Neuroprotective effects vary across nonsteroidal antiinflammatory drugs in a mouse model of developing excitotoxic brain injury.

Glutamate excitotoxicity is among the main cellular mechanisms leading to perinatal insults in human newborns. We used intracerebral injection of the glutamatergic glutamate N-methyl-D-aspartate-receptor agonist ibotenate to produce excitotoxic lesions mimicking the acquired white matter lesions seen in human preterm infants. We evaluated whether nonsteroidal antiinflammatory drugs (NSAIDs) protected against glutamate excitotoxicity. Aspirin (0.01-100 microg/d), indomethacin (0.1-10 microg/d), paracetamol (10-100 microg/d), or NS-398 (12.5 microg/d) was given daily before ibotenate (P1 to P5) or after ibotenate (P5 to P9). Lesion size was measured on Cresyl Violet-stained brain sections collected on P10. None of the drugs tested alone or in combination increased lesion size. Pretreatment with low- or high-dose aspirin and post-treatment with paracetamol or NS-398 protected against white matter lesions, whereas cortical lesions were decreased by pretreatment with low- or high-dose aspirin or post-treatment with NS-398. The corticosteroid betamethasone (0.18 microg/d) was neuroprotective when given before or after ibotenate and this effect was reversed by concomitant aspirin therapy (10 microg/d). In conclusion, perinatal NSAID administration may have beneficial effects on brain injury if appropriately timed.

Using molecular markers and multivariate methods to study the genetic diversity of local European and Asian chicken breeds.

French and Asian subsets of chicken breeds were first analysed using 22 microsatellites and then compared to the AVIANDIV European set using 14 loci. Positive correlations were observed between F(IT) or F(ST) and typological values or variance of markers using the multivariate analysis mcoa. The first axis of the multivariate representation separated Asian from European breeds, revealing breeds with Asian ancestor. Using all or 14 loci, correct assignation rate was always higher than 93%. The Weitzman index and the aggregate diversity D were calculated using 22 loci within French and Asian breeds. The French breed Coucou de Rennes and the Hua-Tung breed seemed to contribute the most to the global diversity of each subset. This approach on French-only breeds and then on French with AVIANDIV domestic breeds (14 loci) showed that the Marans breed contributed the most. The AVIANDIV framework could be useful to evaluate the genetic diversity of local breeds and to help in connecting national and regional conservation policies.

The extent of linkage disequilibrium in a large cattle population of western Africa and its consequences for association studies.

Several previous studies concluded that linkage disequilibrium (LD) in livestock populations from developed countries originated from the impact of strong selection. Here, we assessed the extent of LD in a cattle population from western Africa that was bred in an extensive farming system. The analyses were performed on 363 individuals in a Bos indicus x Bos taurus population using 42 microsatellite markers on BTA04, BTA07 and BTA13. A high level of expected heterozygosity (0.71), a high mean number of alleles per locus (9.7) and a mild shift in Hardy-Weinberg equilibrium were found. Linkage disequilibrium extended over shorter distances than what has been observed in cattle from developed countries. Effective population size was assessed using two methods; both methods produced large values: 1388 when considering heterozygosity (assuming a mutation rate of 10(-3)) and 2344 when considering LD on whole linkage groups (assuming a constant population size over generations). However, analysing the decay of LD as a function of marker spacing indicated a decreasing trend in effective population size over generations. This decrease could be explained by increasing selective pressure and/or by an admixture process. Finally, LD extended over small distances, which suggested that whole-genome scans will require a large number of markers. However, association studies using such populations will be effective.

Role of tissue-plasminogen activator (t-PA) in a mouse model of neonatal white matter lesions: interaction with plasmin inhibitors and anti-inflammatory drugs.

