EARLY TOXIC EFFECTS OF ZINC ON PSII OF SYNECHOCYSTIS AQUATILIS F. AQUATILIS (CYANOPHYCEAE)1

Zinc toxicity on photosynthetic activity in cells of Synechocystis aquatilis f. aquatilis Sauvageau was investigated by monitoring Hill activity and fluorescence. The oxygen‐evolving activity decreased to about 80% of the initial value after exposure to 0.1 mM ZnSO₄ for 1 h. The PSII activity was inhibited by 40% in the presence of zinc concentrations ranging from 0.5 to 5.0 mM, suggesting that the metal effect is limited by zinc uptake. The fluorescence capacity (F_max–F/F_max) decreased from 0.57 to 0.35 and 0.20 in Zn‐treated cells for 15 and 60 min, respectively, thus providing evidence for rapid inactivation of electron transport at PSII. Zinc treatment promoted a rapid increase in PSII fluorescence that was counteracted by addition of 1,4‐benzoquinone, indicating that electron transfer at the reducing side of the PSII reaction center is arrested by zinc. Furthermore, a decline in the fluorescence yield could be observed after 1 h of zinc treatment as well as when Zn‐treated cells were excited in presence of 3‐(3′,4′‐dichlorophenyl)‐1,1‐dimethylurea. Under these conditions, zinc did not affect energy transfer from phycobilisomes to PSII, and the gradual quenching of PSII fluorescence may be due to a decrease in electron flow on the donor side of PSII. However, the 20% increase in the minimal fluorescence intensity (F_o) in parallel to the absence of changes in the maximal fluorescence intensity (F_max), observed in the first hour of zinc treatment, could also suggest a metal‐induced decline in the energy transfer from PSII‐chl a antenna to the PSII reaction center.     

Evolution of rDNA FISH patterns in the Fagaceae

The Fagaceae is one of the most important plant families in European forest ecosystems, and it includes several genera distributed in the Northern hemisphere. In this work we studied the genome organization and evolution within the family, by karyotyping and physically mapping rDNA in ten European and Asian species of the genera Fagus, Quercus, and Castanea. All of the species studied had a chromosome number of 2n=2x=24, except for the first report of a single individual of Quercus suber which proved to be triploid (2n=3x=36). The rDNA physical mapping revealed several patterns: the dominant one is present in European and Asian Quercus subgenus Quercus, and in Castanea sativa and Castanea crenata, consisting of two 18S–25S rDNA loci (one subterminal major and one pericentromeric minor) and one 5S rDNA pericentromeric locus. In Fagus sylvatica and in Quercus sessilifolia, different patterns were observed: four terminal 18S–25S rDNA loci and two 5S rDNA pericentromeric loci in the former, and five 18S–25S rDNA loci (three terminal and two intercalary) and one 5S rDNA pericentromeric locus in the latter. In Castanea mollissima a distinct rDNA distribution pattern with two intercalary 18S–25S rDNA loci and two 5S rDNA was found. These findings suggest rDNA loci restructuring during Castanea evolution, and variability of 18S–25S loci between Quercus and Cyclobalanopsis subgenera.     

B cell response during infection with the MAT a and MAT alpha mating types of Cryptococcus neoformans

In the present study, we compared the B cell response of BALB/c and C57Bl/6 mice during Cryptococcus neoformans infection. This response was investigated using virulent serotype D forms of mating types alpha and a (MAT alpha and MAT a). C57Bl/6 mice showed massive (mainly cerebral) infection by both types, while BALB/c were resistant to infection. Some resistance of C57Bl/6 mice was induced by previous immunization with the capsular polysaccharide from MAT alpha. Passive immunization of C57Bl/6 mice with purified antibody (Ab) obtained from capsular polysaccharide-immunized mice also increased resistance to infection. Both mouse strains showed comparable low IgM response to the capsular polysaccharide from MAT alpha, and only C57Bl/6 mice produced IgM to the polysaccharide of MAT a. Comparable levels of different immunoglobulin (Ig) isotypes against capsular components of MAT alpha and MAT a were detected, and the response of C57Bl/6 mice was higher when compared to that of BALB/c mice. FACS analysis indicated an increase in the percentage of a high-granulosity (side-scatter) splenic subpopulation and in the percentage of splenic Gr-1+ cells in infected C57Bl/6 mice. In addition, the percentage of follicular splenic B cells was decreased after C. neoformans infection of C57Bl/6 mice. This response was more pronounced when we investigated infection induced by the MAT a mating type. Taken together, our results indicate that capsular polysaccharide derived from MAT alpha and MAT a types of C. neoformans have a stimulatory effect upon B cells but that there is no correlation between resistance of BALB/c mice and Ab production. However, the increase in resistance of C57Bl/6 mice parallels the production of Abs and a major change in splenic cell populations.     

