Characterisation Of The Membrane Transport Of Pilocarpine In Cell Suspension Cultures Of Pilocarpus Microphyllus.

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.

Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

OBJECTIVES: This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. MATERIAL AND METHODS: Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized pre-surgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). RESULTS: As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm² in the test group and 2 mm² in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). CONCLUSIONS: The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions.

Forced convection to laminar flow of liquid egg yolk in circular and annular ducts

The steady-state heat transfer in laminar flow of liquid egg yolk - an important pseudoplastic fluid food - in circular and concentric annular ducts was experimentally investigated. The average convection heat transfer coefficients, determined by measuring temperatures before and after heating sections with constant temperatures at the tube wall, were used to obtain simple new empirical expressions to estimate the Nusselt numbers for fully established flows at the thermal entrance of the considered geometries. The comparisons with existing correlations for Newtonian and non-Newtonian fluids resulted in excellent agreement. The main contribution of this work is to supply practical and easily applicable correlations, which are, especially for the case of annulus, rather scarce and extensively required in the design of heat transfer operations dealing with similar shear-thinning products. In addition, the experimental results may support existing theoretical analyses.

Lysine and zinc chelate in diets for brown laying hens: effects on egg production and composition

It was evaluated the effects of levels of digestible lysine and chelate zinc combined in the diet for laying on the egg quality. It was used 720 birds, from 48 to 60 weeks of age, distributed in a completely randomized design in a 3 × 5 factorial scheme with three levels of zinc and five levels of lysine, applied into six replicates in the experimental units of eight birds per plot. The levels were: 137, 309 and 655 ppm zinc and 0.482, 0.527, 0.582, 0.644 and 0.732% digestible lysine. It was not observed any interaction among digestible lysine and zinc for the primary variables of fractions and egg composition. Levels of zinc reduced egg weight, suggesting the lowest efficiency in nutrient intake. At the highest dietary concentration of zinc, the addition of digestible lysine coincided with a linear increase in shell weight. However, zinc addition, regardless of lysine level in the diet, resulted in the reduction of egg weight and of the percentage of mineral matter in the yolk, limiting the efficiency of mineral deposition in this fraction of the egg. Concentration of zinc that produced the best results was 137 ppm inasmuch as higher quantities limit the use of digestible lysine, with effects harming composition and egg quality. The study indicates the following requirement for digestible lysine: 0.639% from the 48th to the 52nd week, 0.679% from the 52nd to 56th week, and 0.635% from the 56th to the 60th week. Considering the total period from 48th to the 60th week, the level 0.638% of lysine or the daily intake of 707 mg of the amino acid met the requirement for egg quality of semi-heavy layers used in this study.

Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/ disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

Galectin-1 Induces Reversible Phosphatidylserine Exposure at the Plasma Membrane

Cells normally undergo physiological turnover through the induction of apoptosis and phagocytic removal, partly through exposure of cell surface phosphatidylserine (PS). In contrast, neutrophils appear to possess apoptosis-independent mechanisms of removal. Here we show that Galectin-1 (Gal-1) induces PS exposure independent of alterations in mitochondrial potential, caspase activation, or cell death. Furthermore, Gal-1-induced PS exposure reverts after Gal-1 removal without altering cell viability. Gal-1-induced PS exposure is uniquely microdomain restricted, yet cells exposing PS do not display evident alterations in membrane morphology nor do they exhibit bleb formation, typically seen in apoptotic cells. Long-term exposure to Gal-1 prolongs PS exposure with no alteration in cell cycle progression or cell growth. These results demonstrate that Gal-1-induced PS exposure and subsequent phagocytic removal of living cells represents a new paradigm in cellular turnover.

ASSOCIATION OF A MYOSIN MOTOR WITH MEMBRANE-BOUNDED PIGMENT GRANULES IN FRESHWATER SHRIMP CHROMATOPHORES: EVIDENCE FROM THE NITELLA ACTIN-CABLE ASSAY

