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Biomedical subjects

M Ares

Publications and source records attributed to M Ares.

At least 37 records · Page 2Linked to original sources
PubMed1998

Lipoprotein-like phospholipid particles inhibit the smooth muscle cell cytotoxicity of lysophosphatidylcholine and platelet-activating factor.

Oxidation of LDL is associated with degradation of phosphatidylcholine into platelet-activating factor (PAF)-like phospholipids and lysophosphatidylcholine (LPC). Exposure of cultured human smooth muscle cells to PAF and LPC in a concentration of 25 micromol/L was found to result in complete cell death, as assessed by the MTT cytotoxicity assay and cell counting. Addition of 50 microg/mL apolipoprotein A-I- and apolipoprotein A-I(Milano)-containing phospholipid particles completely inhibited this cytotoxicity. Phospholipid complexes alone were almost as effective, whereas free apolipoprotein A-I(Milano) and albumin were without effect, suggesting that the effect was phospholipid dependent. Experiments using [14C]LPC demonstrated that apolipoprotein A-I- and apolipoprotein A-I(Milano)-containing phospholipid particles effectively bind LPC. The results show that HDL-like phospholipid particles effectively inhibit the toxic effect of phospholipids and other lipid-soluble factors. The ability of HDL to inhibit the proinflammatory and toxic effects of phospholipids generated during oxidation of LDL may be responsible for part of the antiatherogenic properties of HDL.

Apolipoprotein A-I↗
PubMed1998

Conservation of structure and subunit interactions in yeast homologues of splicing factor 3b (SF3b) subunits.

Human SAP 49, a subunit of the multimeric splicing factor 3b (SF3b), contains two RNA recognition motifs (RRMs) and binds another SF3b subunit called SAP 145, whose yeast homologue is CUS1. Here we show that the predicted yeast open reading frame YOR319w (HSH49) encodes an essential yeast splicing factor. Using bacterially expressed proteins, we find that yeast HSH49 binds CUS1. Mutations that alter putative RNA-binding residues of either HSH49 RRM are lethal in vivo, but do not prevent binding to CUS1 in vitro, suggesting that the predicted RNA-binding surfaces of HSH49 are not required for interaction with CUS1. In vivo interaction tests show that HSH49 and CUS1 associate primarily through the N-terminal RRM of HSH49. Recombinant HSH49 protein has a general RNA-binding activity that does not require CUS1. The parallels in structure and interaction between two SF3b subunits from yeast implies that the mechanism of SF3b action is highly conserved.

Amino Acid Sequence↗
PubMed1997

Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA.

Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA-RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5' splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5' splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing.

Exons↗
PubMed1997

Alternative 3'-end processing of U5 snRNA by RNase III.

The cellular components required to form the 3' ends of small nuclear RNAs are unknown. U5 snRNA from Saccharomyces cerevisiae is found in two forms that differ in length at their 3' ends (U5L and U5S). When added to a yeast cell free extract, synthetic pre-U5 RNA bearing downstream genomic sequences is processed efficiently and accurately to generate both mature forms of U5. The two forms of U5 are produced in vitro by alternative 3'-end processing. A temperature-sensitive mutation in the RNT1 gene encoding RNase III blocks accumulation of U5L in vivo. In vitro, alternative cleavage of the U5 precursor by RNase III determines the choice between the two multistep pathways that lead to U5L and U5S, one of which (U5L) is strictly dependent on RNase III. These results identify RNase III as a trans-acting factor involved in 3'-end formation of snRNA and show how RNase III might regulate alternative RNA processing pathways.

Base Sequence↗
PubMed1996

RNase III cleaves eukaryotic preribosomal RNA at a U3 snoRNP-dependent site.

A yeast gene homologous to bacterial RNase III (RNT1) encodes a double-strand-specific endoribonuclease essential for ribosome synthesis. Two rRNA processing events are blocked in cells temperature sensitive for RNT1: cleavage at the snoRNA-dependent AO site in the 5' ETS and cleavage in the 3' ETS. Recombinant RNT1 protein accurately cleaves a synthetic 5' ETS RNA at AO site in vitro, in the absence of snoRNA or other factors. A synthetic 3' ETS substrate is specifically cleaved at a site 21 nt downstream of the 3' end 28S rRNA. These observations show that a protein endonuclease collaborates with snoRNAs in eukaryotic rRNA processing and exclude a catalytic role for snoRNAs at certain pre-rRNA cleavage.