Ibotenic acid injected intracerebrally over a broad dose range to 5-day-old mice induces cystic white matter (WM) lesions that mimic periventricular leukomalacia (PVL) of preterm infants. With both low (0.1 mug) and high (5 mug) ibotenic acid doses, tissue-plasminogen activator (t-PA) is involved in cyst formation. Subsequent cyst growth depends on high doses. We evaluated the effects of human recombinant tissue-plasminogen activator (hrt-PA), plasmin inhibitors (tranexamic acid, alpha2-antiplasmin, and aprotinin), and anti-inflammatory drugs (betamethasone, NS-398) in wild-type and t-PA(-/-) mice given high-dose or low-dose ibotenic acid. Intracerebral hrt-PA induced WM cystic lesions in t-PA(-/-) mice and had an additive effect when co-injected with high-dose ibotenic acid. Plasmin inhibitors reduced lesion growth in wild-type mice given high-dose, but not low-dose, ibotenic acid but had no effect in t-PA(-/-) mice. Similarly the anti-inflammatory drugs betamethasone and NS-398 (a cyclooxygenase 2 and NFkappaB inhibitor) were neuroprotective in wild-type animals exposed to high-dose, but not low-dose, ibotenic acid. Thus, the t-PA-dependent effect of low-dose ibotenic acid on cyst formation appeared independent from plasmin activity or inflammation. Conversely, a t-PA-dependent inflammatory process occurred with high-dose ibotenic acid. Potential strategies for PVL in preterm neonates may include fibrinolytic monitoring for prevention and anti-inflammatory agents for treatment.

[Assessment of the in vitro and in vivo toxicity of 3,4-dihydroxyphenylacetaldehyde (DOPAL)]. / Recherche d'une toxicité du 3,4-dihydroxyphénylacétaldéhyde (DOPAL) in vitro et in vivo.

This work was carried out in order to evaluate the in vitro and in vivo toxicity of 3,4-dihydroxyphenylacetaldehyde (DOPAL). This aldehyde is formed from dopamine (DA) by monoamine oxidases (MAO) and is mainly oxidised to 3,4-dihydroxyphenylacetic acid by brain aldehyde dehydrogenases (ALDH), or eventually reduced to 3,4-dihydroxyphenylethanol by aldose/aldehyde reductases. In vitro, catecholaminergic SH-SY5Y cells were incubated with DA and disulfiram (DSF), an irreversible inhibitor of ALDH. As evidenced by MTT assays, a 24-h treatment with 10(-4) M DA and/or 10(-6) M DSF followed by a 24-h incubation in a drug-free medium evidenced that the toxicity of each of these drugs was potentiated by the second drug. HPLC measurements demonstrated that this drug association induced an early DOPAL production that could result in a delayed cell toxicity. For in vivo studies, male Sprague-Dawley rats were treated with L-DOPA-benserazide, which increases the production of DOPAL by MAO, and DSF. An acute injection of DSF (100mg/kg i.p.) and L-DOPA/benserazide (100mg/kg+25mg/kg, 24h later) significantly increased the DOPAL striatal level. However, a 30-day treatment with DSF (100mg/kg i.p., once every two days) and L-DOPA/benserazide (100mg/kg+25mg/kg, twice a day) did not affect both indexes used to assess the integrity of the nigro-striatal dopaminergic terminals (i.e. the striatal content in DA and the binding to the vesicular monoamine transporter on striatal membranes). These results do not support the hypothesis of a DOPAL toxicity and argue against the toxicity of L-DOPA therapy.

Gas chromatography/mass spectrometric identification of dopaminergic metabolites in striata of rats treated with L-DOPA.

The ex vivo identification of dopaminergic metabolites present in rat striata after (L-DOPA + benserazide) treatment is reported. The different metabolites have been identified as the trimethylsilyl derivatives in the striatal extracts by gas chromatography/mass spectrometry.

Distinct patterns of protein binding to the MAT2A promoter in normal and leukemic T cells.

The metabolism of S-adenosylmethionine (AdoMet), a key molecule in regulating T cell differentiation and proliferation, is different in normal and leukemic T cells. To delineate the basis for these differences we studied the transcriptional regulation of human methionine adenosyltransferase II (MAT II), which catalyzes AdoMet synthesis in these cells. Recently, we identified an Sp1 site in the proximal promoter of the MAT2A gene, which encodes the alpha2 catalytic subunit of MAT II, that is essential for the in vitro and in vivo promoter activity in Jurkat leukemic T cells, and that involves binding of the nuclear factors Sp2 and Sp3, but not Sp1. Here, the in vitro and in vivo activity of the proximal MAT2A promoter in normal resting, PHA-stimulated, and leukemic human T cells was compared. Significantly different patterns of protein factor interaction in the proximal region of the MAT2A promoter were found. Normal resting and activated T cells produced complexes of significantly lower molecular weight than those formed in leukemic T cells. Supershift studies coupled with analysis of proteins bound to the proximal promoter suggest that low levels of expression of Sp2 and Sp3 in normal T cells may be responsible for the difference in the in vitro promoter activity between normal and leukemic cells. Mutation of the key Sp1 site equally reduced the in vivo promoter activity in normal and malignant T cells; by contrast, it had significantly different effects on protein-DNA interactions in normal and leukemic T cells. Together, the data support the idea that differences in protein-DNA interactions may contribute to significant differences in MAT2A regulation in normal and malignant cells.

