Biomedical subjects

Y Henry

Publications and source records attributed to Y Henry.

At least 19 recordsLinked to original sources

PubMed2001

Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p.

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. How box H/ACA snoRNPs are assembled remains unknown. Here we show that yeast Nhp2p, a core component of these particles, directly binds RNA. In vitro, Nhp2p interacts with high affinity with RNAs containing irregular stem-loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs. The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative beta-strand element within Nhp2p central domain impairs the ability of the protein to interact with H/ACA snoRNAs in cell extracts. In vivo, this deletion prevents cell viability and leads to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p. These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is required for the assembly of H/ACA snoRNPs and hence for the stability of some of their components. In addition, we show that converting a highly conserved glycine residue (G(59)) within Nhp2p central domain to glutamate significantly reduces cell growth at 30 and 37 degrees C. Remarkably, this modification affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA. Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleolus, suggesting that only the assembled H/ACA snoRNP particles can be efficiently retained in the nucleolus.

Amino Acid Substitution↗

PubMed2001

Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p.

Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.

DNA, Recombinant↗

PubMed2001

Prevalence of HFE mutations in people from North Africa living in southern France.

The two main mutations of the HFE (haemochromatosis) gene, C282Y and H63D, were found previously to be rare or absent among Africans. Dried blood samples of 1276 newborns from southern France were analysed for both HFE mutations, and the origins of the four grandparents of each newborn were recorded. The allele frequency of C282Y and H63D was 3.0% +/- 0.7% and 16.9% +/- 1.5% respectively. In a subgroup of 171 newborns with four North African ancestries (mainly from Morocco and Algeria) the allele frequency was 0.9%+2.5%-0.2% for the C282Y and 13.2% +/- 3.6% for H63D. HFE mutations are not absent in individuals with North African origins living in southern Europe. This finding has implications for the diagnosis and screening of hereditary haemochromatosis in these populations.

Africa, Northern↗

PubMed2001

The protein kinases AtMAP3Kepsilon1 and BnMAP3Kepsilon1 are functional homologues of S. pombe cdc7p and may be involved in cell division.

We identified an Arabidopsis thaliana gene, AtMAP3Kepsilon1, and a Brassica napus cDNA, BnMAP3Kepsilon1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT-PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kepsilon1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.

Amino Acid Sequence↗

PubMed2000

Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit.

Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.

Amino Acid Sequence↗

PubMed2000

Spin accumulation and domain wall magnetoresistance in 35 nm Co wires

An enhancement of the resistance due to the presence of only one or two isolated domain walls is clearly evidenced by transport measurements in 35 nm epitaxial Co wires, 20 &mgr;m long. The deduced relative change in the resistivity is at least 1 order of magnitude larger than the one predicted from a model based on the mixing of spin channels occurring over the length scale of the domain wall width [P. M. Levy and S. Zhang, Phys. Rev. Lett. 79, 5110 (1997)]. This inconsistency can be resolved by taking the effect of spin accumulation into account, which scales in the case of Co over the much larger distance of the spin diffusion length.

Journal Article↗

PubMed2000

Nuclear control of mitochondrial genome reorganization characterized using cultured cells of ditelosomic and nullisomic-tetrasomic wheat lines.

In vitro-mediated reorganization of the mitochondrial genome is governed by information contained in the nuclear genome. Here we show, from the study of tissue cultures initiated from an almost complete collection of ditelosomic and nullisomic-tetrasomic wheat lines, that nuclear control of the mitochondrial genome structure is a highly complex process. Whereas information responsible for the amplification of defined molecular configurations has been found to be located in either a single or a few chromosomal arms, other rearrangements such as changes in the relative amounts of interconverting subgenomic structures are governed by a number of genes scattered over most of the wheat chromosomes.

Aneuploidy↗

PubMed2000

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2.

Chemical modifications and processing of the 18S, 5.8S, and 25S ribosomal RNAs from the 35S pre-ribosomal RNA depend on an important set of small nucleolar ribonucleoprotein particles (snoRNPs). Genetic depletion of yeast Gar1p, an essential common component of H/ACA snoRNPs, leads to inhibition of uridine isomerizations to pseudo-uridines on the 35S pre-rRNA and of the early pre-rRNA cleavages at sites A1 and A2, resulting in a loss of mature 18S rRNA synthesis. To identify Gar1p functional partners, we screened for mutations that are synthetically lethal with a gar1 mutant allele encoding a Gar1p mutant protein lacking its two glycine/arginine-rich (GAR) domains. We identified a previously uncharacterized Saccharomyces cerevisiae open reading frame, YDR083W (now designated RRP8), that encodes a highly conserved protein containing motifs found in methyltransferases. Rrp8p localizes to the nucleolus. A yeast strain lacking this protein is viable at 30 degrees C but displays strong growth impairment at lower temperatures. In this strain, cleavage of the pre-rRNA at site A2 is strongly affected whereas cleavages at sites A0 and A1 are only slightly inhibited or delayed.

