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M Miranda

Publications and source records attributed to M Miranda.

At least 91 records · Page 5Linked to original sources
PubMed1997

Comparative analysis on the distribution of heterochromatin in Citrus, Poncirus and Fortunella chromosomes.

Double fluorochrome staining with chromomycin A3 (CMA) and 4'-6-diamidino-2-phenylindole (DAPI) was used to characterize and compare the distribution of constitutive heterochromatin along chromosomes of Citrus, Poncirus and Fortunella species. Only CMA-positive bands were distinguishable in metaphase chromosomes. Preferential distribution of heterochromatin in terminal regions, mainly of the long arm, and centromeric regions of a few long chromosomes was a common feature of these genera. Heteromorphism between possible homologous chromosomes was present in the majority of species. Citrus and Poncirus revealed some remarkably uniform chromosomes without any intensively fluorescing region, whereas Fortunella cultivars were differentiated by the presence of CMA bands in all chromosomes. Through measurements assisted by a computer, amounts of CMA-positive regions were shown to be highest in Fortunella. Similarities between Citrus and Poncirus suggest little heterochromatin diversification among karyotypes of these genera, whereas Fortunella, with higher amounts and more homogenous distribution of heterochromatin, is more divergent.

Chromomycin A3↗
PubMed1997

Sister chromatid exchange (SCE) rates in human melanoma cells as an index of mutagenesis.

Melanomas are highly clonogenic. Genetic variability and polymorphism of tumour cell populations have been reported. However, no direct evidence of mutator activity as a source of genetic polymorphism for melanoma cells has been described. Some intermediates of melanin synthesis are cytotoxic and genotoxic and their mutagenic power has been described. We show here that the rate of sister chromatid exchange (SCE) of the line of human melanoma cells used varies with the concentration of the melanin precursor L-tyrosine, in the culture medium. An increase of melanin synthesis results in increased SCE rates. The highest values of SCEs are found in melanotic melanoma cells compared with the amelanotic ones. Indeed we present evidence that melanoma cells show higher levels of SCE when compared with normal human lymphocytes, and to the SCE frequencies derived from the literature on the lymphocytes of familial malignant melanoma, sporadic malignant melanoma patients and the lymphocytes of relatives of familial and sporadic melanoma patients.

Culture Media↗
PubMed1997

Biochemical basis for glucose-induced inhibition of malolactic fermentation in Leuconostoc oenos.

The sugar-induced inhibition of malolactic fermentation in cell suspensions of Leuconostoc oenos, recently reclassified as Oenococcus oeni (L. M. T. Dicks, F. Dellaglio, and M. D. Collins, Int. J. Syst. Bacteriol. 45:395-397, 1995) was investigated by in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and manometric techniques. At 2 mM, glucose inhibited malolactic fermentation by 50%, and at 5 mM or higher it caused a maximum inhibitory effect of ca. 70%. Galactose, trehalose, maltose, and mannose caused inhibitory effects similar to that observed with glucose, but ribose and 2-deoxyglucose did not affect the rate of malolactic activity. The addition of fructose or citrate completely relieved the glucose-induced inhibition. Glucose was not catabolized by permeabilized cells, and inhibition of malolactic fermentation was not observed under these conditions. 31P NMR analysis of perchloric acid extracts of cells obtained during glucose-malate cometabolism showed high intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate, and glycerol-3-phosphate. Glucose-6-phosphate, 6-phosphogluconate, and NAD(P)H inhibited the malolactic activity in permeabilized cells or cell extracts, whereas NADP+ had no inhibitory effect. The purified malolactic enzyme was strongly inhibited by NADH, whereas all the other above-mentioned metabolites exerted no inhibitory effect, showing that NADH was responsible for the inhibition of malolactic activity in vivo. The concentration of NADH required to inhibit the activity of the malolactic enzyme by 50% was ca. 25 microM. The data provide a coherent biochemical basis to understand the glucose-induced inhibition of malolactic fermentation in L. oenos.

