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P Gonzalez

Publications and source records attributed to P Gonzalez.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns.

The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division.

Base Sequence↗
PubMed1999

Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita.

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5' region) and a gene for tRNA(Asn) (3' region), and is separated from these genes by two A + T-rich intergenic regions of 1048 (5' region) and 3864 (3' region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3'-->5' exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol gamma in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol gamma from yeast to human.

Agaricales↗
PubMed1999

Evolution of sea urchin retroviral-like (SURL) elements: evidence from 40 echinoid species.

We conducted a phylogenetic survey of sea urchin retroviral-like (SURL) retrotransposable elements in 33 species of the class Echinoidea (sea urchins, sand dollars, and heart urchins). A 263-bp fragment from the coding region of the reverse transcriptase (RT) gene was amplified, cloned, and sequenced. Phylogenetic relationships of the elements isolated from independent clones, along with those from seven additional echinoid species obtained earlier by Springer et al., were compared with host phylogeny. Vertical transmission and the presence of paralogous sequences that diverged prior to host speciation can explain most of the phylogenetic relationships among SURL elements. Rates of evolution were estimated from cases in which SURL and host phylogenies were concordant. In agreement with conclusions reached previously by Springer et al., average rates of synonymous substitution were comparable with those of single-copy sea urchin DNA. High ratios of synonymous to nonsynonymous substitution suggest that the RT of the elements is under strong purifying selection. However, a high proportion (approximately 15%) of elements with deleterious frameshifts and stop codons and an increase of the ratio of synonymous to nonsynonymous substitutions with divergence time show that in the short term this selection is relaxed. Despite the predominance of vertical transmission, sequence similarity of 83%-94% for SURL elements from hosts that have been separated for 200 Myr suggests four cases of apparent horizontal transfer between the ancestors of the extant echinoid species. In three additional cases, elements with identical RT sequences were found in sea urchin species separated for a minimum of 3 Myr. Thus, horizontal transfer plays a role in the evolution of this retrotransposon family.

Animals↗
PubMed1999

Identification and isolation of differentially expressed genes from very small tissue samples.

Identification of differentially expressed genes from tissue samples weighing only a few milligrams has remained a major challenge. Here, we describe a novel and simple strategy that uses standard molecular biology equipment and commercially available kits. The approach combines isolation of total RNA by silica-gel binding, reverse transcription using anchored modified, 5' end enhancers oligonucleotides, exponential amplification of the single-stranded cDNA and hybridization to high-density cDNA filter arrays. The method was tested by comparing genes expressed on freshly isolated human trabecular meshwork tissue with those expressed in corresponding primary cells at third passage. Validation was achieved by using two biological properties: (i) hybridization, to identify the differentially expressed genes, and (ii) PCR amplification, to confirm their distinct expression. The strategy presented allows the identification of differentially expressed genes and/or uncharacterized expressed sequence tags (ESTs) in very small tissue samples, including those from clinical specimens.

Biotechnology↗
PubMed1999

Current applications and perspectives of diagnostic nuclear medicine in oncology. Task Group of Oncology.

The characteristic of nuclear medicine is that it gives images of organs, structures and physiological or pathological processes, detecting the distribution of several radio-pharmaceuticals according to their uptake and metabolism. Its imaging can provide morphological information while at the same time containing data on cellular activity and functions. Such molecular imaging fulfils the modern orientations of oncology, where there is a need to define the presence of a malignancy in the earliest and most effective way, to characterise the neoplasm in terms of biological characteristics (e. g., proliferation, aggressiveness, differentiation, receptor status) and to obtain fundamental issues in patient management such as evaluation of disease extent, monitoring of therapies and study of treatment-induced side effects. The aim of this position paper is to discuss the main indications of nuclear medicine in diagnostic oncology reporting the most recent developments in nuclear medicine, and an extensive list of the major indications for nuclear medicine procedures. The techniques have been labelled as when considered essential in the diagnostic process, when they can give useful additional or information in combination with other instrumental diagnostic approaches, and alternative when they may be performed instead of other tests. These indications were derived by a general consensus within the Task Group of Oncology of the World Federation of Nuclear Medicine and Biology, and it should be stressed that only the current role of nuclear medicine was considered. The Task Group does not claim to have covered the entire range of nuclear medicine indications; in fact it was agreed to include only the most widely used techniques. The highly specific or very exceptionally used applications, or those still in development or under evaluation in clinical trials were not taken into consideration. The conclusion is that the current relationship between nuclear medicine and oncology can be defined not only as satisfactory but also as extremely promising, as nuclear medicine has the potential to compete with the most advanced radiological techniques of imaging, perhaps not so much in terms of sensitivity but more so in terms of specificity and biological characterisation. Several novel diagnostic procedures are able to solve clinical problems for which traditional radiology has shown clear limitations. Nuclear medicine remains today a dynamic medical specialty because it includes a combination of advances in basic science research, technology, cell biology and medical practice and it cannot be excluded that the new lines of research both towards new radiopharmaceuticals and advanced instrumentation will offer in the future new stimulating opportunities.

