Biomedical subjects

J C David

Publications and source records attributed to J C David.

At least 19 recordsLinked to original sources

PubMed1994

Expression and distribution of hsp71 and hsc73 messenger RNAs in rat brain following heat shock: effect of dizocilpine maleate.

Induction of hsp71 mRNA, coding for the inducible heat shock protein HSP71, and the constitutively expressed, hsc73 mRNA coding for the heat shock cognate protein HSC73 were studied after heat shock using northern blotting and in situ hybridization. Two cDNA oligonucleotides complementary to the messenger RNAs for hsp71 and hsc73 and suitable for in situ hybridization were designed and shown to hybridize selectively to their respective mRNAs. The inducible hsp71 was rapidly synthesized in brain after heat shock and disappeared in 6 h. A high level of expression was observed first in the dentate granule cell layer and then slightly later in the hippocampal pyramidal cell layer. There was also a dramatic increase in the granular layer of the cerebellar cortex. There was a more modest increase in expression of hsc73 after heat shock in forebrain and in cerebellum (granular layer). The N-methyl-D-aspartate receptor antagonist dizocilpine maleate (0.5 mg/kg, i.p.), given 15 min before heat shock, significantly reduced the expression of hsp71 but had no effect on hsc73. These results suggest that glutamate plays a role in heat shock-induced expression of hsp71 in brain.

Animals↗

PubMed1992

D1-like and D2-like dopamine receptors synergistically activate rotation and c-fos expression in the dopamine-depleted striatum in a rat model of Parkinson's disease.

Selective agonists for D1-like and D2-like dopamine receptors can interact synergistically to enhance each other's actions on locomotion and behavior in experimental animals. Clinically, the combination of the D2 agonist bromocriptine with L-dopa (which has pronounced D1 effects) is a highly effective treatment for Parkinson's disease. The mechanisms underlying this important receptor interaction are poorly understood and are the subject of intense study in vitro. In rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway, D1-selective (but not D2-selective) dopamine agonists produce a marked increase in expression of the immediate-early gene c-fos in the striatum ipsilateral to the 6-OHDA lesion. In the experiments reported here, we have used this in vivo model to explore the possibility that combinations of D1-selective and D2-selective agonists might have effects on c-fos transcription that are different from those exhibited by D1 or D2 agonists administered alone. We examined the effects of the D1-selective agonist SKF-38393 and the D2-selective agonist quinpirole (LY 171555) on the expression of Fos-like protein and c-fos mRNA in the caudoputamen and made parallel behavioral observations in the same animals. A low dose of SKF-38393 produced little contraversive rotation and little induction of Fos-like immunoreactivity in the striatum. A low dose of quinpirole elicited contralateral rotation but little or no induction of Fos-like immunoreactivity in the caudoputamen; there was, however, induction of Fos in the globus pallidus ipsilateral to the 6-OHDA lesion. Combination of the low dose of SKF-38393 and quinpirole produced a synergistic effect on rotation and elicited, in the dopamine-depleted caudoputamen, a striking pattern of Fos-like protein expression in which Fos-positive neurons were concentrated in striosomes and in the dorsolateral caudoputamen. Northern blot analysis showed that c-fos mRNA was expressed following combined agonist treatment but was not detectable after the single-agonist treatments. Both the contraversive rotation and the induction of Fos-like immunoreactivity were blocked by the preadministration of the D1-preferring antagonist SCH-23390 and the D2-selective antagonist raclopride in combination. Pretreatment with the glutamate NMDA receptor antagonist MK-801 also blocked the induction of Fos-like immunoreactivity, and it reversed the rotation. These findings suggest a D1/D2 synergistic mechanism that involves the participation of D1-responsive striatonigral and D2-responsive striatopallidal output pathways, and that is sensitive to glutamatergic modulation.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1991

The LCK gene is involved in the t(1;7)(p34;q34) in the T-cell acute lymphoblastic leukemia derived cell line, HSB-2.