Adenosine Triphosphate↗
PubMed1997

GSH transport in mitochondria: defense against TNF-induced oxidative stress and alcohol-induced defect.

Mitochondria generate reactive oxygen species (ROS) as byproducts of molecular oxygen consumption in the electron transport chain. Most cellular oxygen is consumed in the cytochrome-c oxidase complex of the respiratory chain, which does not generate reactive species. The ubiquinone pool of complex III of respiration is the major site within the respiratory chain that generates superoxide anion as a result of a single electron transfer to molecular oxygen. Superoxide anion and hydrogen peroxide, derived from the former by superoxide dismutase, are precursor of hydroxyl radical through the participation of transition metals. Glutathione (GSH) in mitochondria is the only defense available to metabolize hydrogen peroxide. A small fraction of the total cellular GSH pool is sequestered in mitochondria by the action of a carrier that transports GSH from the cytosol to the mitochondrial matrix. Mitochondria are not only one of the main cellular sources of ROS, they also are a key target of ROS. Mitochondria are subcellular targets of cytokines, especially tumor necrosis factor (TNF); depletion of GSH in this organelle renders the cell more susceptible to oxidative stress originating in mitochondria. Ceramide generated during TNF signaling leads to increased production of ROS in mitochondria. Chronic ethanol-fed hepatocytes are selectively depleted of GSH in mitochondria due to a defective operation of the carrier responsible for transport of GSH from the cytosol into the mitochondrial matrix. Under these conditions, limitation of the mitochondrial GSH pool represents a critical contributory factor that sensitizes alcoholic hepatocytes to the prooxidant effects of cytokines and prooxidants generated by oxidative metabolism of ethanol. S-adenosyl-L-methionine prevents development of the ethanol-induced defect. The mitochondrial GSH carrier has been functionally expressed in Xenopus laevis oocytes microinjected with mRNA from rat liver. This critical carrier displays functional characteristics distinct from other plasma membrane GSH carriers, such as its ATP dependency, inhibitor specificity, and the size class of mRNA that encode the corresponding carrier, suggesting that the mitochondrial carrier of GSH is a gene product distinct from the plasma membrane transporters.

Animals↗
PubMed1997

Changes in histone synthesis and modification at the beginning of mouse development correlate with the establishment of chromatin mediated repression of transcription.

The transition from a late 1-cell mouse embryo to a 4-cell embryo, the period when zygotic gene expression begins, is accompanied by an increasing ability to repress the activities of promoters and replication origins. Since this repression can be relieved by either butyrate or enhancers, it appears to be mediated through chromatin structure. Here we identify changes in the synthesis and modification of chromatin bound histones that are consistent with this hypothesis. Oocytes, which can repress promoter activity, synthesized a full complement of histones, and histone synthesis up to the early 2-cell stage originated from mRNA inherited from the oocyte. However, while histones H3 and H4 continued to be synthesized in early 1-cell embryos, synthesis of histones H2A, H2B and H1 (proteins required for chromatin condensation) was delayed until the late 1-cell stage, reaching their maximum rate in early 2-cell embryos. Moreover, histone H4 in both 1-cell and 2-cell embryos was predominantly diacetylated (a modification that facilitates transcription). Deacetylation towards the unacetylated and monoacetylated H4 population in fibroblasts began at the late 2-cell to 4-cell stage. Arresting development at the beginning of S-phase in 1-cell embryos prevented both the appearance of chromatin-mediated repression of transcription in paternal pronuclei and synthesis of new histones. These changes correlated with the establishment of chromatin-mediated repression during formation of a 2-cell embryo, and the increase in repression from the 2-cell to 4-cell stage as linker histone H1 accumulates and core histones are deacetylated.

Acetylation↗
PubMed1997

[Stress test in the elderly. Clinical, hemodynamic, metabolic and electrocardiographic variables].

