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Biomedical subjects

G Lazar

Publications and source records attributed to G Lazar.

At least 19 recordsLinked to original sources
PubMed2000

The Arabidopsis splicing factor SR1 is regulated by alternative splicing.

The serine-arginine (SR)-rich splicing factors play essential roles in general splicing and regulate alternative splice site utilization in a concentration-dependent manner. SR1 is a plant homologue of the human general/alternative splicing factor SF2/ASF. We report here that alternative splicing regulates SR1 itself. Of the five detected SR1 transcripts only one encodes the full-length protein, while the other four are different variants of the essential arginine-serine-rich domain. The data suggest that SR1 pre-mRNA could be committed to two alternate splicing pathways. One, dependent on the alternative utilization of competing 3' splice sites in intron 9, generates SR1, SR1B and SR1C. The other, regulated by suppression of intron 9 5' splice site utilization, generates SR1D and SR1E. The splicing pattern and molecular structure of SR1D indicates an evolutionary conservation of splicing-based regulation between plants and vertebrates and suggests that the various isoforms perform important functions. Results from transient gene expression assays indicate that alternative splicing is not an autoregulatory mechanism used to control the transcript level of the full-length protein. The ratio of SR1/SR1B transcripts, which are generated by alternative 3' splice site utilization in intron 9, is under temperature control. The temperature-dependent increase in SR1B/SR1 ratio suggests a role of SR1B in the adaptation to high-temperature environments. In addition, based on the regulated co-expression of SR1 transcripts, it is possible that some SR1 functions could be determined by the combinatorial action of the various isoforms.

Alleles↗
PubMed1996

Removal of cobalt-labeled neurons and nerve fibers by microglia from the frog's brain and spinal cord.

We investigated the microglial reaction around cobalt-labeled degenerating neurons and nerve fibers in the frog central nervous system. The aim of these studies was to reveal the routes of migrating microglial cells during debris removal and the effect of seasonal changes on this process in a cold-blooded animal. Oculomotor and spinal motoneurons were filled with cobaltous-lysine complex through their axons. In the torus semicircularis and the isthmic nucleus, neurons were labeled with iontophoretically applied cobaltous-lysine complex through their injured dendrites and axons. The animals were left to survive for 1 to 50 days. During the summer, oculomotor neurons disintegrated by the seventh postoperative day. The debris from the neurons were phagocytosed by microglia-like cells identified by the presence of cobalt in their cytoplasm. Some of these cells were wedged between ependymoglial cells of the cerebral aqueduct, others appeared at the pial surface of the mesencephalon. The speed of this process was twice as fast during the summer as during the winter. Part of cobalt-labeled microglial cells in the torus semicircularis and the isthmic nucleus moved toward the ependyma of the optic ventricle and the cerebral aqueduct, respectively. Cobalt-loaded microglial cells did not move toward the surface in the spinal cord and the deep part of mesencephalic tegmentum, and left the brain probably via blood vessels. We conclude that microglial cells loaded with phagocytosed tissue debris may leave the brain tissue via three routes and their activity depends on the environmental temperature.

Animals↗
PubMed1995

Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.

Amino Acid Sequence↗
PubMed1994

Properties of the mineralocorticoid receptor immunopurified from bovine kidney.

The mineralocorticoid receptor (MCR) from bovine kidney was purified on an affinity column containing covalently linked polyclonal IgG raised in the rabbit against rat kidney protein purified in the presence of RU 26752 that is specific to the MCR. The immuno-affinity eluate was excluded as a single peak during gel permeation chromatography and could be resolved as a single band of approximately 98 kDa by western blot and gel electrophoresis. Immunohistochemistry revealed MCR-specific staining in both the cortical and glomerular regions of bovine kidney. Interestingly, the purified MCR could not be activated in the presence of the specific ligand RU 26752 whereas binding to DNA-cellulose increased by 100% when crude cytosol was left at room temperature for 45 min. The binding of calcium to the MCR resulted in an increase in the fluorescence signal that could be partially reversed by EDTA. By a calcium-specific fluorescence dye technique, 1.13 nM of ionized Ca2+ was bound per 0.01 nM MCR. The binding of ATP32 to the immunopurified receptor was observed following chromatography on P-10 columns. The fluorescence signal of etheno-ATP was maximally attenuated by the receptor at 1/1 stoichiometry of the ATP-MCR complex. Asparagine-linked complex chain N-glycosylation of the purified MCR was also observed. Analysis by far-UV circular dichroism spectra showed that MCR contains 33% alpha helices and 30% beta sheets, compatible with a relatively flat conformation of the native protein. These data provide experimental proof for the predicted computer simulation regarding the structural features of the steroid receptor superfamily and suggest crosstalk between several protein families.

