Evaluation of Functional Stability and Batch-to-Batch Reproducibility of a <Emphasis Type="Italic">Castanea sativa</Emphasis> Leaf Extract with Antioxidant Activity

A growing body of evidence suggests that free radicals are generated by UV irradiation being responsible for skin injury. In this regard, the topical use of formulations composed of plant extracts with antioxidant activity could represent a useful strategy for the prevention of photoaging and oxidative-stress-mediated diseases. The aim of this study was to assess the reproducibility of the extraction method and the functional stability of a Castanea sativa leaf extract in view of its application as topical antioxidant. Measurements of 1,1-diphenyl-2-picryl hydrazyl (DPPH) scavenging activity, total phenols (measured by the Folin Ciocalteu assay) and phenolic composition (high-performance liquid chromatography unit coupled to a UV detector) were carried out on three different batches. The influence of pH and temperature on the extract’s DPPH scavenging activity was assessed in aqueous and glyceric solutions (0.025% w/v) over a 3-month period. Minor differences were found between the three extract batches for all the evaluated parameters, and therefore the reproducibility of the extraction method can be inferred. pH presented a great influence in the extract functional stability. Major antioxidant activity decrease was found at pH 7.1, while lower changes were observed at pH 5. Glyceric solutions were stable throughout the test period. At 40°C and pH 5, a marked decrease of activity was observed. Again, glyceric solutions were the most stable, even at 40°C. Proper selection of pH and solvent is mandatory to ensure the stability of the studied extract after being incorporated in semisolid forms. In view of these results, glycerine is proposed as the best vehicle for topical formulations incorporating C. sativa leaf extract, which should have a pH around 5.     

Origin of Embryo-Like Structures in Soybean Anther Culture Investigated Using SSR Marker

The Satt418 microsatellite locus was examined in order to investigate the origin of embryo-like structures (ELS) obtained from soybean anther culture. Four heterozygous plants were used as anther donors. A total of 7000 anthers were placed on the induction medium under culture conditions known to trigger androgenic response. After 60 days of culture, upper portion of 216 ELS were carefully removed and transferred to a proliferation medium, in order to obtain sufficient tissue for DNA extraction. Callogenic masses originated from 114 ELS were screened for the Satt418 microsatellite locus. Heterozygous and homozygous ELS were identified, suggesting occurrence of somatic embryogenesis and androgenesis in the same system. This unexpected morphogenic response seems to be genotype-dependent.     

Genotype by Sex and Genotype by Age Interactions with Sedentary Behavior: The Portuguese Healthy Family Study

Sedentary behavior (SB) expression and its underlying causal factors have been progressively studied, as it is a major determinant of decreased health quality. In the present study we applied Genotype x Age (GxAge) and Genotype x Sex (GxSex) interaction methods to determine if the phenotypic expression of different SB traits is influenced by an interaction between genetic architecture and both age and sex. A total of 1345 subjects, comprising 249 fathers, 327 mothers, 334 sons and 325 daughters, from 339 families of The Portuguese Healthy Family Study were included in the analysis. SB traits were assessed by means of a 3-d physical activity recall, the Baecke and IPAQ questionnaires. GxAge and GxSex interactions were analyzed using SOLAR 4.0 software. Sedentary behaviour heritability estimates were not always statistically significant (p>0.05) and ranged from 3% to 27%. The GxSex and GxAge interaction models were significantly better than the single polygenic models for TV (min/day), EEsed (kcal/day), personal computer (PC) usage and physical activty (PA) tertiles. The GxAge model is also significantly better than the polygenic model for Sed (min/day). For EEsed, PA tertiles, PC and Sed, the GxAge interaction was significant because the genetic correlation between SB environments was significantly different from 1. Further, PC and Sed variance heterogeneity among distinct ages were observed. The GxSex interaction was significant for EEsed due to genetic variance heterogeneity between genders and for PC due to a genetic correlation less than 1 across both sexes. Our results suggest that SB expression may be influenced by the interactions between genotype with both sex and age. Further, different sedentary behaviors seem to have distinct genetic architectures and are differentially affected by age and sex.     

Cytoskeleton structure,pattern of mitochondrial activity and ultrastructure of frozen or vitrified sheep embryos

Even though sheep embryo cryopreservation is a commonly used procedure the survival and pregnancy outcomes can vary greatly. This study investigated whether cryopreservation was causing subtle changes in ultrastructure, mitochondrial activity or cytoskeletal integrity. Sheep embryos were either slow cooled in 1.5 M EG (n = 22), or vitrified in 20% EG + 20% DMSO with 0.5 M sucrose in Open Pulled Straws (OPS) (n = 24). One hour after warming the cryopreserved embryos differed from control embryos in that they had no mitochondrial activity combined with cytoskeletal disorganization and large vesicles. Vitrified embryos also showed many points of cytoskeleton disruption. Ultrastructural alterations resulting from actin filaments disorganization were observed in both cryopreserved groups. This includes areas presenting no cytoplasmic organelles, Golgi complex located far from the nucleus and a decrease of specialized intercellular junctions. Additionally, large vesicles were observed in vitrified morulae and early blastocysts. The alterations after cryopreservation were proportional to embryo quality as assessed using the stereomicroscope. Even in the absence of mitochondrial activity, grade I and II cryopreserved embryos contained mitochondria with normal ultrastructure. Embryos classified as grade I or II in the stereomicroscope revealed mild ultrastructural alterations, meaning that this tool is efficient to evaluate embryos after cryopreservation.     