PURPOSE: To identify in the elderly adaptations imposed by exercise in both sexes. METHODS: 1528 stress tests were performed on subjects divided in: group I (GI) (90%) between 65 to 75 years old, and group II (GII) more than 75 years old. Protocols applied were Bruce (72%), and modified Naughton (28%). Clinical, hemodynamic and electrocardiographic variables were estimated as recommended by the World Health Organization, and the metabolic variables in the adapted Naughton protocols by the American College of Sports Medicine standards. RESULTS: Analysis of GI and GII, respectively disclosed: 1) stress electrocardiogram (ECG): normal, 36 and 35%; ST depression, 20 and 22%; ST elevation, 6 and 1%; ventricular ectopic beats, 11 and 14%; supra ventricular ectopic beats, 5 and 6%; 2) metabolic and hemodynamic variables: the double-product: 26636 (+/-1539) and 23133 (+/-3218) mmHg X bpm (p < 0.0001). Maximum oxygen uptake measured in METS: GI, men, 7.7 (+/-1.9), women 5.4 (+/-0.8) (p < 0.0001); GII, NS, curve of systolic blood pressure: GI, men, 8.4 +/- (0.5), women, 10.6 (+/-1.8) mmHg/Met (p = 0.03); GII- NS. Difference of diastolic blood pressure and heart rate during exercise were similar between the two groups; 3) chest pain was the main clinical variable. CONCLUSION: The more frequent indication for stress testing to evaluate chest pain in GI, did not correspond to a predominance of this symptom in this group, during exercise; in GI, in contrast to what is seen in the young, the curve of systolic blood pressure was greater in women; despite the greater prevalence of coronary artery disease in aged subjects, it was not observed significative differences between the two groups, to ischaemic ST depression.

Aged↗
PubMed1996

Uncoupling of transcription and translation during zygotic gene activation in the mouse.

Zygotic gene expression in mice is delayed by a time-dependent mechanism until the two-cell stage in development. To investigate the basis of this 'zygotic clock', the firefly luciferase gene was injected into mouse embryos, and quantitative assays were used to monitor luciferase gene transcription and translation in individual embryos from single mothers. These studies confirmed, at the mRNA level, previous conclusions about the relative capacities of paternal and maternal pronuclei to transcribe genes, and the requirements for promoters and enhancers during zygotic gene activation. Furthermore, these studies revealed that fertilized mouse eggs can delay expression of zygotic genes by uncoupling translation from transcription. An RNA polymerase II-dependent gene could be translated until zygotic gene expression began (a delay of up to 15 h after injection). The time course for nascent mRNA accumulation was biphasic, with the second phase occurring during zygotic gene expression. If the luciferase gene was injected after zygotic gene expression had begun, then translation was tightly linked to transcription. If the second phase of mRNA accumulation was repressed, then luciferase was not produced. Therefore, translation was linked to the accumulation of mRNA during the onset of zygotic gene expression. Similar biphasic time courses also were observed for RNA polymerase I- and III-dependent transcription. These and other results reveal that the zygotic clock regulates the onset of both transcription and translation of zygotic genes.

Amanitins↗
PubMed1996

[The molecular pathology of RET protooncogene in families with multiple endocrine neoplasia type 2A].

BACKGROUND: Multiple endocrine neoplasm type 2A (MEN 2A) is an autosomal dominantly inherited disease characterized by medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism. Mutations have been identified in the extracellular domain of the RET proto-oncogen product (10q11.2) in MEN 2A patients. In each case a single base pair substitution results in replacement of cysteine with another amino acid. Most MEN 2A patients have mutations of codon 634. PATIENTS AND METHODS: Sixty-five unrelated MEN 2A patients from seven families were studied. Polymerase chain reaction, segregation, sequence analysis and restriction enzyme digestion were performed. RESULTS: Of seven families, four had the TGC to TAC transition, two families the TGC to TGG transversion and one family the TGC to CGC transition in codon 634 of RET. CONCLUSIONS: We found all the mutations in codon 634. The characterization of MEN 2A mutations allows early and presymptomatic diagnosis in this syndrome.

