Carotenoids as protective response against oxidative damage in Dunaliella bardawil

In the present work the relation between carotenoids production and cell response mechanisms to oxidative damage was studied. High light intensity and nitrogen starvation, both conditions, which may increase the oxidative damage in microalgae, significantly increased total carotenoids content in Dunaliella bardawil, the effect of N-starvation being more noticeable when acting synergetically with light on carotenoid production. S-starvation stimulated carotenoids production as much as N-starvation. The use of norflurazon, inhibitor of phytoene desaturase that blocks formation of epsilon-carotene from phytoene, caused a decrease of carotenoid content down to 5% that of the control cells incubated without the inhibitor. The decrease in the oxygen consumption rate of D. bardawil cells exposed to norflurazon suggests a connection between carotenoids desaturation and chloroplastic oxygen species dissipation processes reported in the literature for other algae. It is an indication of the carotenoids involvement in chloroplastic response mechanisms to oxidative damage.     

Molecular cloning and characterization of a sodium-pump ATPase of the moss Physcomitrella patens

Physcomitrella patens grew slowly at 600 mm Na+, pH 6.0, affected by the low water potential but without signs of suffering Na+ toxicity. At pH 8.0, tolerance seemed to be lower but it grew at 200 mm Na+, again without signs of Na+ toxicity. The resistance of Physcomitrella cells to the toxic effects of Na+ can be accounted for by their capacity to keep high K+:Na+ ratios and to extrude Na+ by a system that is not dependent on DeltapH. Physcomitrella expresses two P-type ATPases similar in sequence to fungal ENA-type Na+-ATPases. A functional study in yeast demonstrated that one of these ATPases, PpENA1, is an Na+-pump. We also found that P. patens has a plant-type SOS1 Na+/H+ antiporter. We discuss that Na+-ATPases existed in early land plants but that they were lost during the evolution of bryophytes to flowering plants.     

Alpha4beta1 integrin/ligand interaction inhibits alpha5beta1-induced stress fibers and focal adhesions via down-regulation of RhoA and induces melanoma cell migration

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4-5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4-5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10+FNIII4-5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.     

A redescription of Cryptosporidium galli Pavlasek, 1999 (Apicomplexa: Cryptosporidiidae) from birds

Cryptosporidium galli Pavlasek, 1999, described from the feces of birds, is redescribed with additional molecular and biological data. Oocysts are ellipsoidal, are passed fully sporulated, lack sporocysts, and measure 8.25 x 6.3 microm (range 8.0-8.5 x 6.2-6.4 microm) with a length-width ratio of 1.30 (n = 50). Oocysts are structurally similar to those of Cryptosporidium baileyi described from chickens, but in addition to being considerably larger than oocysts of C. baileyi, these oocysts infect the proventriculus in a variety of birds and not the respiratory tract. Oocysts were successfully transmitted from chickens to chickens, and morphologically similar oocysts also were observed in a variety of exotic and wild birds (Order Passeriformes, Phasianidae, Fringillidae, and Icteridae). Molecular and phylogenetic analyses at the 18S rRNA, HSP70, and actin gene loci demonstrate that this species is genetically distinct from all known species and genotypes of Cryptosporidium and, thus, was named C. galli.     

A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity

Staining bacteria with esterified fluorogenic substrates followed by flow cytometric analysis offers a means for rapid detection of metabolically active bacteria. Flow cytometry (FCM) was used to assess carboxyfluorescein diacetate (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) as indicators of bacterial activity for cultured bacteria, including Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and bacteria from environmental waters. In theory, CFDA/SE should be a better indicator of metabolic bacterial activity compared to CFDA due to greater intracellular retention of the fluorescent product. Qualitative and quantitative analysis of exponential phase cultures, mixtures of active and inactive cells and bacteria from environmental waters revealed CFDA was successful in detecting active bacteria, whereas CFDA/SE was not. CFDA/SE labelled inactive cells with intensities equal to that of the active population and could not even discriminate between bacteria in exponential phase growth and a fixed cell preparation. We propose that the specific mode of action of the succinimidyl ester (SE) group in combination with the nonenzymatic aqueous hydrolysis of the CFDA moiety results in the nonspecific labelling of all cells, irrespective of their metabolic state. This study shows that CFDA/SE is a poor marker of bacterial activity.     

Chromosomal Location of 46 New RAPD Markers in Rye (Secale Cereale L.)

The polymerase chain reaction (PCR) was used to locate RAPD markers using disomic wheat–rye addition lines in order to develop a set of molecular markers distributed on the seven rye chromosomes. We carried out RAPD amplifications on genomic DNA of wheat Chinese Spring (CS), rye Imperial (I), the amphiploid wheat–rye and the seven disomic wheat–rye addition lines (1R–7R) using 140 different 10-mer oligonucleotides. Forty six new RAPD markers were located on the seven rye chromosomes and all the disomic wheat–rye addition lines were identified on the basis of their amplification patterns. The number of RAPD bands located on 1R, 2R, 3R, 4R, 5R, 6R and 7R chromosomes were 5, 8, 11, 8, 8, 10 and 6, respectively. The seven wheat–rye addition lines can be distinguished using only the following three 10-mer oligonucleotides: OPA16, OPF19 and GEN3-605, the other RAPD primers being useful for this purpose. The use of these RAPDs as a source of molecular markers that could be linked to interesting genes or other important agronomic traits is discussed.     

Control of the annual testicular cycle of the marbled-newt by p53, p21, and Rb gene products

p53, p21, and Rb are proteins with an important role in cell-cycle control. The expression and distribution of these gene products and the apoptotic rate were studied in the marbled-newt testis along the annual cycle to know the role of these factors in the control of spermatogenesis and glandular tissue formation. The study was carried out using Western blot analysis and immunohistochemistry. The results differed from those, previously reported in mammals showing constant spermatogenesis. Greater expression of p53 and p21 was found in the quiescence period and was detected in PCGs (primordial germ cells), spermatogonia, follicular, interstitial cells, and glandular tissue. Greater expression of Rb and phosph-Rb was present in the proliferation period, in PCGs, and spermatogonia. Apoptosis was only detected in secondary spermatogonia (quiescence and spermiogenesis periods) and primary spermatocytes (proliferation and spermiogenesis periods). In the quiescence period, the increase in p53 expression activates p21 expression, which inhibits Rb phosphorylation and arrests the cell cycle in G1. In the proliferation period and, in a lesser degree, in the spermiogenesis period, the expressions of p53 and p21 decrease and phosph-Rb increases, enhancing cell proliferation. These gene products do not seem to be related to apoptosis.     

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.     

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.     

Role of the endocannabinoid system in Alzheimer's disease: new perspectives

The role of the endocannabinoid system in several diseases is currently under intense study. Among these, Alzheimer's disease may be a new promising area of research. We have recently reported the existence of profound changes in the location and density of several elements of this system in Alzheimer's disease tissue samples, indicating that a non-neuronal endocannabinoid system is up-regulated in activated glia. Additional data from other groups suggest that glial cells may be important elements in the regulation of endocannabinoid system activity, both in health as in disease. Some of these aspects are briefly discussed in the present review.