HSB-2 is a cell line derived from a patient who had T-cell acute lymphoblastic leukemia (T-cell ALL) with a t(1;7)(p34;q34). We used a genomic probe from the T-cell receptor beta (TCR beta) locus (7q34) to identify DNA rearrangements in HSB-2. Two rearranged BglII DNA fragments were cloned, and one of these clones was shown to contain the translocation breakpoint on the derivative chromosome I [der(I)]. We used a probe derived from this clone to isolate an unrearranged phage clone encompassing the breakpoint at Ip34. The restriction map of this clone was compared to the published maps of known protooncogenes located at Ip32-34. By restriction mapping, Southern blot analysis, and DNA sequencing we showed that the translocation breakpoint on chromosome I is located within the first intron of the LCK gene. The LCK gene codes for p56lck, a member of the SRC family of cytoplasmic tyrosine protein kinases. There are two classes of LCK transcripts (type I and type II), each expressed from a distinct promoter, and each having a unique 5' untranslated region (UTR); the protein coding regions of the two classes are identical. The breakpoint in the t(1;7) separates the two LCK promoters and juxtaposes the constant region of the TCR beta locus with the proximal promoter and with the protein-coding region of the LCK gene on the der(I) chromosome.

Base Sequence↗

PubMed1991

Mechanism of inhibition of DNA ligase in Ara-C treated cells.

The activity of DNA ligase, the enzyme involved in ligation of DNA fragments and also in DNA repair is inhibited by Ara-C. The exposure of two human leukemic cell lines, K562 and HL-60 to 10(-5) M Ara-C for 3 h, induces a decrease of DNA ligase activity by 40% in K562 and 92% in HL-60. This decreased activity is due to an inhibition by Ara-CTP of the ligase-adenylate complex generation, the crucial step in the action of this enzyme. The activity of the semi-purified ligase as well as the formation of ligase-adenylate complex are decreased in the presence of Ara-CTP. These results demonstrate that Ara-C via its active form Ara-CTP inhibits DNA ligase activity through the inhibition of the ligase-adenylate complex. Other inhibitors of DNA synthesis, such as hydroxyurea, do not exert the same inhibitory effect. The inhibition of DNA ligase activity may be partly responsible for the cytotoxicity of Ara-C.

Adenosine Monophosphate↗

PubMed1991

Rapid deterioration of lens fibers in GSH-depleted mouse pups.

Lens opacities developed within 48-72 hr in mice that received a series of eight injections of L-buthionine sulfoximine, a specific inhibitor of glutathione (GSH) biosynthesis, on postnatal days 8 and 9. Initial histopathologic features consisted of swollen fibers in the central anterior cortex and displacement of cell nuclei from the bow region to the posterior cortex. These aberrations suggest early fiber cell membrane and/or cytoskeletal dysfunction. A massive wave of fiber cell lysis then engulfed the entire lens cortex and nucleus within 24 hr and left only epithelial cells intact, suggesting a concerted mechanism of cataract generation. The acellular core of the mature cataract seen on postnatal day 16 consisted of a granular matrix in which pycnotic and fragmented cell nuclei were located near the terminus of the lens epithelium. The epithelium displayed increased mitotic activity and meridional row disorganization. During the next two weeks, rapid regeneration of lens fibers, displacement of the acellular necrotic cytoplasm to the center and rear of the lens, and vacuole formation were observed. As new fibers were differentiated, partial regeneration of the bow was seen. However, the cataract was irreversible.

Animals↗

PubMed1990

Gene expression of tyrosine hydroxylase in the developing fetal brain.

Tyrosine hydroxylase, aromatic L-amino-acid decarboxylase, and dopamine beta-hydroxylase activities were studied in the developing fetal rat brain. A delay of 2-3 days between the detection of the tyrosine hydroxylase and the aromatic L-amino-acid decarboxylase and dopamine beta-hydroxylase activities was observed. For this reason, the expression of tyrosine hydroxylase mRNA was studied. Tyrosine hydroxylase mRNA was visualized in the whole brain from 13 days of gestation, but the largest increase of the expression was observed in the hypothalamus. These results are discussed in terms of the relative gene expressions of the three enzymes involved in the biosynthesis of catecholamines and phenolamines in nervous tissues.

Actins↗

PubMed1989

A study of the mechanisms of cytotoxicity of Ara-C on three human leukemic cell lines.