Amino Acid Sequence↗

PubMed1999

Plant MAP kinase kinase kinases structure, classification and evolution.

The increasing number of reports describing plant MAP kinase signalling components reflects the cardinal role that MAP kinase pathways are likely to play during plant growth and development. Relationship and structural analyses of plant MAP kinase kinase kinase related cDNAs and genes established, on one hand, the PMEKKs, which may be distinguished into the alpha, beta, gamma, and zeta groups, and, on the other hand, the PRAFs that consist of the delta, eta and theta groups. Plant MAP3Ks are characterized by different primary structures, but conserved within a single group. A relationship analysis, which included animal, fungal and plant MAP3Ks, revealed a high degree of diversity among this biochemically established set of proteins, thus suggesting a range of biological functions. Four major families emerged, namely the MEKK/STE11, including the PMEKKs, the RAF, including the PRAFs, as well as the MLK and CDC7 families. These four families showed phylum-dependent distributions. Signature sequences characterizing the RAF family and the RAF subfamilies have been evidenced. However, no equivalent sequence motifs were identified for the MEKK/STE11 family, which is highly heterogeneous.

Evolution, Molecular↗

PubMed1999

Characterisation of novel plant genes encoding MEKK/STE11 and RAF-related protein kinases.

Various elements of the MAP kinase module have been isolated in plants. We describe here the characterisation of 14 new plant cDNAs and genes encoding putative MAP kinase kinase kinases (MAP3Ks) related to the MEKK/STE11 and RAF protein kinases. Plant MAP3Ks are characterised by a variety of primary structures conserved within closely related proteins. Southern blot analysis suggests that plant MAP3Ks are heterogenous in their genomic structure, existing either as single copy genes or as small gene families. An RT-PCR analysis showed that in Arabidopsis thaliana, all organs studied contain detectable levels of transcripts of each of the MAP3K genes identified; however, signals obtained with mature pollen were weak or non-existent except for AtMAP3Kgamma. None of the reported genes share a cell-cycle or a cold stress regulated expression.

Amino Acid Sequence↗

PubMed1999

Molecular characterisation of plant cDNAs BnMAP4Kalpha1 and BnMAP4Kalpha2 belonging to the GCK/SPS1 subfamily of MAP kinase kinase kinase kinase.

Several yeast and mammal MAP kinase modules require, upstream of their MAP kinase kinase kinase (MAP3K), a MAP3K kinase (MAP4K). An Arabidopsis thaliana EST clone, sharing identity to MAP4Ks from yeast and mammals, has been used to isolate cDNA clones from a Brassica napus microspore-derived embryo cDNA library. The BnMAP4Kalpha1 and BnMAP4K-alpha2 clones encode putative proteins possessing the 12 subdomains of the serine/threonine protein kinase catalytic domain. A detailed analysis showed that they belong to the GCK/SPS1 subfamily of MAP4K proteins which possess an amino terminal catalytic domain and a long carboxy terminal tail. A Southern blot analysis suggested that the two proteins are encoded by a small multigene family. Expression studies revealed the presence of BnMAP4Kalpha1 and -alpha2 transcripts in all the tissues examined; however, they are most abundant in roots, siliques and flower buds. The expression of BnMAP4Kalpha1 and -alpha2 at the three main developmental stages of microspore-derived embryos (i.e., globular/heart, torpedo and cotyledonary) was confirmed by northern blot and RT-PCR analysis. An expression analysis of the above genes using synchronised Arabidopsis thaliana cell suspensions showed that the homologues genes are cell cycle regulated.

Amino Acid Sequence↗

PubMed1999

Influence of the antioxidant quercetin in vivo on the level of nitric oxide determined by electron paramagnetic resonance in rat brain during global ischemia and reperfusion.