Quantitation of drug content in a low dosage formulation by transmission near infrared spectroscopy

A transmission near infrared (NIR) spectroscopic method has been developed for the nondestructive determination of drug content in tablets with less than 1% weight of active ingredient per weight of formulation (m/m) drug content. Tablets were manufactured with drug concentrations of ∼0.5%, 0.7%, and 1.0% (m/m) and ranging in drug content from 0.71 to 2.51 mg per tablet. Transmission NIR spectra were obtained for 110 tablets that constituted the training set for the calibration model developed with partial least squares regression. The reference method for the calibration model was a validated UV spectrophotometric method. Several data preprocessing methods were used to reduce the effect of scattering on the NIR spectra and base the calibration model on spectral changes related to the drug concentration changes. The final calibration model included the spectral range from 11 216 to 8662 cm⁻¹ the standard normal variate (SNV), and first derivative spectral pretreatments. This model was used to predict an independent set of 48 tablets with a root mean standard error of prediction (RMSEP) of 0.14 mg, and a bias of only −0.05 mg per tablet. The study showed that transmission NIR spectroscopy is a viable alternative for nondestructive testing of low drug content tablets, available for the analysis of large numbers of tablets during process development and as a tool to detect drug agglomeration and evaluate process improvement efforts. Published: March 24, 2006     

Calpain-mediated proteolysis of talin regulates adhesion dynamics

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.     

Biodiversity and Land uses at a regional scale: Is agriculture the biggest threat for reptile assemblages?

The human exploitation of land resources (land use) has been considered the major factor responsible for changes in biodiversity within terrestrial ecosystems given that it affects directly the distribution of the fauna. Reptiles are known to be particularly sensitive to habitat change due to their ecological constraints. Here, the impact of land use on reptile diversity was analysed, choosing Catalonia (NE Iberia) as a case study. This region provides a suitable scenario for such a biogeographical study since it harbours: 1) a rich reptile fauna; 2) a highly diverse environment showing strong variation in those variables usually shaping reptile distributions; and 3) good species distribution data. Potential species richness was calculated, using ecological modelling techniques (Ecological Niche Factor Analysis – ENFA). The subtraction of the observed from the potential species richness was the dependent variable in a backwards multiple linear regression, using land use variables. Agriculture was the land use with the strongest relation with the non-fulfilment of the potential species richness, indicating a trend towards a deficit of biodiversity. Deciduous forest was the only land use negatively related with the subtracted species richness. Results indicate a clear relationship between land use and biodiversity at a mesoscale. This finding represents an important baseline for conservation guidelines within the habitat change framework because it has been achieved at the same spatial scale of chorological studies and management policies.     

An assessment of time-dependent effects of lead exposure in algerian mice (Mus spretus) using different methodological approaches

Time-dependent effects of lead (Pb) toxicity were studied in Algerian mice (Mus spretus) treated with Pb acetate via drinking water (1 g Pb acetate/L) for different periods of exposure (15, 45, and 90 d). End points included the determination of hepatic Pb concentration and the assessment of some morphophysiological, biochemical and cytogenetical parameters. A control group receiving distilled water was also monitored for comparative purposes. Hepatic Pb accumulation increased with the time of exposure and was significantly higher in treated mice when compared to controls. In association with significant body mass loss in Pb-exposed mice, for 15 and 45 d, a significant increase in the relative spleen mass was observed after 45 d of intoxication. Pb-exposed mice also showed significant decreases in red blood cells, hematocrit, and mean corpuscular hemoglobin. On the contrary, changes in plasma transferases (aspartate aminotransferase and alanine aminotransferase) and hepatic superoxide dismutase activities did not reach statistical significance. A significant increase in the frequency of micronucleated polychromatic bone marrow erythrocytes was also found in the 90-d-exposed mice, compared to nontreated mice and the other exposed groups. Exposure to Pb acetate resulted also in a slight time-dependent decrease of the polychromatic-normochromatic ratio. These results support the concept that a long-term chronic exposure to Pb induced alterations upon some morphophysiological and genetic paramaters in Algerian mice.     

Characterization of the residues in helix 8 of the human beta1-adrenergic receptor that are involved in coupling the receptor to G proteins

Several key amino acids within amphipathic helix 8 of the human beta1-adrenergic receptor (beta1-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly389 or Arg389 allelic variants of the wild type beta1-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type beta1-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp382 and Arg384 within the hydrophilic interface might serve as a tethering site for the G protein.     

Detection of Mycobacterium leprae DNA in clinical and environmental samples using serological analysis and PCR

Molecular Biology Reports - Leprosy is a chronic infectious disease caused by Mycobacterium leprae and persists as a serious public health problem in Brazil. This microorganism is inculturable,...