Adenosine Triphosphate↗
PubMed1993

The origin of the bifunctional dihydrofolate reductase-thymidylate synthase isogenes of Arabidopsis thaliana.

In most prokaryotic and eukaryotic organisms dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are encoded by independent genes. Evidence is presented here that the higher plant Arabidopsis thaliana has two bifunctional DHFR-TS genes. The structure of the genes, DHFR at the amino terminus and TS at the carboxy terminus, is identical to their organization in protozoa, the only other known organisms with bifunctional genes. Sequence alignments suggest that the bifunctional genes from protozoa and higher plants may have different evolutionary origins. The positions of the introns support the complementary hypothesis that the DHFR domain of the bifunctional plant genes and the monofunctional DHFR gene of vertebrates derive from a common, intron-containing progenitor, although the structure (bifunctional or monofunctional) of the ancestral gene remains indeterminate. Comparison of the two bifunctional genes of Arabidopsis indicates that the DHFR and TS domains evolved at different rates; each following the evolutionary history of their monofunctional counterparts. In contrast to the DHFR domain, the evolution of the TS domain shows a higher level of nucleotide and amino acid sequence conservation, but a remarkable variability in the intron positions.

Amino Acid Sequence↗
PubMed1992

Central projections and motor nuclei of the facial, glossopharyngeal, and vagus nerves in the mormyrid fish Gnathonemus petersii.

Most of the information about the anatomy of the fish's cranial nerves was collected in the first two decades of this century. Experimental analysis of the VIIth, IXth, and Xth cranial nerves by modern tract tracing techniques started about 20 years ago. Several species have been investigated to date, including one species of Agnatha (Myxinoidea), two species of elasmobranchs, and species of some orders of Teleostei like Cyprinidae, Siluriformes, Perciformes, and Gadidae. The sensory and motor nuclei of the VIIth, IXth, and Xth cranial nerves of Gnathonemus petersii were studied by anterograde and retrograde axoplasmatic transport of horseradish peroxidase and cobaltous lysine complex. The sensory nuclei form a continuous column of cells in the brain stem extending caudal to the obex. The rostral one-fourth of this column is occupied by the overlapping terminals of the VIIth and IXth nerves. The vagus nerve has 5 roots. The first 4 of these innervate the gills and the fifth supplies viscera. Afferents from the gills terminate ipsilaterally rostral to the obex in topographic order and their terminal fields overlap. Viscerosensory fibers terminate ipsilaterally in the obex region and bilaterally in the commissural nucleus of Cajal. The facial motor nucleus is located rostral to the sensory nucleus. Facial motoneurons have pear-shaped and multipolar perikarya. Their axons form a rostrally directed knee before leaving the brain. The motoneurons of the IXth and Xth nerves have a common cell column. The vagal motoneurons form a periventricular, a medial, and an intermediate cell group rostral to the obex. In the obex region and also caudal to it, a lateral and a caudal group can be distinguished. Vagal motoneurons show a topographic arrangement that is similar to that of the sensory vagal projections. The majority of motoneurons have pear-shaped perikary and ventrolaterally oriented dendrites. In the caudal nucleus the dendrites extend dorsally and overlap the terminals of sensory fibers. The axons form a dorsolaterally directed arch before joining the sensory roots. Since G. petersii uses its electrosensory system primarily for detection of food, its gustatory system is less developed than in other fishes, which possess a large number of taste buds.

Animals↗
PubMed1992

Immunophotochemical analysis of mineralocortin by polyclonal antibodies against the native receptor from rat kidney.