We characterized the changes in nitric oxide (NO) levels in the brain during global forebrain ischemia and reperfusion and tested the ability of the natural flavonoid, quercetin, and a synthetic flavonoid, FB277, to increase the amount of available NO by elimination of the superoxide radicals produced during reperfusion. In Sprague-Dawley rats, we used a four-vessel occlusion model of forebrain ischemia (15 min) and reperfusion (30 min). Brain NO was measured on samples of cerebral cortex and cerebellum ex vivo by electron paramagnetic resonance (EPR) spectroscopy. The spin trap used was diethyldithiocarbamate sodium salt (DETC) associated with ferrous citrate. The complex Fe(DETC)2NO was detected at 77 K as a triplet signal at g = 2.035. Groups of animals were treated with quercetin or FB277 (3-morpholinomethyl-3',4',5,7tetramethoxyflavone) or polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). In control (intact anesthetized animals), the signal was about 3 times greater in the cortex than in the cerebellum. During ischemia, the signal rose to 110% in cortex (NS) and 283% in cerebellum (P < 0.05). In reperfusion, it fell again to 91% of control in cerebellum (NS) and 35% in cortex (P < 0.05). Treatment by quercetin (5 mg/kg i.v.) of intact and ischemia-reperfusion groups did not significantly change the signal amplitude in the cerebellum, but did double it in the cortex (to 76% of control) for the ischemia-reperfusion group (P < 0.05). In contrast, FB277 (3.75 mg/kg i.v.) did not increase the signal in the cortex during ischemia-reperfusion, but did do so in the cerebellum (to 152% of control, P < 0.05). The results obtained for PEG-SOD (10,000 U/kg i.v.) were similar to those for FB277. In separate in vitro measurements, we found that quercetin but not FB277 efficiently scavenged superoxide. We hypothesize that quercetin but not FB277 scavenged superoxide anions released in the cortex during reperfusion, thus diminishing the amount of NO removed by the formation of peroxynitrite. The lack of effect of PEG-SOD may be related to the need for chronic treatment to obtain protection.

Animals↗

PubMed1999

Interactions of nitric oxide with hemoproteins: roles of nitric oxide in mitochondria.

Nitric oxide (NO) binds to metalloproteins, and particularly to hemoproteins in both ferrous and ferric states, with association and dissociation rate constants which cover many orders of magnitude. These chemical properties often provide clear explanations of enzymatic specificity. A basic and straightforward description of the versatility of NO chemistry and of the biological relevance of NO effects, as understood by biochemists as opposed to physiologists, is presented. NO effects on hemoglobin and soluble guanylate cyclase, two proteins directly involved in arterio-venous oxygen transport at quite different biological levels, are compared. NO and other N-oxides also play primary roles in several mitochondrial functions. Specific interactions with cytochrome c oxidase and cytochrome c are reviewed, and the effects of NO and other N-oxides on other iron-cluster-containing components of mitochondrial respiration are discussed.

Animals↗

PubMed1999

Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast.

Through a computer search of the genome of the yeast Saccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII. The corresponding snoRNAs have been positively identified in yeast cells. Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested. The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA. Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p. After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated. In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated. This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs. In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor.

Base Sequence↗

PubMed1998

Microsomal cytochrome P450 dependent oxidation of N-hydroxyguanidines, amidoximes, and ketoximes: mechanism of the oxidative cleavage of their C=N(OH) bond with formation of nitrogen oxides.

Oxidation by rat liver microsomes of 13 compounds involving a C=N(OH) function (including N-hydroxyguanidines, amidoximes, ketoximes, and aldoximes) was found to occur with the release of nitrogen oxides such as NO, NO2-, and NO3-. The greatest activities were observed with liver microsomes from dexamethasone-treated rats (up to 8 nmol of NO2- nmol of P450(-)1 min-1). A detailed study of the microsomal oxidation of some of these compounds was performed. Oxidation of N-(4-chlorophenyl)-N'-hydroxy-guanidine led to the formation of the corresponding urea and cyanamide in addition to NO, NO2-, and NO3-. Formation of all these products was dependent on NADPH, O2, and cytochromes P450. Oxidation of two arylamidoximes was found to occur with formation of the corresponding amides and nitriles in addition to nitrogen oxides. Oxidation of 4-(chlorophenyl)methyl ketone oxime gave the corresponding ketone and nitroalkane as well as NO, NO2-, and NO3-. These reactions were also dependent on cytochromes P450 and required NADPH and O2. Mechanistic experiments showed that microsomal oxidations of amidoximes to the corresponding nitriles and of ketoximes to the corresponding nitroalkanes are not inhibited by superoxide dismutase (SOD) and are performed by a cytochrome P450 active species, presumably the high-valent P450-iron-oxo complex. On the contrary, microsomal oxidation of N-hydroxyguanidines to the corresponding cyanamides was greatly inhibited by SOD and appeared to be mainly due to O2*- derived from the oxidase function of cytochromes P450. Similarly, microsomal oxidations of N-hydroxyguanidines and amidoximes to the corresponding ureas and amides were also found to be mainly performed by O2*-, as shown by the great inhibitory effect of SOD (70-100%) and the ability of the xanthine-xanthine oxidase system to give similar oxidation products. However, it is noteworthy that other species, such as the P450 Fe(II)-O2 complex, are also involved, to a minor extent, in the SOD-insensitive microsomal oxidative cleavages of compounds containing a C=N(OH) bond. Our results suggest a general mechanism for such oxidative cleavages of C=N(OH) bonds with formation of nitrogen oxides by cytochromes P450 and NO-synthases, with the involvement of O2*- and its Fe(III) complex [(FeIII-O2-) or (FeII-O2)] as main active species.

