Association between estrogen receptor gene polymorphisms and breast density in postmenopausal women.

Objectives The aim of this study was to evaluate the association between clinical characteristics and polymorphisms HaeIII, MspI and XbaI of the estrogen receptor gene alpha with postmenopausal mammographic density. Methods A prospective study was performed with 120 women who were not users of hormones and had no identified breast lesions. All of them underwent bilateral mammography; the radiological density was determined by three independent observers, with two subjective evaluations based on the ACR-BIRADS(R) classification of mammographic patterns, 2003, and one computerized evaluation using the gray-scale histogram tool of the Adobe Photoshop(R) 7.0 software. Peripheral blood samples were obtained for DNA extraction, performed according to the GFX(R) Kit protocol (Amersham-Pharmacia). Polymerase chain reaction restriction fragment length polymorphism was carried out for an analysis of the polymorphisms present in intron 1 (HaeIII and XbaI) and in exon 1 (MspI) of the estrogen receptor gene. Results There was a high degree of concordance among the observers in the determination of mammary density (Kappa, Pearson and Spearman, p < 0.001). The associations of clinical characteristics with mammary density were: age (p = 0.04), body mass index (p < 0.0001) and age at menarche (p = 0.02). The relationship between the allele distribution of the polymorphisms and density was: XbaI (p = 0.02), HaeIII (p = 0.65) and MspI (p = 0.65). Conclusions Our data suggested that the polymorphism XbaI may be strongly related to mammographic density.

Encuesta de pacientes en la consulta del reumatólogo: informe preliminar / Patient syrvery in a rheumatologistïs daily practice: preliminary report

Con propósito de conocer las características demográficas, epidemiológicas y de la terapéutica empleada en pacientes que acuden a consulta del reumatólogo en México, se hizo este estudio multicéntrico formal, en 12 ciudades del país, con colaboración de 17 especialistas certificados (en su mayoría egresados del Centro Médico Nacional. "20 de Noviembre", ISSSTE). Los pacientes fueron elegidos aleatoriamente, de la consulta diaria privada e institucional de cada participante durante 3 meses.Material y método. Empleando un mismo cuestionario diseñado para el estudio, los participantes hicieron el registro en una sola ocasión de datos demográficos, aspectos socioculturales y económicos, así como los inherentes al padecimiento y sus repercusiones, régimen terapéutico vigente en el momento de la entrevista y su costo. además, antecedentes, complicaciones y enfermedades concomitantes. Resultados. Este informe preliminar comprende sólo la información obtenida de 1958 pacientes por los 17 participantes y ofrece datos demográficos, diagnósticos, y de apego al seguimiento por parte del especialista (consultas por primera vez y subsecuentes). Distribución por sexo 7:3 (74 por ciento y 26 por ciento) para mujeres y varones respectivamente; la edad media de la población fue 50.5 y 5l.6 años en el mismo orden anotado para sexo. Se identificaron 12 entidades nosológicas distribuidas entre 1717 pacientes (88 por ciento), 141 quedaron agrupadas como otras enfermedades del tejido conjuntivo (33: 1.7 por ciento), otras enfermedades reumáticas (81: 4.1 por ciento) y sin enfermedad reumática (127= 6 por ciento). Según el número identificado las 10 enfermedades más comunes fueron, en orden decreciente: artritis reumatoide, osteoartrosis, reumatismo no articular, gota, lupus eritematoso sistémico, artritis séptica y reactiva, espondilitis anquilosante, otras enfermedades del tejido conuntivo, artritis crónica juvenil y síndrome de Sjögren. La proporción entre consultas de primera vez y subsecuentes varió entre 1:1 y 1:5 para polimialgia reumática y artritis reumatoide respectivamente y para los diagnósticos más frecuentes fue: AR=l:4.9, OA=1:1.5, RNA=1:1.2, gota, 1.2:1, LES=1:2.8, AS/Re 1:2.2, EA 1:4.7, ACJ 1:3.1 y SS 1:1. La distribución por sexo F:M varió entre 1:0 y 14.2:1, y para las diez primeras entidades en orden de frecuencia fue: AR 6.9:1, OA 3.4:1, RNA 3.5:1, gota 1:24.5, LES 14:2:1, AS/RE 1:2.2, EA 1:4.7. ACJ 1:3.1 y SS 1:0.

