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T Ruiz

Publications and source records attributed to T Ruiz.

At least 19 recordsLinked to original sources
PubMed2006

The three-dimensional structure of complex I from Yarrowia lipolytica: a highly dynamic enzyme.

The structure of complex I from Yarrowia lipolytica was determined by three-dimensional electron microscopy. A random conical data set was collected from deep stain embedded particles. More than 14000 image pairs were analyzed. Through extensive classification combined with three-dimensional reconstruction, it was possible for the first time to show a much more detailed substructure of the complex. The peripheral arm is subdivided in at least six domains. The membrane arm shows two major protrusions on its matrix facing side and exhibits a channel like feature on the side facing the cytoplasm. Structures resembling a tether connecting the subunits near the catalytic center with the protrusions of the membrane arm provide a second connection between matrix and membrane domain.

Cell Membrane↗
PubMed2003

Nutritional assessment, health markers and lipoprotein profile in postmenopausal women belonging to a closed community.

OBJECTIVE: To assess the dietary characteristics of a closed community and their relationship with several health markers and lipid and lipoprotein values in postmenopausal women. DESIGN: Energy and nutrient intake, serum lipids, lipoproteins, antioxidants, peroxides and low-density lipoprotein (LDL) peroxides in addition to several health markers were measured in a closed, postmenopausal female community consuming a diet without meat, meat products and alcoholic beverages. SETTING: Departamento de Nutrición and Sección Departamental de Química Analítica and Escuela de Especialización de Análisis Clínicos, Universidad Complutense de Madrid, Spain and Lerma, Burgos, Spain. RESULTS: Cereals, vegetables, legumes and fruit, together with milk and eggs, constituted the most important ingredients of the diet consumed. Dietary carbohydrates contributed 42%en and lipids 46.4%en. The SFA/MUFA/PUFA ratio was 1/2/1 and the n-3/n-6 ratio 0.05 (SFA=saturated fatty acids, MUFA=monounsaturated fatty acids, PUFA=polyunsaturated fatty acids). The study community diet was monotonous and made for possible deficiencies of iron, magnesium, zinc, vitamin B(6) and vitamin D, in variable proportions. Routine biochemical and haematological normality markers indicate that only one woman presented hyperglycaemia and hyperuricaemia. Two women had haemoglobin levels <12 g/dl, but their mean corpuscular volume or mean corpuscular haemoglobin was normal. The prevalence of high cholesterol values (>6.21 mmol/l) was 42.8%, while that of high LDL-cholesterol levels (>3.88 mmol/l) was 35.7%, but none of the women displayed levels of high-density lipoprotein (HDL)-cholesterol <1.16 mmol/l, triglycerides >1.2 mmol/l or an LDL-cholesterol/HDL-cholesterol ratio>3. Only one woman had apolipoprotein (Apo) B levels >1.5 g/l, while most of the women presented Apo B values <1.2 g/l and an ApoA-1/ApoB ratio &<1.1. Plasma and LDL-peroxide levels, together with the tocopherol and carotene intakes, suggest a good antioxidant status in this population. CONCLUSIONS: The diet of the study group seems compatible with a healthy life-profile and permits a more-than-acceptable degree of cardiovascular disease protection. However, the consumption of certain nutrients should be improved.

Aged↗
PubMed2001

Two-dimensional structure of the native light-harvesting complex LH2 from Rubrivivax gelatinosus and of a truncated form.

The light-harvesting complex LH2 of Rubrivivax gelatinosus has an oligomeric structure built from alpha-beta heterodimers containing three bacteriochlorophylls and one carotenoid each. The alpha subunit (71 residues) presents a C-terminal hydrophobic extension (residues 51-71) which is prone to attack by an endogenous protease. This extension can also be cleaved by a mild thermolysin treatment, as demonstrated by electrophoresis and by matrix-assisted laser desorption-time of flight mass spectrometry. This cleavage does not affect the pigment binding sites as shown by absorption spectroscopy. Electron microscopy was used to investigate the structures of the native and thermolysin cleaved forms of the complexes. Two-dimensional crystals of the reconstituted complexes were examined after negative staining and cryomicroscopy. Projection maps at 10 A resolution were calculated, demonstrating the nonameric ring-like organization of alpha-beta subunits. The cleaved form presents the same structural features. We conclude that the LH2 complex is structurally homologous to the Rhodopseudomonas acidophila LH2. The hydrophobic C-terminal extension does not fold back in the membrane, but lays out on the periplasmic surface of the complex.