Cloning, expression, and functional characterization of the beta regulatory subunit of human methionine adenosyltransferase (MAT II).

MAT II, the extrahepatic form of methionine adenosyltransferase (MAT), consists of catalytic alpha(2)/alpha(2') subunits and a noncatalytic beta subunit, believed to have a regulatory function. The full-length cDNA that encodes the beta subunit of human MAT II was cloned and found to encode for a 334-amino acid protein with a calculated molecular weight of 37,552. Analysis of sequence homology showed similarity with bacterial enzymes that catalyze the reduction of TDP-linked sugars. The beta subunit cDNA was cloned into the pQE-30 expression vector, and the recombinant His tagged protein, which was expressed in Escherichia coli, was recognized by antibodies to the human MAT II, to synthetic peptides copying the sequence of native beta subunit protein, and to the rbeta protein. There is no cross-reactivity between the MAT II alpha(2) or beta subunits. None of the anti-beta subunit antibodies reacted with protein extracts of E. coli host cells, suggesting that these bacteria have no beta subunit protein. Interestingly, the rbeta subunit associated with E. coli as well as human MAT alpha subunits. This association changed the kinetic properties of both enzymes and lowered the K(m) of MAT for L-methionine. Together, the data show that we have cloned and expressed the human MAT II beta subunit and confirmed its long suspected regulatory function. This knowledge affords a molecular means by which MAT activity and consequently the levels of AdoMet may be modulated in mammalian cells.

Core rotational dynamics and geological events

A study of Earth's fluid core oscillations induced by lunar-solar tidal forces, together with tidal secular deceleration of Earth's axial rotation, shows that the rotational eigenfrequency of the fluid core and some solar tidal waves were in resonance around 3.0 x 10(9), 1.8 x 10(9), and 3 x 10(8) years ago. The associated viscomagnetic frictional power at the core boundaries may be converted into heat and would destabilize the D" thermal layer, leading to the generation of deep-mantle plumes, and would also increase the temperature at the fluid core boundaries, perturbing the core dynamo process. Such phenomena could account for large-scale episodes of continental crust formation, the generation of flood basalts, and abrupt changes in geomagnetic reversal frequency.

Inhibition of methionine adenosyltransferase by the polyamines.

The effect of the polyamines, putrescine, spermine, and spermidine, on the activity of extrahepatic methionine adenosyltransferase (MAT II) was studied. The polyamines inhibited MAT II activity at concentrations equal to or greater than 5 mm. Combinations of polyamines were more effective than individual polyamines in inhibiting MAT activity; maximum inhibition approached 80% with combinations of all three polyamines. S-Adenosylmethionine (AdoMet), Pi, and PPi, the products of the MAT reaction, are known to be synergistic inhibitors of the nonhepatic form of the enzyme. Combinations of polyamines plus Pi and/or PPi induced an additive inhibition of the enzyme. AdoMet plus polyamines also resulted in significant inhibition, but inhibition plateaued at about 80%, indicating the presence of a protective mechanism to maintain AdoMet synthesis. Extrahepatic MAT from human and rat tissues was inhibited by the polyamines, indicating that this phenomenon is not species specific. In addition, we examined the effect of polyamines on MAT activity in resting and activated human lymphocytes that were shown to differ in the relative expression of MAT II subunits. Although MAT from mitogen (phytohemagglutinin, PHA)- and superantigen (Staphylococcal enterotoxin B, SEB)-stimulated lymphocytes were similarly inhibited by 10 mM polyamines, at lower concentrations of polyamines (1-5 mM), MAT from SEB-stimulated cells appeared to be more susceptible to inhibition by the polyamines. Inasmuch as SEB is a more physiological stimulator of T cells than PHA, the data suggest a possible role of polyamines in regulating MAT activity.

Differential regulation of methionine adenosyltransferase in superantigen and mitogen stimulated human T lymphocytes.