The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport.

Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize L-leucine as sole nitrogen source and very low levels of [14C]L-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [14C]L-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic.

GAP1 activity is dependent on cAMP in Saccharomyces cerevisiae.

General amino acid permease (GAP1) activity was evaluated in adenylate cyclase-deficient Saccharomyces cerevisiae to determine the effect of cAMP levels on GAP1 activity. Lowering cAMP concentrations in the culture media led to a decrease in the initial rates of L-citrulline uptake. Kinetics of the amino acid transport system showed a partial loss of transport capacity, with no apparent modifications in permease affinity.

RAS2/PKA pathway activity is involved in the nitrogen regulation of L-leucine uptake in Saccharomyces cerevisiae.

The aim of the present work is to study the participation of RAS2/PKA signal pathway in the nitrogen regulation of L-leucine transport in yeast cells. The study was performed on Saccharomyces cerevisiae isogenic strains with the normal RAS2 gene, the RAS2val19 mutant and the disrupted ras2::LEU2. These strains bring about different activities of the RAS2/PKA signal pathway, L-(14C)-Amino acid uptake measurements were determined in cells grown in a rich YPD medium with a mixed nitrogen source or in minimal media containing NH4+ or L-proline as the sole nitrogen source. We report herein that in all strains used, even in those grown in a minimal proline medium, the activity of the general amino acid permease (GAP1) was not detected. L-Leucine uptake in these strains is mediated by two kinetically characterized transport systems. Their KT values are of the same order as those of S1 and S2 L-leucine permeases. Mutation in the RAS2 gene alters initial velocities and Jmax values in both high and low affinity L-leucine transport systems. Activation of the RAS2/PKA signalling pathway by the RAS2val19 mutation, blocks the response to a poor nitrogen source whereas inactivation of RAS2 by gene disruption, results in an increase of the same response.

Isolation of a trifluoroleucine-resistant mutant of Saccharomyces cerevisiae deficient in both high- and low-affinity L-leucine transport.

A yeast mutant defective in permeases S1 and S2 which transport L-leucine was isolated from a parental strain already deficient in the general amino acid permease, GAP1. The mutant was selected as a spontaneous, trifluoroleucine-resistant (TFLR) strain. Full resistance depended upon the presence of two unlinked mutant genes designated let1 and let2. The let1 mutation completely inactivates the high-affinity leucine transport system defined kinetically as S1. Although the let2 mutation caused a marked decrease in the Jmax of the low-affinity transport system, S2, residual leucine transport in the let1 let2 gap1 mutant had the same KT as in the LET1 LET2 gap1 parent. The mutant exhibited a marked decrease in growth on minimal medium containing leucine, isoleucine or valine as a sole nitrogen source. Moreover, assimilation of methionine, phenylalanine, serine and threonine was decreased, whereas basic and acidic amino acids supported normal growth. This indicates that at least one of the leucine permeases has a fairly broad, but still limited, specificity. Reversion of the gap1 gene restored leucine transport. The revertant was sensitive to TFL when grown on proline but resistant when NH4+ was the nitrogen source. The previously published mutations (shr3, aat1, lup1 or raa) would not be related to either LET1 or LET2.

Why Saccharomyces cerevisiae can oxidize but not decarboxylate external pyruvate.