Forecasting↗
PubMed1998

Citrate uptake into tonoplast vesicles from acid lime (Citrus aurantifolia) juice cells.

Citrate transport into the vacuoles of acid lime juice cells was investigated using isolated tonoplast vesicles. ATP stimulated citrate uptake in the presence or in the absence of a Delta mu H+. Energization of the vesicles only by an artificial K+ gradient (establishing an inside-positive Delta psi) also resulted in citrate uptake as was the case of a Delta pH dominated Delta mu H+. Addition of inhibitors to endomembrane ATPases showed no direct correlation between the inhibition to the tonoplast bound H+/ATPase and citrate uptake. The data indicated that, although some citrate uptake can be accounted for by Delta psi and by a direct primary active transport mechanism involving ATP, under in vivo conditions of vacuolar pH of 2.0, citrate uptake is driven by Delta pH.

Adenosine Triphosphate↗
PubMed1998

Molecular analysis of the split cox1 gene from the Basidiomycota Agrocybe aegerita: relationship of its introns with homologous Ascomycota introns and divergence levels from common ancestral copies.

The Basidiomycota Agrocybe aegerita (Aa) mitochondrial cox1 gene (6790 nucleotides), encoding a protein of 527aa (58377Da), is split by four large subgroup IB introns possessing site-specific endonucleases assumed to be involved in intron mobility. When compared to other fungal COX1 proteins, the Aa protein is closely related to the COX1 one of the Basidiomycota Schizophyllum commune (Sc). This clade reveals a relationship with the studied Ascomycota ones, with the exception of Schizosaccharomyces pombe (Sp) which ranges in an out-group position compared with both higher fungi divisions. When comparison is extended to other kingdoms, fungal COX1 sequences are found to be more related to algae and plant ones (more than 57.5% aa similarity) than to animal sequences (53.6% aa similarity), contrasting with the previously established close relationship between fungi and animals, based on comparisons of nuclear genes. The four Aa cox1 introns are homologous to Ascomycota or algae cox1 introns sharing the same location within the exonic sequences. The percentages of identity of the intronic nucleotide sequences suggest a possible acquisition by lateral transfers of ancestral copies or of their derived sequences. These identities extend over the whole intronic sequences, arguing in favor of a transfer of the complete intron rather than a transfer limited to the encoded ORF. The intron i4 shares 74% of identity, at the nucleotidic level, with the Podospora anserina (Pa) intron i14, and up to 90.5% of aa similarity between the encoded proteins, i.e. the highest values reported to date between introns of two phylogenetically distant species. This low divergence argues for a recent lateral transfer between the two species. On the contrary, the low sequence identities (below 36%) observed between Aa i1 and the homologous Sp i1 or Prototheca wickeramii (Pw) i1 suggest a long evolution time after the separation of these sequences. The introns i2 and i3 possessed intermediate percentages of identity with their homologous Ascomycota introns. This is the first report of the complete nucleotide sequence and molecular organization of a mitochondrial cox1 gene of any member of the Basidiomycota division.

Animals↗
PubMed1998

Cholestasis and alterations of glutathione metabolism induced by tacrolimus (FK506) in the rat.

BACKGROUND: Tacrolimus (FK506) is an immunosuppressive agent used for the prevention of allograft rejection after organ transplantation. The aim of this study was to investigate the effects of chronic tacrolimus treatment on bile secretion in rats. METHODS: Tacrolimus was administered intraperitoneally at doses of 0.2, 0.5, and 0.8 mg/kg/day for 6 weeks. RESULTS: Bile flow was significantly reduced at doses of 0.5 mg/kg and 0.8 mg/kg (-25% and -32%, respectively). Bile acid secretion was not significantly modified, but bicarbonate secretion decreased at doses of 0.5 mg/kg and 0.8 mg/kg (-23% and -29%, respectively). Glutathione secretion was significantly reduced at doses of 0.5 mg/kg (-29%) and 0.8 mg/kg (-49%). Liver glutathione concentration was reduced at the higher dose (-17%). Liver gamma-glutamyl-cysteinyl synthetase activity was elevated (+22%, +10, and +15%) and gamma-glutamyl transpeptidase activity was reduced (-18%, -40%, and -25%) at all doses. Dichlorofluorescein and thiobarbituric acid-reactive substance concentrations were not significantly modified. Liver glutathione peroxidase activity increased at doses of 0.5 mg/kg (+65%) and 0.8 mg/kg (+56%). Kidney concentration of thiobarbituric acid-reactive substances was significantly increased at doses of 0.5 mg/kg (+17%) and 0.8 mg/kg (+12%). CONCLUSIONS: Our data indicate that tacrolimus at high doses induces cholestasis by inhibiting primarily biliary excretion of glutathione and, to a lesser extent, bicarbonate. The decrease in biliary glutathione secretion is not due to a lower synthesis or degradation and could be related to its increased sinusoidal efflux.