Base Sequence↗
PubMed1996

The dinuclear copper site structure of Agaricus bisporus tyrosinase in solution probed by X-ray absorption spectroscopy.

We have measured the x-ray absorption near edge structure (XANES) spectra of the enzyme tyrosinase from the mushroom Agaricus bisporus in solution in the oxy and deoxy forms. The spectra, obtained under the same conditions as the analogous forms of mollusc hemocyanin (Hc), show that the oxidation state of copper changes from Cu(II) (oxy form) to Cu(I) (deoxy form), and the copper active site(s) of A. bisporus tyrosinase in solution undergoes the same main conformational changes as Hc. We have applied the multiple scattering theory to simulate the XANES spectra of various alternative geometries of the copper site, accounting for the residual differences between Hc and tyrosinase. While oxy-Hc is reasonably fitted only by the pseudo-square-pyramidal geometry reported by its crystallographic data, oxytyrosinase can be fitted, starting from the Hc coordinates, either by distortions toward a pseudo-tetrahedral geometry, with inequivalent copper sites, or by an apically distorted square-pyramidal geometry (with an elongation of the apical distance of no more than 0.2 A).

Agaricus↗
PubMed1996

Alteration of glutathione transferase subunits composition in the liver of young and aged rats submitted to hypoxic and hyperoxic conditions.

In the present work, we have studied glutathione transferase (GST) activity and GST subunits distribution in the liver of young and aged rats kept under hypoxic or hyperoxic normobaric conditions as model of oxidative stress. A significant decrease of GST activity was detected in young hypoxic rat liver, whereas a significant increase occurred in aged hypoxic liver. No significant alteration of activity was obtained in both young and aged rat livers subjected to hyperoxic treatment. Substrate specificity measurements, SDS/PAGE analysis and reverse-phase HPLC, of GSH-affinity purified fractions were used to study the changes in the GST subunits pattern occurring in the liver of rat as a consequence of hypoxic and hyperoxic treatment. The results demonstrate that young and aged rat liver has a different constitutive GST subunit pattern which are markedly and differentially altered in hypoxia or hyperoxia. The hyperoxic treatment caused an increase of GST subunit 3 in aged, but not in young liver. In aged liver, both the hypoxic and hyperoxic treatment produced a decrease of GST subunit 4. After hypoxic treatment GST subunit 3 significantly increased in both young and aged liver. GST subunit 1a increased in both young and adult liver after hyperoxia. Following hypoxia a decrease of subunit 1a was seen in both young and aged liver. After hypoxic treatment, subunit 6 doubled in young, but not in aged, livers. It was concluded that the alterations in GST subunit expression occurring in the liver as a consequence of hypoxic or hyperoxic treatment respond to the necessity of a better protection of liver against the products of oxidative metabolism.

Aging↗
PubMed1996

Indoor mite allergens in patients with respiratory allergy living in Porto, Portugal.

