orally projecting interneurone, which we have characterized using retrograde labelling in organ culture, combined with immunohistochemistry, intracellular recording and dye filling. 2. Orally projecting interneurones, previously shown to be immunoreactive for choline acetyltransferase, tachykinins, enkephalin, calretinin and neurofilament protein triplet, have axons up to 14 mm long and are the only class of cells with orally directed axons more than 8.5 mm long. 3. They are all small Dogiel type I neurones with short dendrites, usually lamellar in form, and a single axon which sometimes bifurcates. Their axons give rise to short varicose collaterals in myenteric ganglia more than 3 mm oral to their cell bodies. 4. Orally projecting interneurones receive prominent fast excitatory post synaptic potentials (fast EPSPs). A major source of fast EPSPs is other ascending interneurones located further aborally. They also receive fast EPSPs from circumferential pathways. 5. In the stretched preparations used in this study, orally projecting interneurones were highly excitable, firing repeatedly to depolarizing current pulses and had negligible long after-hyperpolarizations following their action potentials. They did not receive measurable non-cholinergic slow excitatory synaptic inputs. 6. Ascending interneurones had a characteristic inflection in their membrane responses to depolarizing current pulses and their first action potential was typically delayed by approximately 30 ms. Under single electrode voltage clamp, ascending interneurones had a transient outward current when depolarized above -70 mV from more hyperpolarized holding potentials. Ascending interneurones also consistently showed marked inward rectification under both current clamp and voltage clamp conditions. 7. This class of cells has consistent morphological, neurochemical and electrophysiological characteristics and are important in mediating orally directed enteric reflexes.
Functional properties of the separate subunits of human DNA helicase II/Ku autoantigen.
The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.
Induction of activating transcription factor 1 by nickel and its role as a negative regulator of thrombospondin I gene expression.
Thrombospondin I (TSP I) is an extracellular matrix glycoprotein that influences cell adhesion, motility, and growth. On the basis of its effects on endothelial cell proliferation, TSP I has attracted interest as a potential regulator of solid tumor growth through modulation of tumor blood supply. The regulation of TSP I expression is of critical importance for designing new approaches in tumor therapy. Recently, we have shown that TSP I expression is lost in nickel-transformed cells. In this paper, we identified an activating transcription factor (ATF)/cAMP-responsive element-binding protein binding site as a negative regulatory site in the 5'-flanking sequence of mouse TSP I promoter. We identified ATF-1 as a major component of the ATF/cAMP-responsive element-binding protein binding complex. This Mr 35,000 nuclear ATF-1 protein was shown to be present in higher amounts in nickel-transformed 3T3 cells that do not express TSP 1. Acute treatment of 3T3 cells with NiCl2 resulted in the induction of this transcription factor, and this induction was correlated temporally with the suppression of TSP I expression in the same cells. These results show that nickel exposure causes accumulation of the ATF-1 transcription factor, which is responsible for the down-regulation of transcription of TSP I, and possibly other tumor suppressor genes during nickel-induced cellular transformation.
Rules for RNA recognition of GNRA tetraloops deduced by in vitro selection: comparison with in vivo evolution.
Terminal loops with a GNRA consensus sequence are a prominent feature of large self-assembling RNA molecules. In order to investigate tertiary interactions involving GNRA loops, we have devised an in vitro selection system derived from a group I ribozyme. Two selections, destined to isolate RNA sequences that would recognize two of the most widespread loops (GUGA and GAAA), yielded variants of previously identified receptors for those loops, and also some yet unrecognized, high-affinity binders with novel specificities towards members of the GNRA family. By taking advantage of available crystal structures, we have attempted to rationalize these results in terms of RNA-RNA contacts and to expose some of the structural principles that govern GNRA loop-mediated tertiary interactions; the role of loop nucleotide 2 in ensuring specific recognition by receptors is emphasized. More generally, comparison of the products of in vitro and natural selection is shown to provide insights into the mechanisms underlying the in vivo evolution of self-assembling RNA molecules.
Excitatory and inhibitory motor reflexes in the isolated guinea-pig stomach.