Role of Ca2+-calmodulin dependent phospholamban phosphorylation on the relaxant effect of β-adrenergic agonists

The role of the Ca²⁺-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of -adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]₀ from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca²⁺-calmodulin dependent protein kinase activity. 3×10^–8 M isoproterenol increased cAMP levels from 0.613±0.109 pmol/mg wet weight to 1.581±0.123, phospholamban phosphorylation from 36±6 pmol³²P/mg protein to 277±26 and decreased time to half relaxation (t1/2) from 61±2 msec to 39±2. Simultaneous perfusion of isoproterenol with 10^–6 M W7, decreased phospholamban phosphorylation to 170±23 and prolongated t1/2 to 47±3 but did not affect the increase either in cAMP levels or myocardial contractility produced by isoproterenol. Similar effects on phospholamban phosphorylation and myocardial relaxation were obtained when isoproterenol was perfused in low [Ca]₀. Low [Ca]₀ did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the -agonist.The results suggest a physiological role of the Ca²⁺-calmodulin dependent phospholamban phosphorylation pathway as a mechanism that supports, in part, the -adrenergic cardiac relaxant effect.     

Rise and fall of the phacopids: the morphological history of a successful trilobite family

Phacopidae were a successful family of the Silurian–Devonian period. Although their diversity trends are well identified, their shape evolution is unknown; their morphology often considered to be conservative. We have quantified these morphologies using geometric morphometrics (landmarks) and investigated their evolution using morphological disparity indices. Results identified morphological variations between the genera, and through time. Phacopids differ from each other by the position of the facial suture linked to the size of the visual complex, the shape of the genal angle and the elongation of both cephalon and pygidium. The morphological disparity of cephala was high from the Silurian, contrary to that of pygidia. Subsequently, the morphological disparity increased in the Early Devonian with the development of narrow cephala and triangular pygidia. Morphological disparity was greater in the Emsian for both cephala and pygidia, more than 50 myr after the origination of phacopids. It constituted a perfect example illustrating that a peak of biodiversity does not necessarily happen in the early history of a clade. Subsequently, a strong decrease of morphological disparity occurred in the Middle Devonian, in conjunction with sea-level changes and anoxic events. Taxonomic richness and morphological disparity declined strongly in the Givetian, in a non-random extinction affecting particularly blind genera. The morphological disparity remained low in the Frasnian despite progressive eye reduction influenced by environmental changes. An extensive recovery occurred in the Famennian with an important increase of both taxonomic and morphological diversity. The Hangenberg event caused the final extinction of phacopids.     

The trans-sialidase from the african trypanosome Trypanosoma brucei.

Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.     

Effects of acute hypercapnia upon the myocardium: contractility, coronary resistance, oxygen consumption and potassium balance (author's transl)

In the isolated perfused dog heart, at constant coronary blood flow and heart rate, we studied the effect of altering CO2 in the gas mixture from 5 to 15% on contractility, coronary resistance, myocardial O2 consumption and K balance. Contractility, assessed by the developed force, and its derivative through a strain-gauge arch sewed to the left ventricle decreased to 54 +/- 7% (p less than 0.01) and 59 +/- 6% (p less than 0.01), respectively from control values. Coronary resistance decreased to 79 +/- 3% of control ( less than 0.01). The negative inotropic effect of hypercapnia was accompanied by a decrease in myocardial O2 consumption to 68 +/- 11% (p less than 0.01) of control value and a net uptake of K by the heart. The possibility of an exchange of H+ by K+ is suggested as a possible mechanism involved in the negative inotropic effect of hypercapnia.     

Formation and characterization of glutamate dehydrogenase monolayers on silicon supports

In this paper we have tested two different procedures (the "three-step" and the "four-step" procedures) for the covalent immobilization of glutamate dehydrogenase (GDH) onto silicon supports. Atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FT-IR), fluorescence spectroscopy and an enzymatic assay were used to probe the structure and activity of the immobilized enzyme. Our results demonstrate that coupling through the "three-step" procedure does not significantly affect either the fold pattern or the activity of the enzyme, suggesting that this method could be ideally suited to the development of high quality monolayers for use in enzyme-based planar biosensors.     

Inflammatory effects of snake venom metalloproteinases

Metalloproteinases are abundant enzymes in crotaline and viperine snake venoms. They are relevant in the pathophysiology of envenomation, being responsible for local and systemic hemorrhage frequently observed in the victims. Snake venom metalloproteinases (SVMP) are zinc-dependent enzymes of varying molecular weights having multidomain organization. Some SVMP comprise only the proteinase domain, whereas others also contain a disintegrin-like domain, cysteine-rich, and lectin domains. They have strong structural similarities with both mammalian matrix metalloproteinases (MMP) and members of ADAMs (a disintegrin and metalloproteinase) group. Besides hemorrhage, snake venom metalloproteinase induce local myonecrosis, skin damage, and inflammatory reaction in experimental models. Local inflammation is an important characteristic of snakebite envenomations inflicted by viperine and crotaline snake species. Thus, in the recent years there is a growing effort to understand the mechanisms responsible for SVMP-induced inflammatory reaction and the structural determinants of this effect. This short review focuses the inflammatory effects evoked by SVMP.