We have obtained a polyclonal antiserum by immunizing fawn Burgundy rabbits with the mineralocorticoid receptor (MCR) purified biochemically from rat kidneys. High titers of anti-MCR activity were obtained in radioimmunoassays within 3 weeks and increased with a booster shot. In Western blot analysis, the antibody revealed a major band of 94-98 kDa in renal cytosol from rat and beef kidneys. We also developed a fluorographic procedure where the MCR linked covalently to tritiated R-5020, following ultraviolet irradiation, gave imprints superimposable on the Western blot profile. The fluorographic pattern was specific since it was largely abolished in the presence of cold RU 26752 that is specific to MCR, or mineralocortin. The immune IgG precipitated rat renal MCR(-)[3H]RU 26752 complexes in a dose-dependent manner and also recognized MCR bound to the natural hormone aldosterone. During gel permeation chromatography on Sephacryl, the elution profile of [3H]RU 26752 shifted to high-molecular-weight regions in the presence of immune IgG. The receptor protein could be immunolocalized primarily to the principal cells of the collecting duct in rat kidney but the intercalated cells and glomeruli were not labeled, contrary to beef kidney where a uniform pattern of immunostaining was evident. These should permit large-scale purification of the MCR for detailed physicochemical studies and for screening of the MCR-positive tissues during various pathophysiological syndromes.

Adrenalectomy↗
PubMed1992

Effects of angiotensin II and nonpeptide receptor antagonists on transduction pathways in rat proximal tubule.

Because the presence of the angiotensin II (ANG II)-dependent phosphoinositide hydrolysis has been questioned from studies in proximal cells in culture, we looked for this transduction pathway in suspension of freshly isolated rat proximal tubule fragments. ANG II-receptor activation induced a prompt (within 15 s) and sustained increase in [3H]inositol phosphates (IPs; inositol trisphosphate, inositol bisphosphate, and inositol monophosphate). In fura-2-loaded tubules, it elicited a rapid and biphasic rise in cytosolic free calcium ([Ca2+]i) with an early peak (within 15 s) followed by a plateau. The peak was maintained in the absence of extracellular calcium. ANG II-induced inositol trisphosphate and [Ca2+]i rises showed a similar dose dependency, with a 50% effective concentration (EC50) of 2.9 and 5.5 nM, respectively. We checked that ANG II inhibited basal (EC50 4.4 nM) and parathyroid hormone- and forskolin-stimulated cAMP production, the latter effect being inhibited by pertussis toxin pretreatment. The effects of ANG II on IPs and [Ca2+]i were inhibited by the ANG II receptor subtype 1 (AT1) antagonist losartan and not by the ANG II receptor subtype 2 (AT2) antagonists PD 123177 and PD 123319. The effect of ANG II on forskolin-stimulated cAMP was inhibited by losartan and not by PD 123319. In agreement with these results, specific binding of 125I-[Sar1,Ile8]ANG II was markedly inhibited by losartan, whereas PD 123319 had no effect. These results demonstrate that AT1 receptor subtypes are present in intact rat proximal tubule cells and are coupled to both IPs-Ca2+ and cAMP signaling pathways. No evidence for AT2 receptor subtype is found.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1992

Acute variations in extracellular pH modulate transduction pathways of PTH in rat proximal tubule.

An increase in circulating parathyroid hormone (PTH) has been shown to enhance the capacity for the kidney to excrete an acid as well an alkaline load, which suggests that changes in systemic acid-base status may modulate the effect of the hormone on bicarbonate absorption in proximal tubule. In the present study, we tested the possibility that acute variations in extracellular pH (pHe), obtained by modifying bicarbonate concentration at constant PCO2 (40 mmHg), may modulate the responses of intracellular messengers coupled to PTH receptors in a preparation of freshly isolated proximal tubule fragments. Variations in pHe, which induced parallel variations in intracellular pH (pHi), did not affect unstimulated values for adenosine 3',5'-cyclic monophosphate (cAMP) production, inositol trisphosphate accumulation, or cytosolic free Ca2+ concentration. In contrast, reducing pHe from 7.4 to 7.2 elicited a decrease of the PTH-induced cAMP production, whereas increasing pHe from 7.4 to 7.6 enhanced it. The ability for cholera toxin and forskolin (which both bypass PTH receptors) to stimulate cAMP formation was diminished at pHe 7.2 and enhanced at pHe 7.6 (the increase did not achieve statistical significance in the presence of forskolin), suggesting that variations in pHe and/or pHi may affect per se adenylyl cyclase activity. Conversely, reducing pHe from 7.4 to 7.2 enhanced the PTH-induced inositol trisphosphate accumulation and rise in cytosolic free Ca2+ whereas increasing pHe from 7.4 to 7.6 had opposite effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1992

Modification of septic shock in mice by the antiglucocorticoid RU 38486.

