Tuning the Hydrophilic/Hydrophobic Balance to Control the Structure of Chitosan Films and Their Protein Release Behavior

The control over the crystallinity of chitosan and chitosan/ovalbumin films can be achieved via an appropriate balance of the hydrophilic/hydrophobic interactions during the film formation process, which then controls the release kinetics of ovalbumin. Chitosan films were prepared by solvent casting. The presence of the anhydrous allomorph can be viewed as a probe of the hydrophobic conditions at the neutralization step. The semicrystalline structure, the swelling behavior of the films, the protein/chitosan interactions, and the release behavior of the films were impacted by the DA and the film processing parameters. At low DAs, the chitosan films neutralized in the solid state corresponded to the most hydrophobic environment, inducing the crystallization of the anhydrous allomorph with and without protein. The most hydrophilic conditions, leading to the hydrated allomorph, corresponded to non-neutralized films for the highest DAs. For the non-neutralized chitosan acetate (amorphous) films, the swelling increased when the DA decreased, whereas for the neutralized chitosan films, the swelling decreased. The in vitro release of ovalbumin (model protein) from chitosan films was controlled by their swelling behavior. For fast swelling films (DA?=?45%), a burst effect was observed. On the contrary, a lag time was evidenced for DA?=?2.5% with a limited release of the protein. Furthermore, by blending chitosans (DA?=?2.5% and 45%), the release behavior was improved by reducing the burst effect and the lag time. The secondary structure of ovalbumin was partially maintained in the solid state, and the ovalbumin was released under its native form.     

Genetic diversity of <Emphasis Type="Italic">Enterolobium cyclocarpum</Emphasis> in Colombian seasonally dry tropical forest: implications for conservation and restoration

Enterolobium cyclocarpum is a characteristic legume tree species of seasonally dry tropical forests (SDTFs) of Mesoamerica and northern South America typically used in silvopastoral and agroforestry systems. Remaining populations of E. cyclocarpum in Colombia are severely fragmented owing to the highly degraded state of SDTF in the country, posing threats to both their in situ persistence and their usefulness as seed sources for future planting efforts. We genotyped E. cyclocarpum populations at nine sampling sites across a latitudinal gradient of SDTF in Colombia by means of eight nSSR markers to elucidate the species diversity distribution in the country. Our data suggest that a deep divide seems to have existed between Caribbean and Andean populations of E. cyclocarpum in Colombian SDTF that may date back to the last glacial maximum (~21,000 BP), or longer. However, we only found evidence of genetic differentiation between trees from the southern Cauca River valley and populations at more northern locations. All the latter populations showed signs of admixture which may be the result of human-influenced movement of germplasm, particularly after the introduction of cattle by European settlers. Most of the sampled sites showed heterozygosity scores close to Hardy–Weinberg expectations. Only the three southern-most populations displayed significantly positive values of inbreeding coefficient, potentially affecting their in situ maintenance and their use as seed sources. Based on our findings we identify priority areas for the in situ conservation of remaining E. cyclocarpum populations, and propose a strategy for sourcing of appropriate planting material for use in future tree planting efforts.     

Lectin histochemistry for detecting cadmium-induced changes in the glycosylation pattern of rat placenta

Cadmium (Cd) is an industrial and environmental pollutant that produces toxic effects on gametogenesis, pre- and post-implantation embryos, and the placenta. Because the effects of acute Cd intoxication on the placenta are not well understood, we investigated changes in its glycosylated components in Cd treated dams at days 4, 7, 10 and 15 of gestation using lectin histochemistry. CdCl₂ was administered to pregnant rats; control animals received sterile normal saline. Placentas were processed for DBA, Con A, SBA, PNA, UEA-I, RCA-I and WGA lectin histochemistry to evaluate changes in the carbohydrate pattern of the placenta that might modify cell interactions and contribute to embryonic alterations. Lectin binding was analyzed in the yolk sac; trophoblast giant cells; trophoblast I, II and III; spongiotrophoblast cells and endovascular trophoblast cells in the chorioallantoic placenta. Our lectin binding patterns showed that Cd caused alteration of SBA and DBA labeling of trophoblast-derived cells, which suggested increased expressions of α and β GalNAc. Cd also caused decreased UEA-1 binding affinity, which indicated fewer α-L-Fuc residues in placentas of Cd treated dams. The nonreactivity in trophoblast I of the control placentas incubated with Con-A contrasted with the labeling in placentas of experimental dams, which indicated increased expression of terminal α-D-Man, and α-D-Glc residues. We found that Cd altered the reactivity of placenta to several lectins, which indicated modification of the glycotype presented by the fetal component of the placenta. We report that Cd exerts a deleterious effect on the glycosylation pattern of the placenta.     

