A fragment of cDNA containing the xylanase gene (XLN) of Cryptococcus albidus was inserted into the vector pJHS which contains the selectable marker his3. To permit expression of the XLN gene, the xylose reductase promoter XYL1 from Pichia stipitis was introduced in front of the XLN gene. P. stipitis PJH53, a histidine-dependent strain, transformed with this plasmid produced an active extracellular xylanase. The enzyme secreted by P. stipitis had an apparent molecular mass of 50 kDa, which was slightly greater than that of the native 48-kDa xylanase produced by C. albidus. The expression of the XLN gene in P. stipitis allowed it to use xylan as a carbon source and to ferment xylan directly to ethanol under anaerobic conditions.

Base Sequence↗

PubMedDate not supplied

Altered mitochondrial gene expression in human gestational trophoblastic diseases.

To assess the molecular basis of phenotypic alterations present in the gestational trophoblastic diseases (GTDs) and to identify genes whose expression is specifically associated with these placental proliferative disorders we performed differential display (DD) techniques. This strategy resulted in the isolation of four mitochondrial transcripts downregulated in benign, as well as in malignant, trophoblastic diseases encoding the cytochrome oxidase subunit I (COX I), the ATPase subunit 6, the 12S ribosomal RNA (12S rRNA) and the transfer RNA for phenylalanine (tRNA(Phe)). This expression pattern was confirmed by Northern blot in normal early placenta (NEP), complete hydatidiform mole (CHM), persistent gestational trophoblastic disease (PGTD) and the human choriocarcinoma derived cell line JEG-3. Quantification of mitochondrial DNA by dot blot indicated that these changes in expression were not associated with a significant alteration in the number of mitochondrial genome. In addition, a reduction in the mitochondrial transcription factor A (mtTFA) mRNA level was observed in benign as well as in malignant trophoblastic diseases in correlation with the decrease in the mitochondrial transcript levels. Furthermore, Western blot analysis for COX-I showed a close parallelism with the expression level of the cognate RNA. Taken together, these data demonstrate that a significant change in mitochondrial transcription is associated with the phenotypic alteration present in GTDs.

Adenosine Triphosphatases↗

PubMedDate not supplied

Identification of multiple differentially expressed messenger RNAs in normal and pathological trophoblast.

In an attempt to assess the molecular basis of phenotypic alterations present in the gestational trophoblastic diseases (GTDs) and to identify genes whose expression is specifically associated to these placental proliferative disorders we performed differential display techniques. Initially 19 candidate gene fragments were identified and differential expression was confirmed in eight of these fragments by Northern blot analysis. At the mRNA level ribosomal L26 (rL26), ribosomal L27 (rL27), a new Krüppel type zinc finger protein and TIS11d were preferentially expressed in normal early placenta (NEP) relative to complete hydatidiform mole (CHM), persistent gestational trophoblastic disease (PGTD) and choriocarcinoma JEG-3 cell line. In contrast, heterogeneous ribonucleoprotein A1 (hnRNPA1), the ferritin light chain mRNA, and the uncharacterized protein KIAA0992 were predominantly expressed in JEG-3 cell line. Finally, decorin, a prototype member of an expanding family of small leucine-rich proteoglycans, showed high expression in CHM. In addition we demonstrated by immunohistochemistry analysis that increased decorin mRNA in CHM reflected a genuine augmentation in average steady state mRNA levels within cells. Taken together, these findings provide several interesting candidates for regulation of tumorigenic expression as well as early placentation development, including those involved in protein synthesis (rL26 and rL27), metabolism (ferritin light chain), intercellular communication (decorin) and regulation of gene expression (Krüppel-like zinc finger, TIS11d and hnRNPA1). Information about such alterations in gene expression could be useful for elucidating the genetic events associated to gestational trophoblastic pathogenesis, developing new diagnostic markers, or determining novel therapeutic targets.

Adult↗

PubMedDate not supplied

Identification of Trypanosoma cruzi zymodemes by kinetoplast DNA probes.

Analysis of zymograms of extracts of Trypanosoma cruzi isolated from different hosts in Argentina allowed characterization of 12 zymodemes or "isozymic strains," only six of which were found in human patients. Two of these six zymodemes (Z1 and Z12) were widely distributed and found in more than 80% of human patients. These two "major natural clones" differed significantly in pathogenic activity. Because the groupings obtained by studying enzymes and kinetoplast DNA (kDNA) were similar, it is possible to identify the zymodeme by analyzing kDNA. A 290-bp fragment was amplified by PCR using primers for the sequences flanking the hypervariable regions of kDNA minicircles. Labeled probes for this fragment, prepared from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified kDNA from parasite stocks, allowing identification of zymodemes.

Adolescent↗

PubMedDate not supplied

Current-induced vasodilation during water iontophoresis (5 min, 0.10 mA) is delayed from current onset and involves aspirin sensitive mechanisms.

Study of the microcirculation by iontophoresis is potentially confounded by any non-specific effects of current application. Laser Doppler flow (LDF, mean +/- SD; arbitrary units; AU) was recorded on the forearms of healthy volunteers during and 20 min following application of 0.10-mA current for 1, 3 and 5 min, using deionised water as a vehicle. Local heating to 44 degrees C was then applied for 24 min to assess maximal vasodilation. Cathodal current applications resulted in delayed and prolonged vasodilation (peak values: 78 +/- 29, 75 +/- 19, 80 +/- 37 AU) whereas anodal peak LDF was 13 +/- 6, 27 +/- 34 and 72 +/- 40 AU for 1-, 3- and 5-min periods of current applications, respectively. From current onset, inflexion points in the responses to 3- and 5-min anodal current applications occurred at 4.5 and 6.5 min, respectively, and at approximately 1.5 min for all cathodal current applications. For 5-min current applications: a preliminary tourniquet ischaemia neither changed the time course nor the amplitude of the response to current application. In this situation, local anaesthesia abolished the current-induced vasodilation. Chronic capsaicin pretreatment decreased the amplitude of the vasodilation. Pretreatment with 500 mg oral aspirin decreased the cathodal vasodilation and abolished the anodal vasodilation, even in the absence of preliminary ischaemia. We conclude that vasodilation to prolonged application of 0.10-mA continuous monopolar current after transient tourniquet ischaemia cannot be exclusively the result of an axon reflex initiated by current onset. This current-induced vasodilation is at least partly dependent on capsaicin-sensitive afferent fibres and relies on aspirin-sensitive mechanisms at both polarities.

Adult↗

PubMedDate not supplied

[Prenatal diagnosis. Better understanding for better performance].

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Amniocentesis↗