Amino Acid Sequence↗
PubMed1996

CUS1, a suppressor of cold-sensitive U2 snRNA mutations, is a novel yeast splicing factor homologous to human SAP 145.

The function of U2 snRNA in splicing is mediated by the proteins of the U2 small nuclear ribonucleoprotein. To identify proteins that influence the function of U2 snRNA we carried out a screen for mutations in Saccharomyces cerevisiae that suppress the cold-sensitive growth defect of a mutation in U2 stem loop IIa, a structure important for the stable association of the U2 snRNP with pre-mRNA. The screen identified three dominant suppressor genes, one of which, CUS1-54, encodes an essential splicing protein required for U2 snRNP addition to the spliceosome. The suppressor protein rescues the spliceosome assembly defect of the mutant U2 in vitro, indicating that suppression is direct. Allele specificity tests show that the suppressor does not simply bypass the requirement for U2 stem loop IIa. Extra copies of wild-type CUS1, but not CUS1-54, suppress the temperature-sensitive prp11 and prp5 mutations, linking CUS1 protein to a subset of other factors required at the same step of spliceosome assembly. CUS1 is homologous to SAP 145, a component of the mammalian U2 snRNP that interacts with pre-mRNA. The yeast genome also encodes a homolog of human SAP 49, a protein that interacts strongly with both SAP 145 and pre-mRNA, underscoring the conservation of U2 snRNP proteins that function in spliceosome assembly.

Amino Acid Sequence↗
PubMed1996

Differentiation of constrictive pericarditis from restrictive cardiomyopathy: assessment of left ventricular diastolic velocities in longitudinal axis by Doppler tissue imaging.

OBJECTIVES: We sought to determine the utility of left ventricular expansion velocities in differentiating constrictive pericarditis from restrictive cardiomyopathy. BACKGROUND: Several studies have shown that left ventricular diastolic expansion is influenced by the elastic recoil forces of the myocardium. These forces are affected by intrinsic myocardial disease but should be preserved when diastole is impaired as a result of extrinsic causes. METHODS: Using Doppler tissue imaging, we measured peak early velocity of longitudinal axis expansion (Ea) in 8 patients with constrictive pericarditis, 7 patients with restriction and 15 normal volunteers. Transmitral early (E) and late (A) Doppler flow velocities, left ventricular systolic and diastolic volumes, ejection fraction and mitral annular M-mode displacement were also compared between the groups. RESULTS: The Ea value was significantly higher in normal subjects (14.5 +/- 4.7 cm/s [mean +/- SD]) and in patients with constriction (14.8 +/- 4.8 cm/s) than in those with restriction (5.1 +/- 1.4 cm/s, p < 0.001 constriction vs. restriction). There was weak correlation between Ea and the extent of annular displacement (r = 0.55, p = 0.004) and the E/A ratio (r = 0.44, p = 0.03). There was no correlation between Ea and E (r = 0.33, p = 0.07) or ejection fraction (r = 0.21, p = 0.26). By multivariate analysis, Ea was the best variable for differentiating constriction from restriction. CONCLUSIONS: Our study indicates that longitudinal axis expansion velocities are markedly reduced in patients with restrictive cardiomyopathy. The poor correlation found with transvalvular flow velocities suggests that Ea may be relatively preload independent. The measurement of longitudinal axis expansion velocities provides a clinically useful distinction between constrictive pericarditis and restrictive cardiomyopathy and may prove to be valuable in the study of diastolic function.

Adult↗
PubMed1996

Assessment of mitral annular dynamics during diastole by Doppler tissue imaging: comparison with mitral Doppler inflow in subjects without heart disease and in patients with left ventricular hypertrophy.