We investigated the levels of mite allergens (Der p 1, Der f 1, Der 2, and Lep d 1) in dust samples from the homes of 59 patients with asthma, 36 sensitized to house-dust mites (HDM) and 23 to grass pollen (controls), living in Porto, northern Portugal. The relationship between exposure and sensitization to HDM and the influence of housing conditions on mite-allergen levels were also evaluated. Der p 1 (median 9.2 micrograms/g) and Der 2 (4.6 micrograms/g) were the main allergens, while Der f 1 and Lep d 1 levels were always < 1 microgram/g dust and undetectable in 11% and 47% of samples, respectively. All HDM-sensitized asthmatics were exposed to Der p 1 levels > 2 micrograms/g and their homes contained significantly higher levels of Der p 1 (median 12.5 vs 6.4 micrograms/g; P = 0.008) and Der 2 (6.2 vs 3.0 micrograms/g; P = 0.004) when compared to the control group. A significant correlation was observed between the exposure to Der p 1 and the wheal area at skin testing with the Dermatophagoides pteronyssinus (Dp) extract (P = 0.01) as well as with serum specific IgE levels to Dp (P = 0.03). Patients with higher levels of serum specific IgE (> or = 17.5 HRU/ml) were also more frequently exposed to Der p 1 levels > or = 10 micrograms/g (P = 0.002). Old homes, presence of carpets, and signs of dampness were conditions associated with significantly higher levels of mite allergens. In conclusion, we found high levels of Der p 1 and Der 2 particularly in the homes of HDM-sensitized patients and we confirm the relationship between exposure and sensitization to HDM, assessed by both in vivo and in vitro methods. In additional to a favorable outdoor climate, we found in our region housing conditions propitious to mite growth, suggesting that specific geographic characteristics must also be taken into account for the correct planning of mite-avoidance measures.

Adolescent↗
PubMed1996

[Orthostatic tremor as a cause of difficulty in standing still].

Orthostatic tremor is characterized by a fine tremor of lower limb muscles that produces instability while standing still and alleviates on walking or sitting. We report two patients, aged 54 and 72 years old, in whom the tremor caused falls. The clinical features, a negative neurological examination, the alleviation on walking or sitting and the good response to clonazepam allowed the diagnosis. This disease should be considered in the differential diagnosis of standing still instability.

Aged↗
PubMed1995

Nucleotide sequence and chromosomal localization of the gene encoding the Old Yellow Enzyme from Kluyveromyces lactis.

A 6.6 kb genomic DNA fragment from the yeast Kluyveromyces lactis was isolated. Sequence analysis of this fragment revealed the presence of two incomplete open reading frames (ORFs) in one strand, one coding for the carboxyl terminus of the plasma membrane H(+)-ATPase and the other for the amino terminus of an unidentified product. In the complementary strand, a full-length ORF which encodes for a protein homologous to the yeast NADPH-dependent Old Yellow Enzyme was found. The deduced amino acid sequence of this ORF predicts a protein of 398 residues with 84% similarity in its full length to OYE1 from Saccharomyces carlsbergensis and OYE2 from Saccharomyces cerevisiae. In addition, an internal region showed considerable similarity to the bile acid-inducible polypeptide from Eubacterium sp., to the NADH oxidase from Thermoanaerobium brockii, to the trimethylamino dehydrogenase from bacterium W3A1 and to the estrogen-binding protein from Candida albicans, suggesting a functional or structural relationship between them. Inactivation of the KYE1 (Kluyveromyces Yellow Enzyme) gene by deletion of 0.6 kb fragment between positions +358 and +936 produced viable cells with a slight increase in their generation time. Haploid cells carrying the disrupted allele showed one-third of the NADPH oxidase activity, compared to wild-type cells. Southern blotting analysis of digested DNA and chromosomes separated by contour-clamped homogeneous electric field electrophoresis from K. lactis indicated that this is a single-copy gene and it is localized on chromosome II, whose molecular size has been estimated to be approximately 1.3 Mb.

Amino Acid Sequence↗
PubMed1995

Synthesis and anti-HIV-1 activity of 4,5,6,7-tetrahydro-5-methylimidazo [4,5,1-jk][1,4]benzodiazepin-2(1H)-one (TIBO) derivatives. 3.