We have adapted an actin-mosin motility assay to examine the interactions in vitro between actin cables isolated from the giant internodal cells of the freshwater alga, Nitella, and pigment granules extracted from red ovarian chromatophores of the freshwater palaemonid shrimp, Macrobrachium olfersi. The chromatophore pigment mass consists of large (0.5-1.0-mu m diameter) membrane-bounded granules, and small (140-nm diameter), a membranous granules, both structurally continuous with the abundant smooth endoplasmic reticulum. Our previous immunocytochemical studies show a myosin motor to be stably associated with the pigment mass; however, to which granule type or membrane the myosin motor is attached is unclear. Here, we show that sodium vanadate, a myosin ATPase inhibitor, markedly increases the affinity of isolated, large, membrane-bounded granules for Nitella actin cables to which they become permanently attached. This interaction does not occur in granule preparations containing ATP with uninhibited, active myosin without vanadate. We propose that a stable state of elevated affinity is established between the granule-located myosin motor and the Nitella actin cables, resulting from a vanadate-inhibited acto-myosin-ADP complex. This finding provides further evidence for a myosin motor positioned on the surface of the membrane-bounded pigment granules in shrimp ovarian chromatophores.

Effect of zinc insertion and hydrophobicity on the membrane interactions and PDT activity of porphyrin photosensitizers

A series of photosensitizers (PS), which are meso-substituted tetra-cationic porphyrins, was synthesized in order to study the role of amphiphilicity and zinc insertion in photodynamic therapy (PDT) efficacy. Several properties of the PS were evaluated and compared within the series including photophysical properties (absorption spectra, fluorescence quantum yield Phi(f), and singlet oxygen quantum yield Phi(Delta)), uptake by vesicles, mitochondria and HeLa cells, dark and phototoxicity in HeLa cells. The photophysical properties of all compounds are quite similar (Phi(f) <= 0.02; Phi(Delta) similar to 0.8). An increase in lipophilicity and the presence of zinc in the porphyrin ring result in higher vesicle and cell uptake. Binding in mitochondria is dependent on the PS lipophilicity and on the electrochemical membrane potential, i.e., in uncoupled mitochondria PS binding decreases by up to 53%. The porphyrin substituted with octyl groups (TC8PyP) is the compound that is most enriched in mitochondria, and its zinc derivative (ZnTC8PyP) has the highest global uptake. The stronger membrane interaction of the zinc-substituted porphyrins is attributed to a complexing effect with phosphate groups of the phospholipids. Zinc insertion was also shown to decrease the interaction with isolated mitochondria and with the mitochondria of HeLa cells, an effect that has been explained by the particular characteristics of the mitochondrial internal membrane. Phototoxicity was shown to increase proportionally with membrane binding efficiency, which is attributed to favorable membrane interactions which allow more efficient membrane photooxidation. For this series of compounds, photodynamic efficiency is directly proportional to the membrane binding and cell uptake, but it is not totally related to mitochondrial targeting.

Placentation in Sigmodontinae: a rodent taxon native to South America

Background: Sigmodontinae, known as ""New World rats and mice,"" is a large subfamily of Cricetidae for which we herein provide the first comprehensive investigation of the placenta. Methods: Placentas of various gestational ages ranging from early pregnancy to near term were obtained for five genera, i.e. Necromys, Euryoryzomys, Cerradomys, Hylaeamys, and Oligoryzomys. They were investigated by means of histology, immunohistochemistry, a proliferation marker, DBA-lectin staining and transmission electron microscopy. Results: The chorioallantoic placenta was organized in a labyrinthine zone, spongy zone and decidua and an inverted yolk sac persisted until term. The chorioallantoic placenta was hemotrichorial. The interhemal barrier comprised fetal capillary endothelium and three layers of trophoblast, an outermost, cellular layer and two syncytial ones, with interspersed trophoblast giant cells (TGC). In addition, accumulations of TGC occurred below Reichert's membrane. The junctional zone contained syncytial trophoblast, proliferative cellular trophoblast, glycogen cells and TGC that were situated near to the maternal blood channels. In three of the genera, TGC were also accumulated in distinct areas at the placental periphery. PAS-positive glycogen cells derived from the junctional zone invaded the decidua. Abundant maternal uNK cells with positive response to PAS, vimentin and DBA-lectin were found in the decidua. The visceral yolk sac was completely inverted and villous. Conclusion: The general aspect of the fetal membranes in Sigmodontinae resembled that found in other cricetid rodents. Compared to murid rodents there were larger numbers of giant cells and in some genera these were seen to congregate at the periphery of the placental disk. Glycogen cells were found to invade the decidua but we did not identify trophoblast in the walls of the deeper decidual arteries. In contrast these vessels were surrounded by large numbers of uNK cells. This survey of wild-trapped specimens from five genera is a useful starting point for the study of placentation in an important subfamily of South American rodents. We note, however, that some of these rodents can be captive bred and recommend that future studies focus on the study of time dated pregnancies.