The survival rate in murine septic peritonitis was inversely proportional to the size of the needle used for cecal puncture following ligation below the ileocecal valve. The smaller, 20-gauge needle permitted 20% survival. Only 20% of the animals survived 24 hr after cecal puncture with a 20-gauge needle compared to 90% survival after 5 days if mice had been rendered tolerant to lipopolysaccharide (LPS) prior to the induction of peritonitis. A single intravenous injection of 1 mg RU 38486, concurrent with puncture with a 21-gauge needle, lowered survival to only 15% from the control level of 71%. This same dose of the antiglucocorticoid decreased the survival rate to only 35% from 90% in the tolerant group. Tolerance to the lethal effects of endotoxins, possibly responsible for resistance to septic peritonitis, was also abolished by the antiglucocorticoid. Just 1 mg RU 38486 lowered the survival rate to 5% if it was given with 600 micrograms LPS, which permitted 95% survival in LPS-tolerant mice and 60% survival in normal controls. Whereas both 1 mg RU 38486 and 100 micrograms dexamethasone, given alone, sensitized mice to septic peritonitis (15% and 30% survival, respectively), their combined effects neutralized each other, leading to 80% survival vs. 71% in the control group. Thus receptor-mediated glucocorticoid-dependent mechanisms appear important in the pathogenesis of both endotoxin and septic shock, albeit to varying degrees and in a seemingly contradictory manner.

Animals↗
PubMed1990

Purification and characterization of the activated mineralocorticoid receptor from rat myocardium.

Cardiac cytosol from adrenalectomized rats was radiolabelled with 10 nM tritiated RU 26752, R 5020 or aldosterone, to saturate the mineralocorticoid receptor (MCR) in the presence of 1 microM RU 38486 to block the glucocorticoid and progestin receptors. Free steroids were removed by charcoal treatment and the radiolabelled cytosol was passed through a phosphocellulose column. The MCR peak in the phosphocellulose eluate was activated at 25 degrees C for 45 min, adsorbed onto the DNA-cellulose and finally extracted once each with buffers containing 1 M potassium chloride or 25 mM magnesium chloride. The pooled DNA-cellulose extracts, reequilibrated with 10 nM [3H]RU 26752, were resolved as a single, homogeneous band of 78 kDa upon polyacrylamide gel electrophoresis. Ion-exchange analysis of the purified MCR on DEAE-cellulose-52 revealed a single peak in the 0.017 M sodium phosphate region with both RU 26752 and R 5020, but aldosterone dissociated during this procedure. Molecular filtration on Ultrogel AcA-44 columns revealed a major 145 kDa peak, with some smaller components of 40 and 80 kDa. These hydrodynamic properties of the purified MCR are at variance with those of the native receptor in crude myocardial cytosol, and suggest that some post-translational modifications in vivo may be required for the expression of MCR-mediated responses.

Aldosterone↗
PubMed1990

Purification and characterization of the activated mineralocorticoid receptor from rat kidney.

1. The mineralcorticoid receptor (MR) from rat kidney was purified within 8 hr by the following, successive steps: stabilization with synthetic, tritiated steroids (RU 26752 or R 5020), phosphocellulose passage, heat activation (25 degrees C), and DNA-cellulose batch elution. 2. The purified preparation was resolved as a single, 75 KDa band on SDS-PAGE electrophoresis although the exact degree of purity was difficult to assess by the charcoal assay due to denaturation. 3. The natural hormone, aldosterone, was unsuitable for receptor purification and characterization. 4. The MR purified with different ligands behaved identically during ion exchange and gel permeation analyses, suggesting post-translational modifications of the native receptor in whole cytosol that exhibits molecular heterogeneity.

Animals↗
PubMed1989

Coregulation of dihydrofolate reductase and thymidylate synthase in overproducer cell lines of wild carrot.

Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) activities are associated with a 285,000 molecular weight enzyme complex in carrot (Daucus carota L.). Selection for methotrexate (MTX) resistance by stepwise increase of the concentration of MTX results in a high frequency adaptation to MTX with little or no significant increase in DHFR activity. However, when as a second step following MTX selection a specific inhibitor of TS, 5-fluoro-2-deoxyuridine was used, DHFR overproducer lines were obtained. The overproduction phenotype of the lines was almost completely lost after 8 weeks of growth in the absence of selection pressure. Although DHFR and TS are independent gene products, their activities increase in proportion ( approximately 20-fold) in the overproducer lines. This strongly suggests that DHFR and TS are not only functionally and physically linked in the same enzyme complex, but also are coregulated. These cell lines resemble the MTX-induced DHFR overproducer amplified cell lines of mammalian origin in their mode of selection, high frequency of appearance, elevated enzyme activity, and increased specific mRNA levels.

