Involvement of the pyrophosphate and the 2'-phosphate binding regions of ferredoxin-NADP+ reductase in coenzyme specificity

Previous studies indicated that the determinants of coenzyme specificity in ferredoxin-NADP+ reductase (FNR) from Anabaena are situated in the 2'-phosphate (2'-P) NADP+ binding region, and also suggested that other regions must undergo structural rearrangements of the protein backbone during coenzyme binding. Among the residues involved in such specificity could be those located in regions where interaction with the pyrophosphate group of the coenzyme takes place, namely loops 155-160 and 261-268 in Anabaena FNR. In order to learn more about the coenzyme specificity determinants, and to better define the structural basis of coenzyme binding, mutations in the pyrophosphate and 2'-P binding regions of FNR have been introduced. Modification of the pyrophosphate binding region, involving residues Thr-155, Ala-160, and Leu-263, indicates that this region is involved in determining coenzyme specificity and that selected alterations of these positions produce FNR enzymes that are able to bind NAD+. Thus, our results suggest that slightly different structural rearrangements of the backbone chain in the pyrophosphate binding region might determine FNR specificity for the coenzyme. Combined mutations at the 2'-P binding region, involving residues Ser-223, Arg-224, Arg-233, and Tyr-235, in combination with the residues mentioned above in the pyrophosphate binding region have also been carried out in an attempt to increase the FNR affinity for NAD+/H. However, in most cases the analyzed mutants lost the ability for NADP+/H binding and electron transfer, and no major improvements were observed with regard to the efficiency of the reactions with NAD+/H. Therefore, our results confirm that determinants for coenzyme specificity in FNR are also situated in the pyrophosphate binding region and not only in the 2'-P binding region. Such observations also suggest that other regions of the protein, yet to be identified, might also be involved in this process.     

RhoA/ROCK regulation of neuritogenesis via profilin IIa-mediated control of actin stability

Neuritogenesis, the first step of neuronal differentiation, takes place as nascent neurites bud from the immediate postmitotic neuronal soma. Little is known about the mechanisms underlying the dramatic morphological changes that characterize this event. Here, we show that RhoA activity plays a decisive role during neuritogenesis of cultured hippocampal neurons by recruiting and activating its specific kinase ROCK, which, in turn, complexes with profilin IIa. We establish that this previously uncharacterized brain-specific actin-binding protein controls neurite sprouting by modifying actin stability, a function regulated by ROCK-mediated phosphorylation. Furthermore, we determine that this novel cascade is switched on or off by physiological stimuli. We propose that RhoA/ROCK/PIIa-mediated regulation of actin stability, shown to be essential for neuritogenesis, may constitute a central mechanism throughout neuronal differentiation.     

Human leukocyte formin: a novel protein expressed in lymphoid malignancies and associated with Akt

The very large family of Formin proteins is involved in processes such as morphogenesis, embryonic differentiation, cell polarity, and cytokinesis. A novel human gene from the Formin family, denominated human leukocyte formin gene, was cloned. The cDNA of the gene was determined to be 3959bp long with an open reading frame of 3302bp and computational analysis located this gene on chromosome 17, suggesting that it is composed of 27 exons. Northern blot analysis revealed a restricted expression of mRNA in the thymus, spleen, and peripheral blood leukocytes in normal human tissues. Western blot analysis demonstrated that the protein encoded by this gene is overexpressed in lymphoid malignancies; cancer cell lines and peripheral blood leukocyte from chronic lymphocytic leukemia (CLL) patients. Furthermore, the human leukocyte formin protein was observed to associate with Akt, a critical survival regulator in many different cell types.     

An in vitro evaluation of the effects of homocysteine thiolactone on key steps of angiogenesis and tumor invasion

Homocysteine thiolactone is a highly reactive homocysteine derivative that can react easily with proteins. Protein homocysteinylation has been suggested as a possible mechanism underlying the pathological consequences of impaired homocysteine metabolism. Homocysteine inhibits key steps of angiogenesis and tumor invasion. It can be hypothesized that homocysteine thiolactone could mimic the described anti-angiogenic and anti-invasive effects of homocysteine. Therefore, we studied the effects of homocysteine thiolactone on different key steps of angiogenesis and tumor invasion, using model endothelial and tumor cell lines. This study demonstrates that homocysteine thiolactone, in high contrast to homocysteine, is not an anti-angiogenic compound. Furthermore, our results suggest that homocysteine thiolactone could behave as a pro-angiogenic compound.     

Chromatin and histones from Giardia lamblia: a new puzzle in primitive eukaryotes

The three deepest eukaryote lineages in small subunit ribosomal RNA phylogenies are the amitochondriate Microsporidia, Metamonada, and Parabasalia. They are followed by either the Euglenozoa (e.g., Euglena and Trypanosoma) or the Percolozoa as the first mitochondria-containing eukaryotes. Considering the great divergence of histone proteins in protozoa we have extended our studies of histones from Trypanosomes (Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana) to the Metamonada Giardia lamblia, since Giardia is thought to be one of the most primitive eukaryotes. In the present work, the structure of G. lamblia chromatin and the histone content of the soluble chromatin were investigated and compared with that of higher eukaryotes, represented by calf thymus. The chromatin is present as nucleosome filaments which resemble the calf thymus array in that they show a more regular arrangement than those described for Trypanosoma. SDS-polyacrylamide gel electrophoresis and protein characterization revealed that the four core histones described in Giardia are in the same range of divergence with the histones from other lower eukaryotes. In addition, G. lamblia presented an H1 histone with electrophoretic mobility resembling the H1 of higher eukaryotes, in spite of the fact that H1 has a different molecular mass in calf thymus. Giardia also presents a basic protein which was identified as an HU-like DNA-binding protein usually present in eubacteria, indicating a chimaeric composition for the DNA-binding protein set in this species. Finally, the phylogenetic analysis of selected core histone protein sequences place Giardia divergence before Trypanosoma, despite the fact that Trypanosoma branch shows an acceleration in the evolutionary rate pointing to an unusual evolutionary behavior in this lineage.     

Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations

We evaluated freeze-thawing tolerance of heterospermic ram spermatozoa (Pampinta breed) in a base diluent (Tris, citric acid, fructose, egg yolk, glycerol) with the addition of different trehalose concentrations (0-400 mOsm). We chose sperm motility, acrosome integrity and hypo-osmotic swelling test as parameters to evaluate cryopreservation capacity. We obtained the best results for 50 and 100 mOsm trehalose-supplemented extenders, with values (referred to fresh semen values) of 65% for motility, 75% for acrosome integrity and 50% for hypo-osmotic swelling test, while freeze-thawing tolerance diminished significantly for 200 and 400 mOsm of the disaccharide. Fertility values measured at lambing were 47.1 and 44.6% (2 consecutive years), using semen cryopreserved in 100 mOsm trehalose-containing diluent, which is 2.5 times greater than those obtained with the base diluent (18.5 and 14.5%). We conclude that the membrane-protecting disaccharide trehalose confers a greater cryoprotective capacity to the base extender, when added up to 100 mOsm. This action is reflected in the different sperm membranes, the motile activity and in vivo fertility.