Amino Acid Sequence↗
PubMed2001

The DnaB.DnaC complex: a structure based on dimers assembled around an occluded channel.

Replicative helicases are motor proteins that unwind DNA at replication forks. Escherichia coli DnaB is the best characterized member of this family of enzymes. We present the 26 A resolution three-dimensional structure of the DnaB hexamer in complex with its loading partner, DnaC, obtained from cryo-electron microscopy. Analysis of the volume brings insight into the elaborate way the two proteins interact, and provides a structural basis for control of the symmetry state and inactivation of the helicase by DnaC. The complex is arranged on the basis of interactions among DnaC and DnaB dimers. DnaC monomers are observed for the first time to arrange as three dumb-bell-shaped dimers that interlock into one of the faces of the helicase. This could be responsible for the freezing of DnaB in a C(3) architecture by its loading partner. The central channel of the helicase is almost occluded near the end opposite to DnaC, such that even single-stranded DNA could not pass through. We propose that the DnaB N-terminal domain is located at this face.

Bacterial Proteins↗
PubMed2001

Structural and functional effects of hydrostatic pressure on centrosomes from vertebrate cells.

In an attempt to better understand the role of centrioles in vertebrate centrosomes, hydrostatic pressure was applied to isolated centrosomes as a means to disassemble centriole microtubules. Treatments of the centrosomes were monitored by analyzing their protein composition, ultrastructure, their ability to nucleate microtubules from pure tubulin, and their capability to induce parthenogenetic development of Xenopus eggs. Moderate hydrostatic pressure (95 MPa) already affected the organization of centriole microtubules in isolated centrosomes, and also impaired microtubule nucleation. At higher pressure, the protein composition of the peri-centriolar matrix (PCM) was also altered and the capacity to nucleate microtubules severely impaired. Incubation of the treated centrosomes in Xenopus egg extract could restore their capacity to nucleate microtubules after treatment at 95 MPa, but not after higher pressure treatment. However, the centriole structure was in no case restored. It is noteworthy that centrosomes treated with mild pressure did not allow parthenogenetic development after injection into Xenopus eggs, even if they had recovered their capacity to nucleate microtubules. This suggested that, in agreement with previous results, centrosomes in which centriole architecture is impaired, could not direct the biogenesis of new centrioles in Xenopus eggs. Centriole structure could also be affected by applying mild hydrostatic pressure directly to living cells. Comparison of the effect of hydrostatic pressure on cells at the G1/S border or on the corresponding cytoplasts suggests that pro-centrioles are very sensitive to pressure. However, cells can regrow a centriole after pressure-induced disassembly. In that case, centrosomes eventually recover an apparently normal duplication cycle although with some delay.

Animals↗
PubMed2001

Cloning and sequence analysis of the LEU2 homologue gene from Pichia anomala.