Superantigens interact with the T cell receptor for antigen (TCR) and are, therefore, more physiological stimulators of T lymphocytes than nonspecific polyclonal T cell mitogens. The effects of these two classes of T cell stimulators on methionine adenosyltransferase (MAT) and S-adenosylmethionine (AdoMet) levels were investigated. Activation of resting human peripheral blood T lymphocytes by the mitogen phytohemagglutinin (PHA) or the superantigen staphylococcal enterotoxin B (SEB) caused a 3- to 6-fold increase in MAT II specific activity. Although the proliferative response was higher in cultures stimulated with PHA compared with SEB, MAT II activity was comparable in both cultures. Both stimuli caused down-regulation of the MAT 68-kDa lambda subunit expression and induced a comparable increase in the expression of the catalytic alpha2/alpha2' subunit mRNA and protein. However, in superantigen-stimulated cells, the expression of the noncatalytic beta subunit was down-regulated and virtually disappeared by 72 h post-stimulation; whereas, no change in the expression of this subunit was noted in PHA-stimulated cells. Thus, at 72 h following stimulation, PHA-stimulated cells expressed MAT II alpha2/alpha2' and beta subunits while SEB-stimulated cells expressed the alpha2/alpha2' subunits only; the beta subunit was no longer expressed in superantigen-stimulated cells. Kinetic analysis of MAT II in extracts of PHA- and SEB-stimulated cells using reciprocal kinetic plots revealed that in the absence of the beta subunit the Km of the enzyme for L-methionine (L-Met) was 3-fold higher than in the presence of the beta subunit. Furthermore, AdoMet levels were 5-fold higher in cell extracts lacking the beta subunit (SEB-stimulated cell extracts) compared with extracts containing MAT II alpha2/alpha2' and beta subunits. We propose that the increased levels of AdoMet in superantigen-stimulated cells may be attributed to the absence of the beta subunit, which seems to have rendered MAT II less sensitive to product feedback inhibition by (-)AdoMet. The data suggest that the beta subunit of MAT II, which has no catalytic activity, may be a regulatory subunit that imparts a lower Km for L-Met but increases the sensitivity to feedback inhibition by AdoMet. The down-regulation of the beta subunit, which occurred when T cells were stimulated via the TCR, may be an important mechanism to regulate AdoMet levels at different stages of T cell differentiation under physiological conditions.

Chromosomal localization and catalytic properties of the recombinant alpha subunit of human lymphocyte methionine adenosyltransferase.

Human lymphocyte methionine adenosyltransferase (HuLy MAT) consists of heterologous subunits alpha and beta. The cDNA sequence of the alpha subunit of HuLy MAT from Jurkat leukemic T cells was identical to that of the human kidney alpha subunit and highly homologous to the sequence of the extrahepatic MAT from other sources. The 3'-untranslated sequence was found to be highly conserved, suggesting that it may be important in regulating the expression of MAT. The extrahepatic alpha subunit unit of MAT was found to be expressed also in human liver, and no differences were found in the sequence of the alpha subunit from normal and malignant T cells. The sequence of two unspliced introns found in the cDNA clones from the Jurkat library enabled us to isolate genomic clones harboring the human extrahepatic alpha subunit gene and to localize it to the centromere on chromosome arm 2p, an area that corresponds to band 2p11.2. Expression of the alpha subunit cDNA in Escherichia coli yielded two peptides with the immunoreactivity and mobilities of authentic alpha/alpha' subunits from HuLy. The Km of the recombinant alpha subunit was 80 microM, which is 20-fold higher than found for the (alpha alpha')x beta y holoenzyme purified from leukemic lymphocytes and 4-10-fold higher than found for the normal lymphocyte enzyme. The data suggest that the alpha/alpha' subunits mediate the enzyme catalytic activity and that the beta subunit may be a regulatory subunit of extrahepatic MAT.

Characterization of distinct forms of methionine adenosyltransferase in nucleated, and mature human erythrocytes and erythroleukemic cells.

Two peaks of methionine adenosyltransferase (MAT) activity from human erythrocytes were partially purified on a DEAE-cellulose column. Using anti-MAT antibodies, a 60 kDa form of MAT, referred to as rho was identified in peak I. Although rho represented the major MAT protein in crude erythrocyte extracts, the enzyme was very labile and accounted for only 6% of the total MAT activity. Peak II enzyme was stable, and consisted of the previously described catalytic alpha (53 kDa) subunit and the beta subunit (38 kDa), both of which are found in activated human lymphocytes and leukemic cells of lymphoid origin. Mature normal and polycythemic erythrocytes contained predominantly rho as the major MAT protein, while nucleated erythrocytes and reticulocytes contained predominantly the lambda (68 kDa), the major form found in resting human lymphocytes. Human erythroleukemic cells (HEL 92.1.7) contained the alpha, alpha' and beta subunits of MAT, and in this regard was indistinguishable from MAT found in activated lymphocytes and leukemic cells of lymphoid origin (Jurkat). Since rho was generated during the incubation of extracts from resting lymphocytes, which contain predominantly lambda, in the absence of protease inhibitors; the rho form of MAT appears to be derived from the lambda form by proteolytic cleavage. The data indicate that distinct forms of MAT are present at different stages of erythrocyte maturation and reveal the presence of a new form of MAT with reduced activity compared to previously described forms.