4,5,6,7-Tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2 (1H)-ones (TIBO), 1, have been shown to significantly inhibit HIV-1 replication in vitro by interfering with the virus's reverse transcriptase enzyme. They have also demonstrated potential clinical efficacy in combating HIV-1, on the basis of a preliminary study. Our prior publications have discussed the discovery of this series of compounds and reported some preliminary chemical and biological studies around N-6 substitutions and 5-membered ring variations of 1. This manuscript describes our synthetic endeavors around 4, 5, and 7 mono- and disubstitutions of 1 and discusses related HIV-1 inhibitory structure-activity relationships. On the basis of inhibition of HIV-1's cytopathic effects in MT-4 cells, we found that 5-mono-Me-substituted analogues, the original substitution in the early lead compounds, and 7-mono-Me-substituted analogues of 1 were comparable as being consistently the most active compounds. Although generally less active, the 4,5,7-unsubstituted, 4-mono-substituted, cis- and trans-5,7-di-Me-substituted, and cis-4,5-di-Me-substituted analogues of 1 also exhibited some significant desired activity. The remaining trans-4,5-di-Me-substituted, cis- and trans-4,7-di-Me-substituted, and all 4,5-, 5,6-, 6,7-, and 7,8-fused disubstituted analogues of 1 possessed no noticeable desired activity.

Antiviral Agents↗
PubMed1995

Synthesis and anti-HIV-1 activity of 4,5,6,7-tetrahydro-5-methylimidazo-[4,5,1-jk][1,4]benzodiazepin- 2(1H)-one (TlBO) derivatives. 4.

In previous papers, we have described the discovery of a new series of compounds, 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)- ones, TlBO (1 and 1a), with potent anti-HIV-1 activity and the synthesis of analogues to better define the structure-activity relationships (SAR) in terms of changes in substituents at the N-6 position and variations of the five-membered urea ring as well as the seven-membered diazepine ring. This paper describes the synthesis of TlBO analogues with various substitutents on the aromatic ring and their SAR in terms of anti-HIV-1 properties. Substituents on the 8-position furnished the most rewarding results and gave a large improvement in potency versus the parent compound. These included halogen, thiomethyl, and methyl. Analogues like 8-cyano, -methoxy, and -acetylene were equipotent, while 8-amino, -acetylamino, -dimethylamino, and -nitro were inactive (Table 1). Substituents at the 9-position tended to have little effect on activity, and 10-substituents decreased activity. The 8-chloro compound 6a with IC50 = 0.0043 microM is currently under clinical development.

Antiviral Agents↗
PubMed1995

Repression of gene expression at the beginning of mouse development.

The transition from maternal to zygotic gene expression in the mouse occurs in the 2-cell embryo. Previous studies in which DNA was injected into 2-cell embryos revealed that transcription promoters and origins of DNA replication are strongly repressed in cleavage stage embryos unless linked to an embryo-responsive enhancer. Repression also occurs when DNA is injected into the paternal pronucleus of a 1-cell embryo and the injected embryo subsequently undergoes mitosis, except that repression is no longer relieved by enhancers. Here we extend this observation to maternal pronuclei in 1-cell embryos and show that this repression could not be relieved either by linking the promoter to an embryo-responsive enhancer or by inducing hyperacetylation of chromatin inorder to increase its accessibility to transcription factors. However, repression could be relieved by transplanting the injected pronucleus to a 2-cell embryo, even when the recipient cell subsequently underwent mitosis. Both the extent of promoter repression and the ability of enhancers to stimulate promoter activity increased as development proceeded from the early 2-cell stage to the 4-cell stage. Once repression was established in late 2-cell embryos, transplanting an injected 2-cell embryo nucleus back to an early 1-cell embryo failed to restore activity to the injected promoter, even when it was linked to an enhancer. These and other data demonstrate that cytoplasmic factors appear during formation of a 2-cell embryo that can repress promoter activity and activate enhancer activity. These factors are absent from the paternal pronucleus and cytoplasm of early (S-phase arrested) 1-cell embryos. Moreover, the cytoplasm of early 1-cell embryos appears to lack the ability to reprogram expression of genes once they have progressed to the late 2-cell stage in mouse development.

Animals↗