The purpose of this study was to determine the normal pattern and magnitude of mitral annular velocities in diastole by Doppler tissue imaging (DTI) and to assess whether this is altered in patients with left ventricular hypertrophy. Mitral annulus velocities were measured by DTI. Peak and time-velocity integral were measured from the DTI tracings and the timing of the velocities in relation to electrocardiogram. DTI was compared with M-mode echo of the annulus and mitral inflow Doppler velocities. Integrated annular velocities by DTI correlated with the annular displacement. Early diastolic velocities decreased with age and in patients with left ventricular hypertrophy. In the hypertrophy group, early diastolic velocities were significantly lower than normal even after correcting for age. Patients with left ventricular hypertrophy also showed a delay in peak early diastolic mitral annular velocity (5.5 +/- 21 msec after the E wave). In conclusion, mitral annular velocity in diastole is readily recorded by DTI. The magnitude and the pattern of these velocities are significantly altered by age and by left ventricular hypertrophy. This method provides a new insight into diastolic filling events and may prove useful in detecting abnormal diastolic function.

Adult↗
PubMed1996

Myocardial wall velocity assessment by pulsed Doppler tissue imaging: characteristic findings in normal subjects.

To validate the use of pulsed Doppler tissue imaging that measures myocardial wall velocities and to define the characteristics of these velocities in normal subjects, we obtained and compared the anteroseptal and posterior wall velocities in 24 volunteers with pulsed Doppler tissue imaging and digitized M-mode echocardiography. We also studied the relation between velocity components and hemodynamic events timed by standard Doppler flows. There was an excellent correlation between Doppler and M-mode-derived velocities (r = 0.95, p < 0.001), with higher reproducibility for Doppler (r = 0.99) than for M-mode (r = 0.95, p < 0.001). Biphasic velocities that were uniformly present during isovolumic contraction and relaxation were attributed to geometric changes due to asynchronous contraction and ventricular interdependence. We conclude that wall velocities obtained by pulsed Doppler tissue imaging are accurate and reproducible. This method may prove useful for studying the contractile and elastic properties of the myocardium.

Adolescent↗
PubMed1996

Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits.

U2 small nuclear RNA (snRNA) contains a sequence (GUAGUA) that pairs with the intron branchpoint during splicing. This sequence is contained within a longer invariant sequence of unknown secondary structure and function that extends between U2 and I and stem IIa. A part of this region has been proposed to pair with U6 in a structure called helix III. We made mutations to test the function of these nucleotides in yeast U2 snRNA. Most single base changes cause no obvious growth defects; however, several single and double mutations are lethal or conditional lethal and cause a block before the first step of splicing. We used U6 compensatory mutations to assess the contribution of helix III and found that if it forms, helix III is dispensable for splicing in Saccharomyces cerevisiae. On the other hand, mutations in known protein components of the splicing apparatus suppress or enhance the phenotypes of mutations within the invariant sequence that connect the branchpoint recognition sequence to stem IIa. Lethal mutations in the region are suppressed by Cus1-54p, a mutant yeast splicing factor homologous to a mammalian SF3b subunit. Synthetic lethal interactions show that this region collaborates with the DEAD-box protein Prp5p and the yeast SF3a subunits Prp9p, Prp11p, and Prp21p. Together, the data show that the highly conserved RNA element downstream of the branchpoint recognition sequence of U2 snRNA in yeast cells functions primarily with the proteins that make up SF3 rather than with U6 snRNA.

Base Sequence↗
PubMed1994

Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4.

Studies on the function of circular RNA and RNA topology in vivo have been limited by the difficulty in expressing circular RNA of desired sequence. To overcome this, the group I intron from the phage T4 td gene was split in a peripheral loop (L6a) and rearranged so that the 3' half intron and 3' splice site are upstream and a 5' splice site and 5' half intron are downstream of a single exon. The group I splicing reactions excise the internal exon RNA as a circle (RNA cyclase ribozyme activity). We show that foreign sequences can be placed in the exon and made circular in vitro. Expression of such constructs (RNA cyclase ribozymes) in Escherichia coli and yeast results in the accumulation of circular RNA in these organisms. In yeast, RNA cyclase ribozymes can be expressed from a regulated promoter like an mRNA, containing 5' leader and 3' trailer regions, and a nuclear pre-mRNA intron. RNA cyclase ribozymes have broad application to questions of RNA structure and function including end requirements for RNA transport or function, RNA topology, efficacy of antisense or ribozyme gene control elements, and the biosynthesis of extremely long polypeptides.

Bacteriophage T4↗
PubMed1994

Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures.

Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.

Base Sequence↗
PubMed1994

Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.