The Pichia anomala LEU2 gene (PaLEU2) was isolated by complementation of a leu2 Saccharomyces cerevisiae mutant. The cloned gene also allowed growth of a Escherichia coli leuB mutant in leucine-lacking medium, indicating that it encodes a product able to complement the beta-isopropylmalate dehydrogenase deficiency of the mutants. The sequenced DNA fragment contains a complete ORF of 1092 bp, and the deduced polypeptide shares significant homologies with the products of the LEU2 genes from S. cerevisiae (84% identity) and other yeast species. A sequence resembling the GC-rich palindrome motif identified in the 5' region of S. cerevisiae LEU2 gene as the binding site for the transcription activating factor encoded by the LEU3 gene was found at the promoter region. In addition, upstream of the PaLEU2 the 3'-terminal half of a gene of the same orientation, encoding a homologue of the S. cerevisiae NFS1/SPL1 gene that encodes a mitochondrial cysteine desulphurase involved in both tRNA processing and mitochondrial metabolism, was found. The genomic organization of the PaNFS1-PaLEU2 gene pair is similar to that found in several other yeast species, including S. cerevisiae and Candida albicans, except that in some of them the LEU2 gene appears in the reverse orientation.

3-Isopropylmalate Dehydrogenase↗
PubMed2001

The structure of the V(1)-ATPase determined by three-dimensional electron microscopy of single particles.

We determined the structure of the V(1)-ATPase from Manduca sexta to a resolution of 1.8 nm, which for the first time reveals internal features of the enzyme. The V(1)-ATPase consists of a headpiece of 13.5 nm in diameter, with six elongated subunits, A(3) and B(3), of approximately equal size, and a stalk of 6 nm in length that connects V(1) with the membrane-bound domain, V(O). At the center of the molecule is a cavity that extends throughout the length of the A(3)B(3) hexamer. Inside the cavity the central stalk can be seen connected to only two of the catalytic A subunits. The structure was obtained by a combination of the Random Conical Reconstruction Technique and angular refinements. Additional recently developed techniques that were used include methods for simultaneous translational rotational alignment of the 0 degrees images, contrast transfer function correction for tilt images, and the Two-Step Radon Inversion Algorithm.

Animals↗
PubMed2001

The first three-dimensional structure of phosphofructokinase from Saccharomyces cerevisiae determined by electron microscopy of single particles.

Phosphofructokinaseis a key regulatory enzyme of the glycolytic pathway. We have determined the structure of this enzyme from Saccharomyces cerevisiae to a resolution of 2.0 nm. This is the first structure available for this family of enzymes in eukaryotic organisms. Phosphofructokinase is an octamer composed of 4alpha and 4beta subunits arranged in a dihedral point group symmetry D(2). The enzyme has a very open and elongated structure, with dimensions of 24 nm in length and 17 nm in width. The final structure, calculated from 0 degrees tilt projections of the molecule at random orientations using as reference the volume obtained by the random conical reconstruction technique in ice, has allowed us to discern the shapes of the subunits and their mutual arrangement in the octamer.

Microscopy, Electron↗
PubMed2001

Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae results in two different active forms of hypoglycosylated invertase.

The Pichia anomala invertase gene (INV1) was introduced at different copy numbers into a sucrose-nonfermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences. The level reached in the production of invertase by the transformants (up to 540 units/10(10) cells) was in agreement with the INV1 gene dosage. Two forms of multimeric active and glycosylated invertase displaying different subcellular locations and molecular masses could be detected in the transformants. One was found to be present in the culture medium and in the periplasm, and the other could only be detected inside the cell. Each of the two heterologous forms of invertase was shown to be an oligomer composed of identical subunits. The difference found in the apparent molecular masses of their monomers (81.5 and 78.3 kDa, respectively) seems to be due to the size of their N-linked oligosaccharide chains (on average 2.4 and 1.9 kDa, respectively), since the number of sugar chains (9) and the molecular mass of the protein moiety (60.5 kDa) are identical in both forms. The shorter size of their oligosaccharides must also be the reason for the lower apparent molecular masses of the heterologous invertases when compared with the enzyme purified from P. anomala. The hypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/10(10) cells). Such accumulation of active enzyme inside the cells, as well as its underglycosylation, could be due to intrinsic properties of the P. anomala invertase that are determined by the particular primary structure of its protein moiety.

Glycoside Hydrolases↗
PubMed2001

Analysis of a putative voltage-gated prokaryotic potassium channel.

