Molecular Cloning and Characterization of a Superoxide Dismutase (sod) Gene in Pneumocystis carinii

This work reports the isolation and characterization of a gene encoding a superoxide dismutase (SOD. EC.1.15.1.1.) from Pneumocystis carinii derived from rat. Sense and antisense oligonucleotides, deduced from SOD amino acid sequences from a wide variety of organisms, allowed amplification of a 669 bp genomic DNA fragment specific to this P. carinii. RACE-PCR was used to obtain the major pan of the complementary DNA; the 5- and 3'-genomic regions were obtained respectively from a Mbo I subgenomic library and from an amplified fragment using oligonucleotides designed from the cDNA sequence. Comparison of genomic and cDNA sequences showed an open reading frame of 660 bp interrupted by seven small introns. The deduced amino acid sequence contained 220 residues. Protein sequence alignment demonstrated the highest homology (50.5% identity. 70.3% similarity) with Saccharomyces cerevisiae manganese-SOD (MnSOD) suggesting that P. carinii SOD belongs to the mitochondrial MnSOD group. A putative targeting peptide found at the 5'-end of the P. carinii SOD sequence also suggested its mitochondrial localization.     

Anticlastogenic effect of Spirulina maxima extract on the micronuclei induced by maleic hydrazide in Tradescantia

The aim of this investigation was to determine if extracts of Spirulina maxima reduce the genotoxic damage induced by maleic hydrazide (MH) using the Tradescantia biosssay. Two types of extracts from the alga were prepared: an aqueous extract with two different concentrations, 100 and 500 mg/ml, and a second one, the extract of a 1% solution of dimethyl sulfoxide (DMSO) which corresponded to 100 mg/ml of the alga. The capacity of MH to induce micronuclei (MN) was initially established by administering 0.005, 0.01, and 0.015 mg/ml of the chemical to the Tradescantia inflorescences, and observing its effect after 24 h.The results of this experiment showed a significant MN increase with the two high concentrations tested, although no dose-response effect was observed. For the anticlastogenic assay, the extracts of Spirulina were applied to the inflorescences alone or immediately before the application of MH (0.01 mg/ml) and the induced MN were observed 24 h later. We found that none of the extracts increased the MN level with respect to the untreated plants; also, that MH more or less doubled the basal micronuclei frequency, and finally, that all tested extracts reduced the genotoxic damage caused by MH. The inhibitory indices obtained for the aqueous extracts (100 and 500 mg/ml) and for the DMSO extract were respectively 59, 85, and 56.3%. These data indicate that Spirulina is an anticlastogenic agent and suggest that it is advisable to extend studies on this matter using other biological models.     

Inhibition of pea ferredoxin-NADP(H) reductase by Zn-ferrocyanide.

Ferredoxin-NADP(H) reductases (FNRs) represent a prototype of enzymes involved in numerous metabolic pathways. We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn2+ (Ki 1.57 microM). Dichlorophenolindophenol diaphorase activity was also inhibited by Zn2+ (Ki 1.80 microM), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions. Escherichia coli FNR was also inhibited by Zn-ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes. The inhibitor behaves in a noncompetitive manner for NADPH and for artificial electron acceptors. Analysis of the oxidation state of the flavin during catalysis in the presence of the inhibitor suggests that the electron-transfer process between NADPH and the flavin is not significantly altered, and that the transfer between the flavin and the second substrate is mainly affected. Zn-ferrocyanide interacts with the reductase, probably increasing the accessibility of the prosthetic group to the solvent. Ferredoxin reduction was also inhibited by Zn-ferrocyanide in a noncompetitive manner, but the observed Ki was about nine times higher than those for the diaphorase reactions. The electron transfer to Anabaena flavodoxin was not affected by Zn-ferrocyanide. Binding of the apoflavodoxin to the reductase was sufficient to overcome the inhibition by Zn-ferrocyanide, suggesting that the interaction of FNRs with their proteinaceous electron partners may induce a conformational change in the reductase that alters or completely prevents the inhibitory effect.     

Transgene elimination in genetically modified dry bean and soybean lines

Transgene elimination is a poorly studied phenomenon in plants. We made genetic and molecular studies of a transgenic dry bean line immune to bean golden mosaic geminivirus and a soybean line. In both lines, the transgenes were stable during the vegetative phase but were eliminated during meiosis. Due to its potential biotechnological value, this transgenic line was micropropagated by grafting and the vegetative copies were studied for more than two years. More than 300 plants of progeny were obtained during this period, demonstrating that the phenomenon of elimination was consistently repeated and offering an opportunity for detailed study of transgene elimination, including the characterization of the integration sites. Cloning and sequencing of the transgenic loci, reciprocal crosses to untransformed plants, genomic DNA blots, and GUS assays were performed in the transgenic lines. Based on the molecular and genetic characterization, possible mechanisms involved in transgene elimination include intrachromosomal recombination, genetic instability resulting from the tissue culture manipulations, and co-elimination of transgenes, triggered by a process of genome defense.     

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1⁺ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1⁺ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1⁺ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.     

FINE STRUCTURE OF BIFLAGELLATE ZOOSPORES OF ASTEROCOCCUS SUPERBUS (TETRASPORALES, CHLOROPHYCEAE), INCLUDING THE ABSOLUTE CONFIGURATION OF THE FLAGELLAR APPARATUS

The ultrastructure of zoospores of Asterococcus superbus (Cienk.) Scherffel was studied to provide ultrastructural data relevant to the systematic position of the genus. Our results demonstrated that the motile cells of A. superbus were similar to those of the tetrasporalean algae, such as Tetraspora sp. and Tetrasporidium javanicum Moebius . The flagellar apparatus of A. superbus had the same clock-wise orientation of basal bodies and the V-shaped alignment of basal bodies as Tetraspora cylindrica (Wahlb.) Ag. and T. lubrica (Roth) Ag., but differed by having rhizoplasts . The motile cells of A. superbus displayed chlamydomonadal ultrastructure, similar to Chlamydomonas reinhardtii Dangeard , including the absolute configuration of the flagellar apparatus. The pyrenoid matrix in A. superbus, however , showed a large lateral invagination occupied by chloroplast stroma, a characteristic that has never been observed in Chlorophyta.