Animals↗
PubMed1998

Subsolubilizing alterations caused by alkyl glucosides in phosphatidylcholine liposomes.

The subsolubilizing alterations caused by a series of alkyl glucosides (alkyl chain lengths ranging from C8 to C12) in unilamellar phosphatidylcholine (PC) liposomes were investigated. The surfactant to phospholipid molar ratios (RE) and the normalized bilayer/aqueous phase partition coefficients (K) were determined by monitoring the increase of the fluorescence intensity of liposome suspensions due to the 5(6)-carboxyfluorescein (CF) released from the interior of vesicles to the bulk aqueous phase. Given that the free surfactant concentrations was always lower than the critical micelle concentration (CMC) of the surfactant tested we may assume that the surfactant-liposome interactions were mainly ruled by the action of surfactant monomers. In general terms, the decrease in the surfactant alkyl chain length (or the rise in the surfactant CMC) resulted in an increase in the ability of these surfactants to alter the permeability of liposomes and, inversely, in an abrupt decrease in their affinity with these bilayers structures. The overall balance of these opposite tendencies shows that at the two interaction levels studied (50 and 100% of CF release) the nonyl and the octyl glucoside showed, respectively, the highest ability to alter the release of the CF trapped in bilayers (lowest RE values), whereas the dodecyl glucoside showed the highest degree of partitioning into liposomes or affinity with these bilayer structures (highest K values).

Glucosides↗
PubMed1998

Fru 2,6-P2 and citrate: intracellular distribution in citrus tissues and effect on grapefruit leaf PFP.

Grapefruit leaf PFP was studied for its activation by fructose 2,6-bisphosphate (Fru 2,6-P2) in the forward and reverse reactions. In the glycolytic reaction, a Ka of 4.0 +/- 0.12 nM was obtained. This constant is affected by the presence of increasing concentrations of citrate (1, 5 and 20 nM) with a Ka(citrate) of 4.5 +/- 0.09, 6.9 +/- 0.05 and 38.2 +/- 1.4 respectively. The inhibition mode of citrate is competitive with Fru 2,6-P2, but non-linear in relation of increasing concentrations of the inhibitor. The intracellular distribution and concentration of the key regulatory metabolite Fru 2,6-P2 was further investigated in citrus leaves and juice cells. Fru 2,6-P2 was only found in the cytosol of juice cells. Fru 2,6-P2 was detected under both conditions with higher concentrations found under aerobiosis.

Citric Acid↗
PubMed1998

Phase I trial of a 96 h paclitaxel infusion with filgrastim support in refractory solid tumor patients.

A phase I study of a 96 h paclitaxel infusion with filgrastim support was performed to determine the toxicity, maximum-tolerated dose (MTD) and pharmacokinetics in patients with refractory solid tumors. In this phase I trial, the initial paclitaxel dose was 140 mg/m2/96 h followed by filgrastim (5 microg/kg/day s.c.) beginning 24 h after the paclitaxel and continued until granulocyte recovery. Cycles were repeated every 21 days. Patients with refractory solid tumors were eligible; however, only one previous chemotherapy regimen was allowed. The dose of paclitaxel was escalated by 20 mg/m2/96 h in subsequent cohorts until dose-limiting toxicity (DLT) occurred. Pharmacokinetic analysis was performed by quantitating paclitaxel concentrations at baseline, 24, 48, 72 and 96 h after the start of the paclitaxel infusion. Twenty-one patients were entered into this trial of which 19 were evaluable. A total of 52 treatment cycles were administered. DLT was seen in two of four patients at 200 mg/m2/96 h, and consisted of diarrhea, mucositis and granulocytopenic infection. The MTD of the 96 h paclitaxel infusion was 180 mg/m2 with filgrastim support. Mucosal and granulocyte toxicity were correlated with steady-state paclitaxel concentrations (Css) greater than 0.100 micromol/l. In the presence of liver function test 1.5 times or lower than normal, metastatic liver disease did not alter paclitaxel Css. Objective responses were observed in non-small cell lung cancer, small cell lung cancer and melanoma. The recommended phase II dose of paclitaxel infused over 96 h with filgrastim support is 180 mg/m2. Paclitaxel Css correlate with mucosal and granulocyte toxicity. In the presence of normal enzymatic function, metastatic liver disease does not affect paclitaxel clearance.