Amides↗

PubMed1998

Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs.

The small nucleolar ribonucleoprotein particles containing H/ACA-type snoRNAs (H/ACA snoRNPs) are crucial trans-acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site-specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinity-purified complexes containing epitope-tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope-tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively. Both proteins are conserved, essential and present in the dense fibrillar component of the nucleolus. Nhp2p and Nop10p are specifically associated with all H/ACA snoRNAs and are essential to the function of H/ACA snoRNPs. Cells lacking Nhp2p or Nop10p are impaired in global rRNA pseudouridylation and in the A1 and A2 cleavage steps of the pre-rRNA required for the synthesis of mature 18S rRNA. These phenotypes are probably a direct consequence of the instability of H/ACA snoRNAs and Gar1p observed in cells deprived of Nhp2p or Nop10p. Our results suggest that Nhp2p and Nop10p, together with Cbf5p, constitute the core of H/ACA snoRNPs.

Amino Acid Sequence↗

PubMed1998

Differential sensitivity of the tyrosyl radical of mouse ribonucleotide reductase to nitric oxide and peroxynitrite.

Ribonucleotide reductase is essential for DNA synthesis in cycling cells. It has been previously shown that the catalytically competent tyrosyl free radical of its small R2 subunit (R2-Y.) is scavenged in tumor cells co-cultured with macrophages expressing a nitric oxide synthase II activity. We now demonstrate a loss of R2-Y. induced either by .NO or peroxynitrite in vitro. The .NO effect is reversible and followed by an increase in ferric iron release from mouse protein R2. A similar increased iron lability in radical-free, diferric metR2 protein suggests reciprocal stabilizing interactions between R2-Y. and the diiron center in the mouse protein. Scavenging of R2-Y. by peroxynitrite is irreversible and paralleled to an irreversible loss of R2 activity. Formation of nitrotyrosine and dihydroxyphenylalanine was also detected in peroxynitrite-modified protein R2. In R2-overexpressing tumor cells co-cultured with activated murine macrophages, scavenging of R2-Y. following NO synthase II induction was fully reversible, even when endogenous production of peroxynitrite was induced by triggering NADPH oxidase activity with a phorbol ester. Our results did not support the involvement of peroxynitrite in R2-Y. scavenging by macrophage .NO synthase II activity. They confirmed the preponderant physiological role of .NO in the process.

Animals↗

PubMed1998

The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase.

Many or all of the sites of pseudouridine (Psi) formation in eukaryotic rRNA are selected by site-specific base-pairing with members of the box H + ACA class of small nucleolar RNAs (snoRNAs). Database searches previously identified strong homology between the rat nucleolar protein Nap57p, its yeast homolog Cbf5p, and the Escherichia coli Psi synthase truB/P35. We therefore tested whether Cbf5p is required for synthesis of Psi in the yeast rRNA. After genetic depletion of Cbf5p, formation of Psi in the pre-rRNA is dramatically inhibited, resulting in accumulation of the unmodified rRNA. Protein A-tagged Cbf5p coprecipitates all tested members of the box H + ACA snoRNAs but not box C + D snoRNAs or other RNA species. Genetic depletion of Cbf5p leads to depletion of all box H + ACA snoRNAs. These include snR30, which is required for pre-rRNA processing. Depletion of Cbf5p also results in a pre-rRNA processing defect similar to that seen on depletion of snR30. We conclude that Cbf5p is likely to be the rRNA Psi synthase and is an integral component of the box H + ACA class of snoRNPs, which function to target the enzyme to its site of action.

Cell Nucleolus↗