Most of the completely sequenced prokaryotic genomes contain genes of potassium channel homologues, but there is still not much known about the role of these proteins in prokaryotes. Here we describe the large-scale overproduction and purification of a prokaryotic voltage-gated potassium channel homologue, Kch, from Escherichia coli. After successful overproduction of the protein, a specific increase in the potassium permeability of the cells was found. Kch could be purified in large amounts using classical purification methods to prevent aggregation of the protein. The physiological state of the protein was revealed to be a homotetramer and the protein was shown to be localized to the cytoplasmic membrane of the cells. In the course of the localization studies, we found a specific increase in the density of the cytoplasmic membrane on Kch production. This was linked to the observed increase in the protein to lipid ratio in the membranes. Another observed change in the membrane composition was an increase in the cardiolipin to phosphatidylglycerol ratio, which may indicate a specific cardiolipin requirement of Kch. On the basis of some of our results, we discuss a function for Kch in the maintenance of the membrane potential in E. coli.

Cell Membrane↗
PubMed2001

Some predictors of cardiovascular mortality among the elderly population of Botucatu (SP).

OBJECTIVE: To detect factors associated with cardiovascular mortality in the elderly of Botucatu. METHODS: We evaluated 29 variables of interest in a cohort of patients aged 60 using data from a survey conducted between 1983/84. The elderly cohort was analyzed in 1992 to detect the occurrence of cardiovascular deaths. Survival analysis was performed using the Kaplan-Meier method, the log-rank test, and Cox regression analysis. Three models were adapted for each group of variables, and a final model was chosen from those variables selected from each group. RESULTS: We identified predictor for cardiovascular death according to age for elderly males not supporting the family, not possessing a vehicle, and previous cardiovascular disease. In elderly females, the predictor variables were previous cardiovascular disease and diabetes mellitus. CONCLUSION: Socioeconomic indicators (family heading and vehicle ownership) may be added to well stabilished medical factors (diabete mellitus and hypertension to select target groups for programs intended to reduce deaths due to cardiovascular diseases in elderly people.

Aged↗
PubMed2000

Three-dimensional structure and subunit topology of the V(1) ATPase from Manduca sexta midgut.

The three-dimensional structure of the Manduca sexta midgut V(1) ATPase has been determined at 3.2 nm resolution from electron micrographs of negatively stained specimens. The V(1) complex has a barrel-like structure 11 nm in height and 13.5 nm in diameter. It is hexagonal in the top view, whereas in the side view, the six large subunits A and B are interdigitated for most of their length (9 nm). The topology and importance of the individual subunits of the V(1) complex have been explored by protease digestion, resistance to chaotropic agents, MALDI-TOF mass spectrometry, and CuCl(2)-induced disulfide formation. Treatment of V(1) with trypsin or chaotropic iodide resulted in a rapid cleavage or release of subunit D from the enzyme, indicating that this subunit is exposed in the complex. Trypsin cleavage of V(1) decreased the ATPase activity with a time course that was in line with the cleavage of subunits B, C, G, and F. When CuCl(2) was added to V(1) in the presence of CaADP, the cross-linked products A-E-F and B-H were generated. In experiments where CuCl(2) was added after preincubation of CaATP, the cross-linked products E-F and E-G were formed. These changes in cross-linking of subunit E to near-neighbor subunits support the hypothesis that these are nucleotide-dependent conformational changes of the E subunit.

Amino Acid Sequence↗
PubMed1999

In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders.

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

Actin Cytoskeleton↗
PubMed1999

Molecular architecture of Manduca sexta midgut V1 ATPase visualized by electron microscopy.

The structure of the V1 ATPase from the tobacco hornworm Manduca sexta has been determined from electron micrographs of isolated, negatively stained specimens. The resulting images clearly show a pseudohexagonal arrangement of six equal-sized protein densities, presumably representing the three copies each of subunits A and B, which comprise the headpiece of the enzyme. A seventh density could be observed either centrally or asymmetrically to the hexamer. The maximum diameter of the V1 complex in the hexagonal projection is 13 nm with each of the six peripheral densities being 3-4 nm in diameter.