1. We have described and analysed the movements of the isolated stomach during distension by correlating intragastric pressure with video recordings, and investigated the presence of intrinsic inhibitory and excitatory reflexes. 2. Isolated guinea-pig stomachs, placed in an organ bath, were slowly distended with Krebs solution using a syringe pump via a cannula through the pylorus. The changes in intragastric pressure during cycles of distension were monitored by pressure transducers connected to both oesophageal and pyloric cannulae. The resistivity of the gastric wall (change in pressure with volume, delta P/delta V) and the amplitude and frequency of phasic pressure events were calculated from pressure recordings. 3. The movements of the stomach were also recorded onto videotape. The motion of the gastric wall during distension cycles was analysed to establish the patterns of contractions, their propagation and the distribution of fluid in the stomach. During filling, fluid was preferentially accommodated in the fundus. Propagating (peristaltic) contractions, often starting in the fundus, moved aborally towards the pylorus. The peak of the phasic pressure event was observed when a contraction reached the orad antrum. As it reached the pylorus, intragastric pressure was at its minimum. 4. During the initial phase of distension, intragastric pressure increased steeply. Tetrodotoxin and hyoscine reduced both the resistivity and amplitude of phasic pressure events. Hexamethonium had a similar effect. Thus distension appears to activate an excitatory reflex pathway, involving nicotinic ganglionic transmission. This reflex increases wall tension and enhances myogenic peristaltic contractions. 5. In control preparations, with larger distension volumes, the intragastric pressure decreased, despite the continued infusion of Krebs solution. L-NAME and apamin abolished this drop in pressure, indicating that gastric enteric inhibitory mechanisms prevail at larger distension volumes. After blockade of the excitatory reflex, hexamethonium antagonized the inhibitory response, indicating that activation of inhibitory mechanisms involves nicotinic transmission, probably on enteric inhibitory motoneurons. 6. Both the excitatory and inhibitory reflexes in the isolated stomach operate within a physiological range of gastric volumes. The excitatory reflex predominates at small distension volumes, leading to large phasic propagated contractions that mix the contents and may lead to emptying of the stomach. The inhibitory reflex, described previously as adaptive relaxation, can maximally relax the stomach and is activated preferentially at higher distension volumes to accommodate the contents. The interplay of these reflex pathways in the isolated stomach produces a rich repertoire of gastric movements. 7. The isolated stomach preparation, used with a combination of kinematic, kinetic and pharmacological methods, provides a highly suitable means of investigating the mechanisms of gastric motility.
The role of actin filaments in patterning the Caenorhabditis elegans cuticle.
Nematodes are covered by a cuticle with a prominent pattern of circumferentially oriented, parallel furrows. We report here that the pattern of furrows on the first larval cuticle of Caenorhabditis elegans, which is secreted during embryogenesis, is coincident with a pattern of submembranous actin filament bundles in the epithelial cells that secrete the cuticle. We propose that the pattern of cortical actin filaments biases the growth of the epithelial cell membranes, creating a furrowed surface template for deposition of the first cuticle layer. This layer then detaches from the epithelial cell surface as additional, nonpatterned components of the cuticle are secreted. Furrows are present on the surfaces of each of the four larval cuticles in C. elegans and on the adult cuticle. We show that similar ordered arrays of actin filaments appear during each of the postembryonic molts when new cuticles are synthesized. Our analysis suggests that conditions or mutations that affect the pattern of cuticle furrows might cause primary defects in the cytoskeletal organization of the epithelial cells that secrete the cuticle.
Immunohistochemical and electrophysiological characterization of submucous neurons from the guinea-pig small intestine in organ culture.
Immunohistochemical and electrophysiological properties of submucous neurons were investigated in organ cultures of the guinea-pig small intestine. Preparations of submucosa, with or without the myenteric plexus attached, were maintained in vitro for 3 to 5 days. Immunohistochemical labelling for peptides revealed that the cultured submucous plexus remained substantially intact and the immunoreactivity of cell bodies was well preserved. Substantial sprouting of nerve fibers immunoreactive for vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) was evident in submucous ganglia after 5 days in organ culture. Nerve fibers immunoreactive for substance P. somatostatin, 5-hydroxytryptamine or tyrosine hydroxylase were substantially depleted in submucous ganglia or perivascular nerves at 3 days and had virtually disappeared after 5 days in cultures of isolated submucosa. During intracellular recording from submucous neurons, action potentials were initiated by depolarizing current pulses in all neurons cultured with or without the myenteric plexus and muscle layers. Electrical stimulation of internodal strands evoked fast excitatory synaptic potentials (fast EPSPs) in nearly all neurons whether or not the myenteric plexus was present during the culture period up to 5 days. The removal of myenteric plexus and extrinsic nerves did not abolish fast EPSPs from submucous neurons, suggesting that some fast EPSPs may originate from neurons in the submucous plexus, although the possibility that new synapses formed by sprouting, or surviving axons severed from myenteric or sympathetic ganglia may have been functional, cannot be entirely excluded. This work demonstrates that the immunohistochemical and electrophysiological characteristics of submucous neurons are largely maintained in organ cultures of the submucosa.