Base Sequence↗
PubMed1994

[The estimation of the severity of tricuspid insufficiency by Doppler color: the effects of gain, pulse repetition frequency and the echographic view].

INTRODUCTION AND OBJECTIVES: Regurgitant jet area is to date the most widely accepted color Doppler parameter for quantitation of atrium-ventricular regurgitations. In experimental studies, it has been demonstrated that color regurgitant jet area is greatly influenced by technical settings. The present study was aimed to analyze the effect of gain setting, pulse repetition frequency and echocardiographic plane on color jet area in patients with tricuspid regurgitation. METHODS: We studied 64 patients with nontrivial (more than 1 cm2), rheumatic tricuspid regurgitation. Examinations were performed in apical four-chamber and right ventricle inflow-tract views, at two gain settings (subsaturation gain and 1/3 reduced gain) and at two pulse repetition frequencies (3.1 and 3.8 kHz). Therefore, every regurgitant jet was study under 8 different technical conditions. The remaining technical settings were held unchanged included throughout the studies. The traced area induced central varianced and aliased signals, as well as the immediately contiguous nonturbulent velocities that were moving in the same direction as the jet. Measurements were taken from 5 and 3 representative cycles in patients with atrial fibrillation and sinus rythm, respectively. RESULTS: Globally, reduction in gain setting produced a color area decrease by 50%. This reduction was of greater extent for small jets (less than 5 cm2, 45 +/- 17%) with respect to larger jets (more than 5 cm2, 61 +/- 30%, p = 0.0009). Results were similar for both apical four-chamber and right ventricle inflow views and for the two pulse repetition frequencies. At a given color gain level and echocardiographic view, pulse repetition frequency at 3.1 kHz produced greater color areas (by 3-11%) than frequency at 3.8 kHz, but the difference was not significant. Overall, color jet area was significantly greater in right ventricle inflow view than in apical four-chamber view, but substantial individual variability was observed. Difference between both views was greater than 40% in 28% of patients, with the largest color area in right ventricle inflow view in most, especially in those patients with eccentric jets. CONCLUSIONS: Gain setting and echocardiographic view greatly affect regurgitant jet area by color Doppler in patients with tricuspid regurgitation. Therefore, standardization of technical settings is mandatory in order to avoid diagnostic pitfalls.

Aged↗
PubMed1991

Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype.

U2 is a highly conserved small nuclear RNA essential for pre-mRNA splicing in mammals and yeast and for trans-splicing in trypanosomes. To test the function of variant U2 RNA structures from different organisms, we conducted phylogenetic exchanges of U2 domains. Replacing nucleotides 1-120 of yeast U2 with the corresponding region of human U2 generates a U2 RNA that is correctly folded and functions in yeast. In contrast, replacement of the branchpoint interaction region of yeast U2 with the corresponding region from trypanosome is dominant lethal. Using a GAL-U2 promoter fusion, we show that the dominant phenotype can be made conditional and that the accumulation of mutant U2 is followed rapidly by inhibition of nuclear pre-mRNA splicing. The results suggest that U2 small nuclear ribonucleoprotein particles normally participate in stable complexes with a limiting splicing factor prior to formation of U2-intron branchpoint base pairs.

Base Sequence↗
PubMed1991

Depletion of U3 small nucleolar RNA inhibits cleavage in the 5' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA.

Multiple processing events are required to convert a single eukaryotic pre-ribosomal RNA (pre-rRNA) into mature 18S (small subunit), 5.8S and 25-28S (large subunit) rRNAs. We have asked whether U3 small nucleolar RNA is required for pre-rRNA processing in vivo by depleting Saccharomyces cerevisiae of U3 by conditional repression of U3 synthesis. The resulting pattern of accumulation and depletion of specific pre-rRNAs indicates that U3 is required for multiple events leading to the maturation of 18S rRNA. These include an initial cleavage within the 5' external transcribed spacer, resembling the U3 dependent initial processing event of mammalian pre-rRNA. Formation of large subunit rRNAs is unaffected by U3 depletion. The similarity between the effects of U3 depletion and depletion of U14 small nucleolar RNA and the nucleolar protein fibrillarin (NOP1) suggests that these could be components of a single highly conserved processing complex.

Animals↗