Animals↗
PubMed1998

Effects of calcium and nucleotides on the structure of insect flight muscle thin filaments.

The structure of the insect flight muscle thin filament has been studied using a Drosophila mutant (Ifm(2)2) which does not contain thick filaments. Thin filaments that are biochemically identical to those of the wild type can be isolated free from thick filament contamination. We show that isolated thin filaments have different symmetries depending upon the calcium concentration. While the filaments mainly contain 13 subunits in six turns of the 5.9 nm genetic helix in the absence of calcium, 50% of the filaments have 28 subunits in 13 turns of the genetic helix at calcium concentrations equivalent to those present during muscle contraction. We also show that the structure (mainly the helical order) of the thin filaments depends on the nature of the nucleotide bound to the actin monomers. Three-dimensional reconstructions of the thin filaments in the presence and absence of calcium show that tropomyosin moves between two different positions on the actin filament. However, in Drosophila the amplitude of the movement as well as the disorder in the positions of the components (tropomyosin, troponin complex) are larger than those generally observed in other species.

Actin Cytoskeleton↗
PubMed1997

Endothelium-derived nitric oxide-dependent response to hypoxia in piglet intrapulmonary arteries.

The purpose of this study was to determine the involvement of eicosanoids and nitric oxide (NO) in the response to hypoxia in isolated intrapulmonary (third branch) arteries from 10- to 17-day-old piglets. We also compared the response to hypoxia in pulmonary arteries to pulmonary veins, mesenteric arteries and coronary arteries. Hypoxia was generated in vascular rings (under resting force or precontracted with 30 mM KCl) by switching the gas aerating the organ chambers from one composed of 21% O2-5% CO2-balance N2 (pO2 145 +/- 1.27 mm Hg) to a mixture of 5% CO2-balance N2 (pO2 33.87 +/- 0.24 mm Hg). In precontracted rings hypoxia produced a transient vasoconstriction (26 +/- 8% of the precontraction value) reaching a peak in 3-4 min, followed by a relaxation. A similar pattern of response was observed in pulmonary veins, coronary arteries and mesenteric arteries. The contractile phase was not present in endothelium-denuded arteries or after incubation with the NO synthase inhibitor L-NAME (10(-4) M) or the guanylate cyclase inhibitor methylene blue (10(-5) M). No changes in the hypoxia-induced vasoconstriction were observed after preincubation with the NO precursor L-arginine (10(-5) M), the lipoxygenase inhibitor meclofenamate (10(-5) M), the cyclooxygenase inhibitor AA 861 (10(-5) M), or the cytochrome P450 oxidase inhibitor SKF 525A (10(-5) M). These findings demonstrate that the contractile response to hypoxia in the isolated intrapulmonary porcine artery is caused by the loss of the inhibitory effects of endothelium-derived NO on the vascular tone. Eicosanoids do not appear to be involved in this response. Since the response to hypoxia in isolated rings is not specific to pulmonary vessels, any correlation between this response and hypoxic pulmonary vasoconstriction should be avoided.

Animals↗
PubMed1996

Cloning and sequence analysis of the invertase gene INV 1 from the yeast Pichia anomala.

A genomic library from the yeast Pichia anomala has been constructed and employed to clone the gene encoding the sucrose-hydrolysing enzyme invertase by complementation of a sucrose non-fermenting mutant of Saccharomyces cerevisiae. The cloned gene, INV1, was sequenced and found to encode a polypeptide of 550 amino acids which contained a 22 amino-acid signal sequence and ten potential glycosylation sites. The amino-acid sequence shows significant identity with other yeast invertases and also with Kluyveromyces marxianus inulinase, a yeast beta-fructofuranosidase which has a different substrate specificity. The nucleotide sequences of the 5' and 3' non-coding regions were found to contain several consensus motifs probably involved in the initiation and termination of gene transcription.

Amino Acid Sequence↗