DNA-protein cross-links produced by various chemicals in cultured human lymphoma cells.
Chemicals such as cis-platinum, formaldehyde, chromate, copper, and certain arsenic compounds have been shown to produce DNA-protein cross-links in human in vitro cell systems at high doses, such as those in the cytotoxic range. Thus far there have only been a limited number of other chemicals evaluated for their ability to produce cross-links. The purpose of the work described here was to evaluate whether select industrial chemicals can form DNA-protein cross-links in human cells in vitro. We evaluated acetaldehyde, acrolein, diepoxybutane, paraformaldehyde, 2-furaldehyde, propionaldehyde, chloroacetaldehyde, sodium arsenite, and a deodorant tablet [Mega Blue; hazardous component listed as tris(hydroxymethyl)nitromethane]. Short- and long-term cytotoxicity was evaluated and used to select appropriate doses for in vitro testing. DNA-protein cross-linking was evaluated at no fewer than three doses and two cell lysate washing temperatures (45 and 65 degrees C) in Epstein-Barr virus (EBV) human Burkitt's lymphoma cells. The two washing temperatures were used to assess the heat stability of the DNA-protein cross-link, 2-Furaldehyde, acetaldehyde, and propionaldehyde produced statistically significant increases in DNA-protein cross-links at washing temperatures of 45 degrees C, but not 65 degrees C, and at or above concentrations of 5, 17.5, and 75 mM, respectively. Acrolein, diepoxybutane, paraformaldehyde, and Mega Blue produced statistically significant increases in DNA-protein cross-links washed at 45 and 65 degrees C at or above concentrations of 0.15 mM, 12.5 mM, 0.003%, and 0.1%, respectively. Sodium arsenite and chloroacetaldehyde did not produce significantly increased DNA-protein cross-links at either temperature nor at any dose tested. Excluding paraformaldehyde and 2-furaldehyde treatments, significant increases in DNA-protein cross-links were observed only at doses that resulted in complete cell death within 4 d following dosing. This work demonstrates that DNA-protein cross-links can be formed in vitro following exposure to a variety of industrial compounds and that most cross-links are formed at cytotoxic concentrations.
Multiple tertiary interactions involving domain II of group II self-splicing introns.
The ribozyme core of group II introns is organized into six domains of secondary structure. Of these, domain II was long thought to be relatively unimportant for group II self-splicing. However, we now demonstrate the existence, in both major subdivisions of the group II family, of essential tertiary interactions involving domain II. theta-theta' is a novel tertiary interaction between the terminal loop of the IC1 stem of domain I and the basal stem of domain II. The theta-theta' interaction appears to stabilize the group II ribozyme core: it is essential for efficient self-splicing at elevated temperatures but, as shown by the use of a bimolecular reaction system, molecules with a defective theta-theta' contact are not affected in catalysis. An interaction, eta-eta', between domains II and VI of subgroup IIB introns was recently reported to mediate a conformational rearrangement between the two steps of the self-splicing reaction. We now show that domains II and VI of subgroup IIA introns also contact each other, although in a somewhat different way. Reinforcement of the eta-eta' interaction of a subgroup IIA intron prevents the use of a specific 2'-hydroxyl group in domain VI to initiate splicing by transesterification at the 5' splice site; the 5' intron-exon junction is hydrolyzed instead. Since disruption of eta-eta' has exactly opposite effects, and promotes reversal of the first transesterification step, it is concluded that formation of eta-eta' mediates a conformational change in subgroup IIA introns as well. Just like the eta-eta' interaction of subgroup IIB introns, the eta-eta' interaction of subgroup IIA introns (and the theta-theta' interaction) involves terminal loops of the GNRA family and their RNA receptors. Therefore, these motifs are used by nature not only to stabilize three-dimensional RNA architectures, but also in situations that require dynamic interactions.
Urokinase-mediated transactivation of the plasminogen activator inhibitor type 2 (PAI-2) gene promoter in HT-1080 cells utilises AP-1 binding sites and potentiates phorbol ester-mediated induction of endogenous PAI-2 mRNA.