Protoplasts of the yeast Saccharomyces cerevisiae oxidized externally added pyruvate by pyruvate oxidase system but were not able to decarboxylate it anaerobically by pyruvate decarboxylase at pH 6.4 in isotonic solutions. The decarboxylation set in hypotonic solutions in which the integrity of the plasma membrane was being impaired. Yeast cells incubated with [1-14C]pyruvate accumulated radioactivity under conditions allowing oxidation of pyruvate, but virtually no pyruvate was taken up when the oxidation had been arrested by inhibition or mutation. In view of a large difference between Km for pyruvate of pyruvate decarboxylase (30 mM) and of pyruvate oxidase (0.16 mM), the results may be accounted for by the assumption that transport of pyruvate across the yeast plasma membrane is trans-inhibited by relatively high concentrations of intracellular pyruvate. This arrangement would allow utilization of external pyruvate by the cell energy-transforming machinery and, at the same time, prevent its wastage by futile decarboxylation.

GABA uptake in a Saccharomyces cerevisiae strain.

The aim of this work was to characterize the 4-aminobutyric acid (GABA) transport in the Saccharomyces cerevisiae D27 strain, followed by the study of the relationship between 5-aminolevulinic acid (ALA) and GABA transport systems. It was found that the general amino acid permease (GAP) is not active in D27 strain, suggesting that GABA incorporation should be mediated by PUT4 and UGA4 permeases. However, after kinetic studies only one system was detected. It was also shown that GABA uptake is competitively inhibited by ALA. GABA incorporation is regulated by the carbon source but not by the nitrogen source. When cells were grown in the presence of GABA, its entrance was very low.

Evidence that 4-aminobutyric acid and 5-aminolevulinic acid share a common transport system into Saccharomyces cerevisiae.

It has been previously reported that 5-aminolevulinic acid (ALA) and 4-aminobutyric acid (GABA) share a common permease in Saccharomyces cerevisiae (Bermúdez Moretti et al., 1993). The aim of the present work was to determine the relationship between the transport of these compounds in isolated cells. Assessment of amino acid incorporation was performed in S. cerevisiae using 14C-ALA or 3H-GABA. Initial rates of ALA incorporation in cells grown in the presence of 5 mM ALA and 5 mM GABA, were three to four times lower than in cells grown without supplements. Kinetic studies indicate that GABA competitively inhibits ALA transport. During the growth phase GABA uptake was also inhibited by 74% and 60% in the presence of ALA and GABA, respectively. These findings indicate that in S. cerevisiae the structurally related compounds, ALA and GABA, may be incorporated into the cells by a common carrier protein. Should this occur in other lukaryotic cells it may explain the neurotoxic effect attributed to ALA in the pathogenesis of acute porphyrias.

L-leucine transport systems in Saccharomyces cerevisiae participation of GAP1, S1 and S2 transport systems.

L-leucine uptake in Saccharomyces cerevisiae is mediated by three different transport systems, S1, S2 and GAP1. Their activities are dependent on the nitrogen source of the culture media. Wild type cells grown in L-proline exhibit a single transport system with high affinity and high Vmax that is partially inhibited by L-citrulline. A gap1 mutant shows two transport systems with Km and Vmax values similar to those previously described as S1 and S2, this transport activity is not inhibited by D-leucine, D-isoleucine or D-valine. Two systems can be also determined in wild type cells grown in rich medium containing a mixed nitrogen source where decreased GAP1 function is observed. In either wild type or gap1 cells grown in medium containing ammonium ions as sole nitrogen source, L-leucine uptake kinetics shows two systems with lower Vmax and similar Km values to those of the S1 and S2 systems. These results show that in S. cerevisiae GAP1, S1 and S2 participate in L-leucine entrance in cells grown in a poor nitrogen source, and that S1 and S2 are two ammonia-sensitive permeases that mediate the uptake in cells grown in a rich nitrogen source.

Inhibitory action of palmitic acid on the growth of Saccharomyces cerevisiae.

High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms. This effect was investigated in Saccharomyces cerevisiae. Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization. Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L. As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation. Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.

Study on fatty acid binding by proteins in yeast. Dissimilar results in Saccharomyces cerevisiae and Yarrowia lipolytica.