Oxidative stress induced by ciprofloxacin in Staphylococcus aureus

Staphylococcus aureus with multiple sensitivity to ciprofloxacin, was investigated to detect alterations in the production of superoxide anion (O(2)(-)), other reactive oxidant species (ROS), and superoxide dismutase (SOD), and to relate them with ciprofloxacin accumulation and sensitivity. Oxidative stress was studied by means of Nitroblue Tetrazolium reaction (NBT) and chemiluminescence (CL); lucigenin was employed to detect O(2)(-), and luminol was used to measure other ROS. Sensitive strains exhibited higher intracellular O(2)(-) increase than resistant ones when incubated with ciprofloxacin. SOD was determined in normal conditions and induction was investigated in the presence of ciprofloxacin. These assays demonstrated that resistant and sensitive strains exported a great amount of SOD and that the induction of SOD intracellular was insufficient to counteract the augment of O(2)(-) in the cytoplasm of sensitive strains. Accumulation of ciprofloxacin, researched by spectrofluorometry, showed high levels of antibiotic in sensitive strains which increased the O(2)(-) causing more oxidative stress than in resistant S. aureus.     

Protein Disulfide Oxidoreductases and the Evolution of Thermophily: Was the Last Common Ancestor a Heat-Loving Microbe?

Protein disulfide oxidoreductases (PDOs) are redox enzymes that catalyze dithiol–disulfide exchange reactions. Their sequences and structure reveal the presence of two thioredoxin fold units, each of which is endowed with a catalytic site CXXC motif. PDOs are the outcome of an ancient gene duplication event. They have been described in a number of thermophilic and hyperthermophilic species, where they play a critical role in the structural stabilization of intracellular proteins. PDOs are homologous to both the N-terminal domain of the bacterial alkyl hydroperoxide reductase (AhpF) and to the eukaryotic protein disulfide isomerase (PDI). Phylogenetic analysis of PDOs suggests that they first evolved in the crenarchaeota, spreading from them into the Bacteria via the euryarchaeota. These results imply that the last common ancestor (LCA) of all extant living beings lacked a PDO and argue, albeit weakly, against a thermophilic LCA. Reviewing Editor: Martin Kreitman     

Increased Na+/Ca2+ Exchanger Expression/Activity Constitutes a Point of Inflection in the Progression to Heart Failure of Hypertensive Rats

Spontaneously hypertensive rat (SHR) constitutes a genetic model widely used to study the natural evolution of hypertensive heart disease. Ca²⁺-handling alterations are known to occur in SHR. However, the putative modifications of Ca²⁺-handling proteins during the progression to heart failure (HF) are not well established. Moreover, the role of apoptosis in SHR is controversial. We investigated intracellular Ca²⁺, Ca²⁺-handling proteins and apoptosis in SHR vs. control Wistar rats (W) from 3 to 15 months (mo). Changes associated with the transition to HF (i.e. lung edema and decrease in midwall fractional shortening), occurred at 15 mo in 38% of SHR (SHRF). In SHRF, twitch and caffeine-induced Ca²⁺ transients, significantly decreased relative to 6/9 mo and 15 mo without HF signs. This decrease occurred in association with a decrease in the time constant of caffeine-Ca²⁺ transient decay and an increase in Na⁺/Ca²⁺ exchanger (NCX) abundance (p<0.05) with no changes in SERCA2a expression/activity. An increased Ca²⁺-calmodulin-kinase II activity, associated with an enhancement of apoptosis (TUNEL and Bax/Bcl2) was observed in SHR relative to W from 3 to 15 mo. Conclusions: 1. Apoptosis is an early and persistent event that may contribute to hypertrophic remodeling but would not participate in the contractile impairment of SHRF. 2. The increase in NCX expression/activity, associated with an increase in Ca²⁺ efflux from the cell, constitutes a primary alteration of Ca²⁺-handling proteins in the evolution to HF. 3. No changes in SERCA2a expression/activity are observed when HF signs become evident.     

