Héctor N. Torres

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Aims:  To establish a reliable and rapid protocol to simultaneously obtain high quality DNA from an infected host plant and the infecting pathogen. To develop an accurate and sensitive low‐cost assay for the quantification and in planta monitoring of Phytophthora infestans growth.

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa.

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca2+ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca2+ and 1 µg/ml of the modulator. The stimulatory effect of the Ca2+-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca2+-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.

Improvement of Aroma in Transgenic Potato As a Consequence of Impairing Tuber Browning

Sensory analysis studies are critical in the development of quality enhanced crops, and may be an important component in the public acceptance of genetically modified foods. It has recently been established that odor preferences are shared between humans and mice, suggesting that odor exploration behavior in mice may be used to predict the effect of odors in humans. We have previously found that mice fed diets supplemented with engineered nonbrowning potatoes (-PPO) consumed more potato than mice fed diets supplemented with wild-type potatoes (WT). This prompted us to explore a possible role of potato odor in mice preference for nonbrowning potatoes. Taking advantage of two well established neuroscience paradigms, the “open field test” and the “nose-poking preference test”, we performed experiments where mice exploration behavior was monitored in preference assays on the basis of olfaction alone. No obvious preference was observed towards -PPO or WT lines when fresh potato samples were tested. However, when oxidized samples were tested, mice consistently investigated -PPO potatoes more times and for longer periods than WT potatoes. Congruently, humans discriminated WT from -PPO samples with a considerably better performance when oxidized samples were tested than when fresh samples were tested in blind olfactory experiments. Notably, even though participants ranked all samples with an intermediate level of pleasantness, there was a general consensus that the -PPO samples had a more intense odor and also evoked the sense-impression of a familiar vegetable more often than the WT samples. Taken together, these findings suggest that our previous observations might be influenced, at least in part, by differential odors that are accentuated among the lines once oxidative deterioration takes place. Additionally, our results suggest that nonbrowning potatoes, in addition to their extended shelf life, maintain their odor quality for longer periods of time than WT potatoes. To our knowledge this is the first report on the use of an animal model applied to the sensory analysis of a transgenic crop.

The NADPH-cytochrome P450 reductase family in Trypanosoma cruzi is involved in the sterol biosynthesis pathway

Trypanosoma cruzi flavoproteins TcCPR-A, TcCPR-B and TcCPR-C are members of the NADPH-dependent cytochrome P-450 reductase family expressed in the parasite. Epimastigotes over-expressing TcCPR-B and TcCPR-C showed enhanced ergosterol biosynthesis and increased NADP(+)/NADPH ratio. Transgenic parasites with augmented ergosterol content presented a higher membrane order with a corresponding diminished bulk-phase endocytosis. These results support a significant role for TcCPR-B and TcCPR-C in the sterol biosynthetic pathway and to our knowledge for the first time reveals the participation of more than one CPR in this metabolic route. Notably, TcCPR-B was found in reservosomes while TcCPR-C localised in the endoplasmic reticulum. In addition, we suggest a different role for TcCPR-A, since its over-expression is lethal, displaying cells with an increased DNA content, aberrant morphology and severe ultrastructural alterations.

A novel stimulator of protein phosphorylation in Neurospora crassa

SummaryAn endogenous thermostable activator of Protein kinase III (PKIII) was purified from 100000 × g supernatants of Neurospora crassa mycelial extracts. This 38 000 dalton polypeptide, clearly separable from calmodulin on P-60 gel filtration, specifically stimulated N. crassa PKIII activity on casein or phosvitin “in vitro” phosphorylation.The factor was only present in the initial growth phase of the fungus. The mechanism of PKIII activation and its possible regulatory role are discussed.

Monoclonal antibodies to neurospora adenylate cyclase

A monoclonal antibody against Neurospora soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.