1. The presence of soluble proteins with fatty acid binding activity was investigated in cell-free extracts from Saccharomyces cerevisiae and Yarrowia lipolytica cultures. 2. No significant fatty acid binding by proteins was detected in S. cerevisiae, even when grown on a fatty acid-rich medium, thus indicating that such proteins are not essential to fatty acid metabolism. 3. An inducible fatty acid binding protein (K0.5 = 3-4 microM) was found in Y. lipolytica which had grown on a minimal medium with palmitate as the sole source of carbon and energy. 4. The relative molecular mass of this protein was 100,000 as inferred from Sephacryl S-200 gel filtration.

[Effect of ammonium ions on the uptake of L-leucine in Saccharomyces cerevisiae. Repression and inhibition of transport systems]. / Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae. Represión e inhibición de los sistemas transportadores.

L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.

Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae. Represión e inhibición de los sistemas transportadores / [Effect of ammonium ions on the uptake of L-leucine in Saccharomyces cerevisiae. Repression and inhibition of transport systems]

L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.

Efecto de iones amonio sobre la entrada de L-leucina en Saccharomyces cerevisiae. Represión e inhibición de los sistemas transportadores. / [Effect of ammonium ions on the uptake of L-leucine in Saccharomyces cerevisiae. Repression and inhibition of transport systems]

L-leucine entrance into Saccharomyces cerevisiae is mediated by the general amino acid permease, GAP and two transport systems, S1 and S2, kinetically characterized. S1 is a high-affinity, low-velocity transport system, operating at lower L-leucine external concentration (0.05-0.1 mM), while S2 is a low-affinity, high-velocity transport system, operating at higher L-leucine external concentration (1.0 mM). In cells grown in minimal medium containing ammonium as sole nitrogen source the values of L-leucine entrance and uptake are smaller than those in cells grown in L-proline containing medium. When GAP is repressed by ammonium, L-leucine entrance is mediate by systems S1 and S2. Both systems are inhibited by ammonium. When GAP is derepressed, in cells grown in L-proline medium, L-leucine is transported by systems S1 and GAP (lower L-leucine external concentration), and mainly by S2 (higher L-leucine external concentration). GAP is the largest system inhibited by ammonium.

Control of leucine transport in yeast by periplasmic binding proteins.

The concentrative inward transport of leucine in Saccharomyces carlsbergensis involves two transport systems (S1 and S2); S1 is a system of high affinity and low translocation velocity, and S2 is a system of low affinity and high translocation velocity. The inward transport process of the amino acid is discriminated into two kinetically defined steps: first, binding to periplasmic proteins and second, translocation across the plasmalemma. When cells were incubated with glucose to increase the metabolic energy charge, we observed that JTmax (maximum flux that each system can exhibit for the translocation step) increased for both systems. This increase in JTmax is due to variations in the parameters defining the initial step (Ks (apparent dissociation constant) and N (concentration of binding sites)): for S1, N1 increases and for S2, KS2 diminishes. Dissipation of the electrochemical proton gradient produced an increase of KS1 and a decrease of N2, resulting in a decrease of JTmax in both systems. Instead, osmotic shock decreases N1 and N2, which suggests that periplasmic components were removed, resulting also in a decrease of JTmax in both systems. These results are consistent with the proposition that the total unidirectional flux of the amino acid proceeds by means of a system of multiple components, with the simultaneous operation of two independent transport processes. We propose that the initial interaction of leucine with components of the cellular envelope might be the essential step for the subsequent translocation of the amino acid across the permeability barrier.

[Determination of intracellular pH by the distribution of benzoic acid in S. cerevisiae. Amino acid transport and proton gradient]. / Determinación del pH intracelular por distribución de ácido benzoico en S. cerevisiae. Transporte de aminoácidos y gradiente de protones.