Chorioallantoic placentation in Galea spixii (Rodentia, Caviomorpha, Caviidae)

Background: Placentas of guinea pig-related rodents are appropriate animal models for human placentation because of their striking similarities to those of humans. To optimize the pool of potential models in this context, it is essential to identify the occurrence of characters in close relatives. Methods: In this study we first analyzed chorioallantoic placentation in the prea, Galea spixii, as one of the guinea pig's closest relatives. Material was collected from a breeding group at the University of Mossoro, Brazil, including 18 individuals covering an ontogenetic sequence from initial pregnancy to term. Placentas were investigated by means of histology, electron microscopy, immunohistochemistry (vimentin, alpha-smooth muscle actin, cytokeration) and proliferation activity (PCNA). Results: Placentation in Galea is primarily characterized by an apparent regionalization into labyrinth, trophospongium and subplacenta. It also has associated growing processes with clusters of proliferating trophoblast cells at the placental margin, internally directed projections and a second centre of proliferation in the labyrinth. Finally, the subplacenta, which is temporarily supplied in parallel by the maternal and fetal blood systems, served as the center of origin for trophoblast invasion. Conclusion: Placentation in Galea reveals major parallels to the guinea pig and other caviomorphs with respect to the regionalization of the placenta, the associated growing processes, as well as trophoblast invasion. A principal difference compared to the guinea pig occurred in the blood supply of the subplacenta. Characteristics of the invasion and expanding processes indicate that Galea may serve as an additional animal model that is much smaller than the guinea pig and where the subplacenta partly has access to both maternal and fetal blood systems.

Deletion of the Basement Membrane Heparan Sulfate Proteoglycan Type XVIII Collagen Causes Hypertriglyceridemia in Mice and Humans

Background: Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism. Methods and Findings: We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia. Conclusions: This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.

Giant Vesicles under Oxidative Stress Induced by a Membrane-Anchored Photosensitizer

We have synthesized the amphiphile photosensitizer PE-porph consisting of a porphyrin bound to a lipid head-group. We studied by optical microscopy the response to light irradiation of giant unilamellar vesicles of mixtures of unsaturated phosphatidylcholine lipids and PE-porph. In this configuration, singlet oxygen is produced at the bilayer surface by the anchored porphyrin. Under irradiation, the PE-porph decorated giant unilamellar vesicles exhibit a rapid increase in surface area with concomitant morphological changes. We quantify the surface area increase of the bilayers as a function of time and photosensitizer molar fraction. We attribute this expansion to hydroperoxide formation by the reaction of the singlet oxygen with the unsaturated bonds. Considering data from numeric simulations of relative area increase per phospholipid oxidized (15%), we measure the efficiency of the oxidative reactions. We conclude that for every 270 singlet oxygen molecules produced by the layer of anchored porphyrins, one eventually reacts to generate a hydroperoxide species. Remarkably, the integrity of the membrane is preserved in the full experimental range explored here, up to a hydroperoxide content of 60%, inducing an 8% relative area expansion.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Source populations in coastal crabs: parameters affecting egg production

Benthic marine invertebrates may form metapopulations connected via propagule dispersal. Conservation efforts often target potential source coastlines to indirectly benefit areas depending on allochthonous offspring production. Besides population density, adult size structure, sex ratio, brooding frequency and the proportion of breeding individuals may significantly influence the reproductive output of benthic populations, but these effects have seldom been tested. We used rocky shore crabs to assess the spatial variability of such parameters at relevant scales for conservation purposes and to test their consistency over 2 consecutive years; we then used the data to address whether bottom-up processes or biological interactions might explain the patterns observed. We decomposed egg production rates into their components for the 2 most abundant brachyuran species inhabiting the intertidal rocky habitat. Adult density and brooding frequency varied consistently among shores for both species and largely explained the overall spatial trends of egg production. Temporally consistent patterns also included among-shore differences in the size of ovigerous females of the grapsid Pachygrapsus transversus and between-bay differences in the fecundity of the spider crab Epialtus brasiliensis. Sex ratio was remarkably constant in both. We found no positive or negative correlations between adult density and brooding frequency to support either the existence of a component Allee effect (lack of mate encounters) or an effect of intra-specific competition. Likewise, shore-specific potential growth in P. transversus does not negatively correlate with frequency of ovigerous individuals, as would be expected under a critical balance between these 2 processes. The patterns observed suggest that bottom-up drivers may best explain spatial trends in the reproductive output of these species.