Adult↗
PubMed1998

Sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6, and V9 domains reveal highly species-specific variations within the genus Agrocybe.

A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The conservation of the sequences of the V4, V6, and V9 domains of the mitochondrial SSU rRNA within species and the high degree of interspecific variation found in the Agrocybe species studied open the way for these sequences to be used as specific molecular markers of the Basidiomycota.

Base Sequence↗
PubMed1997

Guinea pig and bovine zeta-crystallins have distinct functional characteristics highlighting replacements in otherwise similar structures.

zeta-Crystallin, a major cytosolic protein of guinea pig lens, has been characterized as an NADPH:quinone oxidoreductase (EC 1.6.5.5) [Rao et al. (1992) J. Biol. Chem. 267, 97-103]. A bovine lens homolog with 83% sequence identity was found to have very different functional characteristics. While the bovine lens zeta-crystallin exhibits similar physicochemical properties, such as molecular weight, hydropathy profile, and predicted secondary structure, and exhibits strong immunological cross-reactivity with the guinea pig and human lens zeta-crystallins, it shows minimal quinone oxidoreductase activity. On the other hand, bovine lens zeta-crystallin, but not guinea pig zeta-crystallin, showed a strong binding affinity to single-stranded DNA (ssDNA) that could be competed with NADPH, the specific cofactor of zeta-crystallin. NADH and dextran sulfate did not affect this characteristic of bovine zeta-crystallin and the enzyme showed no binding affinity for the heparin-Ultragel A4R. Two-dimensional electrophoresis of bovine lens zeta-crystallin showed a distinct pattern of posttranslational charge modification as compared to the guinea pig protein. Alignment of eight zeta-crystallin sequences, and computer modelling of the bovine and human forms based on the crystallographically analyzed Escherichia coli form, suggest that if loss of a functional residue accounts for the lowered catalytic activity of the bovine protein, Tyr 52 of the E. coli enzyme, and the equivalent Tyr present in all known mammalian forms except the bovine, is the likely candidate. In the bovine form this tyrosine is replaced by histidine.

Amino Acid Sequence↗
PubMed1997

DNA sequence and secondary structure of the mitochondrial small subunit ribosomal RNA coding region including a group-IC2 intron from the cultivated basidiomycete Agrocybe aegerita.

Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles. However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision. The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5'- and 3'-ends were localized by nuclease SI mapping, leading to a size of 3277 nt. The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C. The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases. The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs. Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3'-part of the SSU rRNA. This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility. This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision.

Amino Acid Sequence↗
PubMed1997

Influence of opiate tolerance and calcium channel antagonists on the antinociceptive effects of L-arginine and NG-nitro L-arginine.

1. The antinociceptive effects induced by L-arginine (L-Arg 300-600 mg sc) or NG-nitro-L-arginine (NOArg 20-70 mg sc) in mice were assessed by the hot-plate test. 2. The antinociception induced by both agents was antagonized by naloxone. L-Arg significantly reduced the effects of the largest doses of morphine (3, 5, and 10 mg/kg) or pentazocine (7.5, 15, and 30 mg/kg). 3. Morphine antagonized L-Arg-induced antinociception but did not change the responses to NOArg. 4. Diltiazem (10 mg/kg) or verapamil (10 mg/kg) decreased L-Arg antinociceptive responses, whereas the effects of NOArg were enhanced. 5. The antinociceptive effects of L-Arg and NOArg were also tested in mice rendered tolerant to morphine or pentazocine. Whereas the effect of L-Arg were lower in tolerant animals, the responses to NOArg were unchanged. 6. The results suggest the involvement of opiate mechanisms and NO synthesis in L-ARG-induced antinociception and a lesser influence of opiate mechanisms in the antinociception induced by NOArg.

Analgesia↗
PubMed1997

Littoral cell angioma in a patient with Epstein syndrome.

A 61-year-old male with Epstein syndrome and chronic renal failure was admitted to our institution for kidney transplantation. He was asymptomatic at the time of evaluation. Physical examination revealed pallor and marked splenomegaly measuring approximately 12 x 10 cm below the left costal margin. Because of this finding, and a history of resected non-small lung cancer, a CT scan of the abdomen was obtained. This demonstrated massive, diffusely nodular splenomegaly. This article describes the histopathologic and immunohistochemical findings of littoral cell angioma, a novel splenic tumor. Also, we discuss the differential diagnosis and include a description of the imaging study performed. To our knowledge, this is the first case report of littoral cell angioma occurring in a patient with Epstein syndrome, and the first radiographic appearance of this rare tumor.

Carcinoma, Non-Small-Cell Lung↗