The internal pH (pHi) of Saccharomyces cerevisiae, wild type strain and its mutant rho- has been measured by the intra-extracellular distribution of 14C-benzoic acid. The values of pHi (external pH 4.5) change with the yeast strain and depend on the cellular metabolic conditions. The values of pHi and proton gradient in the wild type yeast are higher in energized than in starved cells: in energized cells pHi, 6.15 to 6.40, delta pH 1.65 to 1.90 or -97 to -112 mV; starved cells pH 5.90, delta pH 1.40 or -82 mV. In the rho- mutant, the values are lower than in the wild type yeast, in the same metabolic conditions. Energized rho- mutant cells, pH 6.05, delta pH 1.55 or -91 mV; starved cells, pHi 5.70, delta pH 1.20 or -71 mV. The proton conductors, DNP and PCP produce a decrease in pHi and delta pH and inhibition of L-leucine entrance by system S1, high affinity and low velocity and system S2, low affinity and high velocity. The obtained values of delta pH decrease and L-leucine transport inhibition, demonstrate that there is no strict relationship between the proton gradient across the cell membrane and the process of transport of L-leucine in yeast.

Determinación del pH intracelular por distribución de ácido benzoico en S. cerevisiae. Transporte de aminoácidos y gradiente de protones. / [Determination of intracellular pH by the distribution of benzoic acid in S. cerevisiae. Amino acid transport and proton gradient]

The internal pH (pHi) of Saccharomyces cerevisiae, wild type strain and its mutant rho- has been measured by the intra-extracellular distribution of 14C-benzoic acid. The values of pHi (external pH 4.5) change with the yeast strain and depend on the cellular metabolic conditions. The values of pHi and proton gradient in the wild type yeast are higher in energized than in starved cells: in energized cells pHi, 6.15 to 6.40, delta pH 1.65 to 1.90 or -97 to -112 mV; starved cells pH 5.90, delta pH 1.40 or -82 mV. In the rho- mutant, the values are lower than in the wild type yeast, in the same metabolic conditions. Energized rho- mutant cells, pH 6.05, delta pH 1.55 or -91 mV; starved cells, pHi 5.70, delta pH 1.20 or -71 mV. The proton conductors, DNP and PCP produce a decrease in pHi and delta pH and inhibition of L-leucine entrance by system S1, high affinity and low velocity and system S2, low affinity and high velocity. The obtained values of delta pH decrease and L-leucine transport inhibition, demonstrate that there is no strict relationship between the proton gradient across the cell membrane and the process of transport of L-leucine in yeast.

Amino acid uptake by yeasts. IV. Effect of thiol reagents on L-leucine transport in Saccharomyces cerevisiae.

(1) N-Ethylmaleimide (a penetrating SH- reagent) inactivated L-[14C]leucine entrance (binding and translocation) into Saccharomyces cerevisiae, the extent of inhibition depending on the time of preincubation with N-ethylmaleimide, N-ethylmaleimide concentration, the amino acid external and internal concentration, and the energization state of the yeast cells. With D-glucose-energized yeast, N-ethylmaleimide inhibited L-[14C]leucine entrance in all the assayed experimental conditions, but with starved yeast and low (0.1 mM) amino acid concentration, it did not inhibit L-[14C]leucine binding, except when the cells were preincubated with L-leucine. With the rho- respiratory-deficient mutant (energized cells), N-ethylmaleimide inhibited L-[14C]leucine entrance as with the energized wild-type, though to a lesser extent. (2) Analysis of the N-ethylmaleimide effect as a function of L-[14C]leucine concentration showed a significant decrease of Jmax values of the high- (S1) and low- (S2) affinity amino acid transport systems, but KT values were not significantly modified. (3) When assayed in the presence of D-glucose, N-ethylmaleimide inhibition of D-glucose uptake and respiration contributed significantly to inactivation of L-[14C]leucine entrance. Pretreatment of yeast cells with 2,4-dinitrophenol enhanced the effect of L-[14C]leucine binding and translocation. (4) Bromoacetylsulfanilic acid and bromoacetylaminoisophthalic acid, two non-penetrating SH- reagents, did not inactivate L-[14C]leucine entrance, while p-chloromercuribenzoate, a slowly penetrating SH-reagent, inactivated it to a limited extent. When compared with the effect of N-ethylmaleimide, these negative results indicate that thiol groups of the L-[14C]leucine carrier were not exposed on the outer surface of the yeast cell permeability barrier.