The main biochemical determinants involved in cytosine arabinoside (Ara-C) metabolism were studied in one lymphoblastic (Reh) and two myeloid (HL60 and K562) human leukemic cell lines exhibiting various sensitivities to Ara-C, Reh being the most and HL60 the least sensitive. The level of intracellular Ara-C accumulation and Ara-CTP formation was far more important in Reh cells than in myeloid cell lines but was not closely related to deoxycytidine kinase activity or to deoxycytidine triphosphate pool size. The level of Ara-C incorporated into DNA was similar in the three cell lines. Ara-CTP formation correlated better with the cytotoxicity to clonogenic cells than did Ara-C incorporation into DNA. DNA polymerase alpha was moderately inhibited to various degrees, depending on the cell line; this moderate inhibition does not seem sufficient to explain the inhibition of DNA synthesis. The activity of DNA ligase, the enzyme joining the Okazaki fragments, which was not detected in Reh cells, was strongly inhibited by Ara-C in HL60 and to a lesser degree, in K562 cells. The inhibition of DNA ligase probably also contributes to the inhibition of DNA synthesis and, thus, to the cytotoxic effect of Ara-C and may explain the smaller size of DNA fragments observed following Ara-C treatment. The variations in each critical determinant observed in these three cell lines increase the complexity and plurality of the mechanisms of Ara-C action.

Arabinofuranosylcytosine Triphosphate↗

PubMed1989

Evidence for a change in expression of DNA ligase genes in the Pleurodeles waltlii germ line during gonadogenesis.

The expression of DNA ligase genes was studied using the nuclear transplantation approach in the germ line of Pleurodeles waltlii (P. waltlii) just before and during gonadogenesis. Germ cell (GC) nuclei were isolated from larvae of P. waltlii and transplanted into unfertilized Ambystoma mexicanum eggs. DNA ligase activity in these eggs was then analyzed after sucrose gradient fractionation. The activity of DNA ligase I (heavy form, 7.5 S) of P. waltlii was present when the transplanted GC nuclei were isolated before the first histological appearance of gonadogenesis. At the beginning of genital ridge formation and thereafter, DNA ligase I activity was replaced by that of DNA ligase II (light form, 7 S). Expression of form I was found to be sensitive to inhibitors of translation and transcription, while that of form II was not. Therefore, the change in DNA ligase activity of the transferred nuclei of P. waltlii germ cells was assumed to be the consequence of a change in gene activity, namely, the repression of the gene encoding DNA ligase I. This change in the gene-regulated state could be linked to protein modifications of the chromatin. These results indicate that, at the beginning of gonadogenesis, germ cells receive information leading to a new state of differentiation.

Ambystoma↗

PubMed1989

Effects of aging on p- and m-octopamine, catecholamines, and their metabolizing enzymes in the rat.

Functions of octopamine in the mammalian brain are still not well known. An important aspect of this problem is the relationship between octopamines and catecholamines. Previous data have shown that their respective ontogenic evolutions are not parallel. Do the changes in brain related to aging also differentially affect these two groups of molecules? In order to check this point, the brain levels of p- and m-octopamine, p-tyramine, noradrenaline, and dopamine, as well as the activities of metabolizing enzymes, were determined in young adult and aging rats (20-26 months). Unlike catecholamines, there is a drastic decrease of p-octopamine after 20 months of age in the hypothalamus and telencephalon. p-Tyramine levels are also lowered. This change appears to be due to a decrease of the aromatic L-amino acid decarboxylase activity. These data, as those of ontogenic studies, confirm that p-octopamine and catecholamine metabolisms may have some independent steps and, moreover, that p-octopamine may have a role in the normal activity of the brain.

2-Hydroxyphenethylamine↗

PubMed1989

Prenatal ontogenesis of brain phenolamines and catecholamines in relation to their metabolizing enzymes in Roman avoider strains of rats.

Phenolamines, particularly octopamines, are of special importance in avoidance behavior. In the Roman low avoidance (RLA) strain, p-octopamine can induce locomotor behavioral activity that is normally observed in the Roman high avoidance (RHA) strain. For these reasons, the levels of prenatal octopamines (para and meta isomers) have been studied in relation to noradrenaline and dopamine levels. In the hypothalamus and brainstem of RHA, a maximum level of the para isomer is observed at 15 days of embryonic development but, unlike in controls and RLA animals, this level remains almost constant until 20 days. For the meta-isomer and catecholamines, there is a 1-2 day delay in detection between controls and RLA or RHA. The study of related enzyme activities reveals that tyrosine hydroxylase displays a 2-day delay in RHA when compared to the control value at 19 days of fetal life. These results are discussed in terms of the role of p-octopamine in avoidance conditioning and of the possible delayed expression of the tyrosine hydroxylase gene in Roman strains of rats.