Changes in the relative amount of subunits of methionine adenosyltransferase in human lymphocytes upon stimulation with a polyclonal T cell mitogen.

Activation of resting human peripheral blood T lymphocytes by the lectin phytohemagglutinin results in an increase in methionine adenosyltransferase (MAT) activity, accompanied by an increase in the amount of the alpha/alpha' catalytic subunits of the enzyme. In contrast, the amount of the noncatalytic beta subunit remains constant throughout the course of the response. Using both polyclonal antibodies to the holoenzyme and monoclonal antibodies to the alpha/alpha' subunits, we detected a cross-reactive 68-kDa protein, which we refer to as lambda. This protein is present in high abundance in resting T cells but decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits increase. The decrease in lambda and increase in alpha/alpha' occurs after interleukin-2 production and before DNA synthesis. lambda virtually disappears when the cells are actively dividing. Several continuous T cell lines (HPB-ALL, MOLT-4, and Jurkat) as well as a freshly isolated T cell leukemia (ALL-2) had no detectable lambda. The Km for L-methionine for enzyme from resting peripheral blood mononuclear cells was 19-23 microM, which is 3-8-fold higher than purified MAT from fresh leukemic cells or enzyme from Jurkat cells, both of which have a Km of 3.5-3.8 microM. Kinetic analysis of enzyme activity from activated peripheral blood mononuclear cells suggested the presence of two forms of enzyme catalyzing the synthesis of AdoMet. After separation of lambda from the alpha and beta subunits by hydrophobic chromatography, it was determined that lambda has MAT activity but that it is significantly less active than the form containing the alpha subunit. It therefore appears that in resting T cells MAT is sequestered as a less active form. We hypothesize that lambda is a precursor to the catalytic subunits of human lymphocyte MAT and propose that the transition from lambda to alpha/alpha' may be important in the response of T cells to mitogenic signals.

Induction of interleukin 2 production but not methionine adenosyltransferase activity or S-adenosylmethionine turnover in Jurkat T-cells.

We have recently reported that methionine adenosyltransferase (MAT) in resting human peripheral blood T-cells is primarily present in the form of a precursor which we named lambda. This protein decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits of the enzyme increase. When resting cells are activated by phytohemagglutinin, the decrease in lambda and increase in alpha/alpha' occurs after interleukin 2 (IL-2) production and before DNA synthesis. The human T-leukemia cell line, Jurkat, is unique in its ability to produce IL-2 in response to exogenous stimuli such as T-cell mitogens and therefore provides a convenient model for studying biochemical reactions involved in T-cell activation. In this study the regulation of MAT activity and S-adenosylmethionine (AdoMet) in resting and activated Jurkat cells was investigated. Here we report that MAT activity in unstimulated Jurkat cells is about 10- and 3-fold higher than the activity in resting and activated peripheral blood mononuclear cells, respectively. Activation of Jurkat cells with phytohemagglutinin resulted in increased IL-2-production, but not an increase in MAT activity. Identical results were obtained using freshly isolated cells from acute lymphoblastic leukemia patients. AdoMet utilization and pool size were approximately 3- and 10-fold higher, respectively, in Jurkat cells compared to peripheral blood mononuclear cells, and both parameters were unaffected by phytohemagglutinin stimulation. Jurkat MAT was determined to be structurally indistinguishable from enzyme from T- or B-leukemia cells but was different from resting, normal T-cells in that it lacked the lambda form. Furthermore, unlike MAT in resting T-cells, the relative amounts of the alpha, alpha', and beta subunits of the enzyme did not change throughout the course of IL-2 induction. We conclude that AdoMet metabolism and MAT activity in Jurkat cells are constitutively high and that induction of IL-2 synthesis in these cells is independent of changes in AdoMet synthesis or turnover. The lack of the lambda form and the difference in MAT regulation between leukemic T-cells and peripheral blood mononuclear cells may be exploited in the design of specific chemotherapeutic agents.