Urokinase-type plasminogen activator (u-PA) bound to its receptor, u-PAR, initiates signal transduction pathways able to induce expression of the activator protein-1 (AP-1) family member c-fos [1]. Since transcription factors bound to AP-1 recognition sequences within the PAI-2 gene promoter play a role in basal and phorbol ester-mediated induction of PAI-2 gene expression, we hypothesised that u-PA/u-PAR-mediated modulation of AP-1 activity would in turn influence constitutive and inducible PAI-2 gene expression. Treatment of HT-1080 or U-937 cells with high molecular weight u-PA (HMW u-PA) resulted in induction of nuclear proteins binding to a functional AP-1 element in the proximal PAI-2 promoter. This increase in AP-1 activity correlated with a transactivation of the PAI-2 gene promoter in transiently transfected HT-1080 cells. We also demonstrate the u-PA treatment potentiated phorbol ester (PMA)-mediated induction of PAI-2 mRNA, indicating that u-PA binding produces a bone fide response in vivo.
Distension-evoked ascending and descending reflexes in the isolated guinea-pig stomach.
Distension-evoked gastric reflexes were studied by intracellular recording from circular muscle cells in the gastric fundus, corpus and antrum in the isolated guinea-pig stomach. Localised electrical stimulation, 2 mm circumferential to the recording electrode, evoked inhibitory junctions potentials in all three gastric regions, sometimes followed by depolarisations in the antrum. In the mid corpus, the inhibitory responses were substantially reduced by Nw-nitro-L-arginine (100 microM), unmasking excitatory junction potentials. Residual hyperpolarisations were blocked by apamin (0.5 microM) which also enhanced the amplitude of excitatory junction potentials. These excitatory junction potentials were abolished by hyoscine (1 microM). Thus transmission from inhibitory motor neurons is mediated by both nitric oxide and an apamin-sensitive mechanism. Transmission from excitatory motor neurons to the circular muscle is mediated by acetylcholine via muscarinic receptors. Balloon distension of 10 s duration of the fundus or antrum elicited inhibitory junction potentials in circular muscle cells of the mid corpus. These inhibitory junction potentials were blocked by tetrodotoxin (0.6 microM) and were greatly reduced by Nw-nitro-L-arginine (100 microM). The residual hyperpolarisations were blocked by apamin (0.5 microM). This indicates the presence of ascending and descending inhibitory reflex pathways in the stomach. In 3 out of 7 experiments, following blockade of inhibitory transmission, small nerve-mediated excitatory junction potentials were evoked by antral distension indicating the presence of an additional ascending excitatory reflex pathway. Distension of the corpus elicited prominent inhibitory junction potentials, sometimes followed by large depolarisations, in circular muscle cells in the fundus, but not in the antrum. This suggests that there is also an ascending inhibitory reflex pathway from the corpus to the fundus but no distension-sensitive descending reflex pathway from the corpus to the antrum. These results demonstrate that within the stomach there are reflex pathways which can be activated by localised distension and project at some distance orally and aborally within the gastric wall. It is likely that the inhibitory reflex pathways are involved in gastric adaptive relaxation which occurs when the intact, isolated stomach is distended. The excitatory reflex pathways from the antrum to the corpus are likely to be involved in the intrinsic excitatory reflex responses observed in the isolated intact stomach to distension and thus be involved in the mixing and emptying of gastric contents.
Carcinogenicity assessment of selected nickel compounds.
The early epidemiological data indicated different carcinogenic risks from inhalation of different nickel compounds, but it was not clear what characteristics governed the intrinsic carcinogenic hazard of the various nickel compounds. Based on the earlier results, all soluble and insoluble nickel compounds were assumed to have the same carcinogenic mechanism albeit different potencies. Recent in vivo and in vitro studies challenged this assumption. In this paper an attempt is made to integrate the most relevant human, animal, and in vitro data into a general model that can help understand the different carcinogenic potentials of the various nickel compounds. In this perspective, it is recognized that there are two main components that could contribute to the development of lung cancer via exposure to certain nickel compounds. The first component corresponds to the heritable changes (genetic or epigenetic) derived from the direct or indirect actions of nickel compounds. The second component may be the promotion of cell proliferation elicited by certain nickel compounds. The different contributions of three nickel compounds to these two components are presented. This paper emphasizes the importance of recognizing the individuality of the different nickel species in reaching regulatory decisions and the fact that different risk assessment considerations may apply for compounds that appear to produce immortality and cancer by genetic/epigenetic mechanisms (like nickel subsulfide), compounds that may present a threshold for the induction of tumors in rats (like high-temperature nickel oxide), or compounds that may only have an enhancing effect on carcinogenicity (like nickel sulfate).