2-Hydroxyphenethylamine↗

PubMed1989

dATP-mediated inhibition of DNA ligase by 2'-deoxycoformycin in T and B cell leukemia.

2'-Deoxycoformycin (dCF), a potent adenosine deaminase inhibitor, has been reported to display greater toxicity for T than for B lymphoblasts. Since this compound can block DNA replication and since this effect is mediated by the intracellular ATP/dATP balance, its possible effect on DNA ligase was investigated. dCF at relatively low concentrations (1 microM), in association with dATP (100 microM), is a strong inhibitor of DNA ligase in T blasts, whereas it has no significant effect in B blasts at this concentration. The AMP-ligase complex is the target of the observed inhibition because the combined presence of the inhibitor and dATP results in a more stable dAMP-ligase complex. Because of this observation and of the greater adenosine deaminase activity observed in T cells, the dATP mediated dCF inhibition of ligase might be the crucial replication target of T cell toxicity. These observations are discussed in terms of T immunodeficiencies including Graft Versus Host Disease and related syndromes.

Adenosine Triphosphate↗

PubMed1988

Association of a possible DNA ligase deficiency with T-cell acute leukemia.

DNA ligase activities were determined in blood samples of 431 different cases of lymphoblastic and nonlymphoblastic leukemia. Less activity was observed in samples from lymphoblastic patients. RNA translation together with ligase immunoprecipitation experiments show that in T-cell acute lymphoblastic leukemic cells, no ligase is translated. This deficiency is correlated with the presence of more breaks in the DNA from these kinds of leukemia which results in altered DNA. This DNA can be ligated by the addition of exogenous ligase. This is the first demonstration of a ligase deficiency in leukemic human cells. These results are discussed in terms of chromosome abnormalities and rearrangements of genes coding for enzymes involved in DNA replication and repair.

Acute Disease↗

PubMed1988

Peplomycin. DNA breakage and in vivo inhibition of DNA polymerases and ligase from human normal and leukemic cells.

Peplomycin, a bleomycin-related cytostatic agent, was tested on DNA polymerases and DNA ligase. These enzymes were purified from normal human immunocompetent cells (thymocytes and lymphocytes) and from peripheral blast cells from different kinds of acute lymphoblastic and acute non-lymphoblastic leukemia. At low concentration ranges (1-25 microM) this compound was found to strongly inhibit polymerase alpha and ligase from leukemic cells while being less effective on the enzyme activity from normal thymocytes and lymphocytes. At the DNA level, low concentrations of peplomycin resulted in the induction of dose-dependent single-stranded breaks. The incubation of peplomycin (5 microM) with plasmid DNA resulted in its degradation as observed by agarose gel electrophoresis. Lowering the peplomycin concentration showed that ligase inhibition takes place prior to this phenomenon. The decreased formation of the ligase--adenylate complex under the effect of peplomycin is consistent with a direct interaction between the drug and the enzyme. These results are discussed in terms of possible selective cytostatic effects of peplomycin on leukemic cells.

Bleomycin↗

PubMed1988

DNA ligation in relation to DNA strand breaks in phytohemagglutinin-stimulated blast cells from acute lymphoblastic and nonlymphoblastic leukemia.

DNA ligase activity was determined in the WBCs from 306 cases of acute lymphoblastic leukemia (ALL) and acute nonlymphocytic leukemia (ANLL). In T-ALL cells this activity was either low or absent. DNA analysis by nucleoid, alkaline elution, and alkaline sucrose centrifugation after cells were embedded in agarose inserts has shown more DNA breaks in T-ALL than in ANLL blasts. Phytohemagglutinin stimulation of T-ALL blasts resulted in the apparent joining of the DNA breaks. Apparent identical results can be obtained by the incubation of DNA with exogenous DNA ligase. The authors suggest that this enzyme is a crucially regulated step of replication and subsequent proliferation in this type of leukemia.