Journal Article↗
PubMed1987

Oculomotor system of the weakly electric fish Gnathonemus petersii.

The peripheral and central aspects of the extraocular system were studied in the weakly electric fish Gnathonemus petersii. All six extraocular muscles show a similar composition of large and small fibers grouped characteristically in the proximal and distal regions respectively. The exit of the three extraocular nerves from the brain is similar to that in other vertebrates. However, the intracephalic and intracranial course of the trochlear nerve is unusual, partly because of the extraordinary hypertrophy of the cerebellum. The three nerves course rostrally on the ventral brain surface; the trochlear nerve penetrates the orbital cavity separately from the two other nerves. The fiber-diameter spectrum of each extraocular nerve is bimodal; unmyelinated fibers were not observed in any of the nerves. The location of the extraocular motor nuclei was established by retrograde axonal transport of HRP or cobaltic-lysine complex. The oculomotor nucleus is situated ventral to the posterior pole of the magnocellular mesencephalic nucleus and the trochlear nucleus is found caudal and dorsal to this. The abducens nucleus is situated at the level of the octavolateral efferent nucleus and consists of a single group of cells on each side of the ventral tegmentum. The oculomotor nucleus of G. petersii shows a somatotopic organization. The superior rectus muscle receives a contralateral innervation whereas the inferior rectus and oblique muscles and the internal rectus muscles receive an ipsilateral innervation. The superior oblique muscle is innervated by contralateral trochlear motoneurons and the external rectus by ipsilateral abducens motoneurons. The majority of extraocular motoneurons have piriform perikarya and long beaded dendrites that extend laterally in the oculomotor and abducens nuclei and rostrally in the trochlear nucleus. The terminal dendritic portions of trochlear motoneurons widely overlap with oculomotor dendrites and perikarya. In all three nuclei the axon originates opposite to the main dendrite. Collaterals of the hairpin-bend abducens axons could be identified in a few cases. The oculomotor system of G. petersii appears basically similar to that of other teleosts; the differences observed concern mainly the structure of the abducens nucleus, the intracranial and intracephalic course of the trochlear nerve, and the relatively small number of axons in each nerve.

Abducens Nerve↗
PubMed1987

Purification and immunochemical characterization of a dihydrofolate reductase-thymidylate synthase enzyme complex from wild-carrot cells.

An enzyme complex with dihydrofolate reductase (DHFR, E.C.1.5.1.3.) activity was purified to apparent homogenity from wild-carrot cells. The complex has a mol. wt of 286 kd and contains five polypeptide chains of 95, 70, 50, 45 and 26 kd. The DHFR enzyme activity and methotrexate-binding site are on the 45-kd subunit. Folate analogs (methotrexate, aminopterin and formylaminopterin) as well as SH-group inhibitors [p-hydroxymercuribenzoate, 5,5' -dithiobis(2-nitrobenzoic acid), or N-ethylmaleimide] inhibit DHFR. Thymidylate synthase (TS, E.C.2.1.1.45) activity co-purified with the enzyme complex through each of seven steps and co-eluted from gel filtration columns with the DHFR activity at the mol. wt of the enzyme complex. Further identification of TS within the complex was achieved using a Leishmania DHFR-TS antisera which specifically inhibited the carrot TS, although it immunoprecipitated both TS and DHFR. Polyclonal antisera, raised against and specific for the complex as judged by Ouchterlony double diffusion tests and Western blot analysis, inhibited and immunoprecipitated both DHFR and TS. The Leishmania antisera also identified the 70-kd polypeptide within the purified complex as TS in a Western blot experiment. The functions of the other three polypeptides have not yet been established.

Journal Article↗
PubMed1987

Cytarabine, cisplatin, and etoposide chemotherapy for refractory non-Hodgkin's lymphoma.

A phase II trial of cytarabine, cisplatin, and etoposide was conducted in 38 patients with refractory stage III and IV non-Hodgkin's lymphoma. There were two complete and nine partial responses (32%) among 35 evaluable patients. Response rate in patients with large cell lymphoma was 45%. The dose-limiting toxic effect was myelosuppression in 66% of patients.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1986

Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases.

We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N. tabacum. Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago. However, the divergence of GapA/B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages. Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes. These results strongly support the symbiotic origin of chloroplasts.

Amino Acid Sequence↗