Centrifugation, Density Gradient↗

PubMed1988

Effects of clinical combinations of antileukemic drugs on DNA ligase from human thymocytes and normal, stimulated, or leukemic lymphocytes.

Human DNA ligase was purified from different kinds of immunocompetent cells: thymocytes, normal and stimulated lymphocytes, blasts from ALL (Burkitt and non-T, non-B) and ANLL (M1, M2, and M5). Based upon the protocol for the treatment of these leukemias, the purified enzymes were assayed in the presence of routinely used combinations of antileukemic drugs. At the range of concentration tested (between 0.1 and 5 microM) some drugs taken separately were totally inactive on the enzyme from the different sources. For those being inhibitory, when used in combination their effect was always different from what was observed when the compound was tested alone. Some combinations were more effective in inhibiting the enzyme from leukemic than from normal cells (vincristine + cyclophosphamide + prednisone in ALL and rubidazone + Ara-C, Ara-C + m-AMSA, in ANLL). However, some combinations of drugs are without effect on ligase from leukemic cells at this dose range (vincristine + rubidazone + Ara-C + prednisone and adriamycin + asparaginase + Ara-C in ALL or etoposide + Ara-C, adriamycin + cyclophosphamide in ANLL). This is the first direct observation of the effect of cytostatic drugs on DNA ligase, a key enzyme of the DNA replication and repair process. The clinical consequences of these observations are discussed in an attempt to selectively inhibit replication, thereby division, of cancer cells.

Adolescent↗

PubMed1987

DNA repair in human promyelocytic cell line, HL-60.

The human promyelocytic cell line, HL-60, shows large changes in endogenous poly(ADP-ribose) and in nuclear ADP-ribosyl transferase activity (ADPRT) during its induced myelocytic differentiation. DNA strand-breaks are an essential activator for this enzyme; and transient DNA strand breaks occur during the myelocytic differentiation of HL-60 cells. We have tested the hypothesis that these post-mitotic, terminally differentiating cells are less efficient in DNA repair, and specifically in DNA strand rejoining, than their proliferating precursor cells. We have found that this hypothesis is not tenable. We observe that there is no detectable reduction in the efficiency of DNA excision repair after exposure to either dimethyl sulphate or gamma-irradiation in HL-60 cells induced to differentiate by dimethyl sulphoxide. Moreover, the efficient excision repair of either dimethyl sulphate or gamma-irradiation induced lesions, both in the differentiated and undifferentiated HL-60 cells, is blocked by the inhibition of ADPRT activity.

Cell Differentiation↗

PubMed1987

Effects of triethyltin on brain octopamines and their metabolism in the rat.

The effects of triethyltin given acutely on the cerebral level of p- and m-octopamines were studied in rats. These octopamines were reduced drastically in hypothalamus and brainstem, while noradrenaline and dopamine were only slightly decreased. No important changes were observed in the activities of tyrosine hydroxylase, dopamine beta-hydroxylase or monoamine oxidase. However, the activity of aromatic L-amino acid decarboxylase was significantly reduced. The addition of the inhibitor of dopa decarboxylase, Ro 44602, caused a total inhibition of this enzyme activity. These results are discussed in terms of the possible use of the triethyltin-induced cerebral oedema as a model for the study of some aspects of the phenolamine changes related to cerebral oedema processes.

Animals↗

PubMed1987

Relationship between thymus ontogeny and DNA ligase expression in the chicken.

DNA ligase expression was studied in the embryonic chicken thymus. As previously reported, during thymus ontogeny two separate and successive (8S and 6S) forms of enzyme are found. The switch from the heavy to the light form takes place at around 18 days of incubation in situ. The demonstration that two successive generations of thymic lymphoid precursor cells develop in the embryonic bird thymus and that the replacement of the first by the second thymocyte generation takes place at around 18 days suggested the possibility that each form of enzyme could be restricted to a given thymocyte generation. By either culturing or transplanting embryonic thymuses of different ages it appears that the switch from the heavy to the light form of DNA ligase is not related to a change over of the first by the second generation of embryonic thymocytes but rather to some maturation process of the thymic lymphocytes starting from 17 days of embryonic life. The influence of extrathymic factors on the turning on of the expression of the 6 S DNA ligase in the post-natal environment is also suggested.

Animals↗