Reliable molecular identification of nine tropical whitefly species

The identification of whitefly species in adult stage is problematic. Morphological differentiation of pupae is one of the better methods for determining identity of species, but it may vary depending on the host plant on which they develop which can lead to misidentifications and erroneous naming of new species. Polymerase chain reaction (PCR) fragment amplified from the mitochondrial cytochrome oxidase I (COI) gene is often used for mitochondrial haplotype identification that can be associated with specific species. Our objective was to compare morphometric traits against DNA barcode sequences to develop and implement a diagnostic molecular kit based on a RFLP‐PCR method using the COI gene for the rapid identification of whiteflies. This study will allow for the rapid diagnosis of the diverse community of whiteflies attacking plants of economic interest in Colombia. It also provides access to the COI sequence that can be used to develop predator conservation techniques by establishing which predators have a trophic linkage with the focal whitefly pest species.     

Variability,genetic structure and phylogeography of the dolomitophilous species Convolvulus boissieri (Convolvulaceae) in the Baetic ranges,inferred from AFLPs,plastid DNA and ITS sequences

Convolvulus boissieri is an edaphic endemic plant which grows in the Baetic ranges always in association with high mountain xeric dolomitic outcrops. As these dolomitic areas appear in a ‘soil‐island’ pattern, the distribution of this species is disjunct. Populations of this species frequently include a low number of individuals, which could have an important impact on their genetic diversity and viability. Convolvulus boissieri provides an excellent opportunity to study the genetic and phylogeographical aspects of species linked to dolomites. We used amplified fragment length polymorphism markers and nuclear (internal transcribed spacer region of the nuclear ribosomal cistron) and plastid sequences (trnL‐trnF, rpl32‐trnL and trnQ‐5′rps16). Data were generated from 15 populations, representing the distribution area of the species. For sequence analysis and estimation of divergence times we also used sequences from other Convolvulus species. Results revealed low intrapopulational genetic diversity and a strong interpopulational structure. Furthermore, we found clear‐cut differentiation caused by the existence of two large population groups separated by the Guadiana Menor river basin. Estimation of divergence times indicated that divergence took place during the Pleistocene glaciations. Genetic diversity and differentiation are similar to those other species exhibiting naturally fragmented distribution with a sky islands pattern. In phylogeographical terms, the successive glaciation–interglaciation cycles caused the species to spread from the western sites to eastern sites, the latter being more exposed to the effects of glaciation. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 506–523.     

Stent implantation in acute myocardial infarction using a heparin-coated stent: a pilot study as a preamble to a randomized trial comparing balloon angioplasty and stenting

Preliminary experience with primary stenting in myocardial infarction has suggested a greater benefit in clinical outcome than has been obtained with direct balloon angioplasty. However, subacute thrombosis (SAT) remains a limitation for this new mode of therapy. In the BENESTENT II Pilot and main trials, the incidence of SAT with the heparin-coated Palmaz-Schatz stent was only 0.15%. Therefore, as a preamble to a large randomized trial, the feasibility and safety of the use of the Heparin-Coated Palmaz-Schatz trade mark Stent in Acute Myocardial Infarction (AMI) was tested in 101 patients enrolled between April and September 1996 in 18 clinical centres. In 101 stent-eligible AMI patients, as dictated by protocol, a heparin-coated stent was implanted. The primary objectives were to determine the in-hospital incidence of major adverse cardiac events (MACE: death, MI, target lesion revascularization) and bleeding complications, while the secondary objectives were the procedural success rate and the MACE, the restenosis and reocclusion rates at 6.5 months. Stent implantation (n 3 129 stents) was successful in 97 patients of the 101 who were included in this trial. During their hospital stay, two patients died and no patient experienced re-infarction, ischaemia prompting re-PTCA or CABG. Four patients suffered a bleeding complication, three major and one minor, of whom three required surgical repair. At 210 days follow-up, 81% of the patients were event free. At 6.5 months restenosis was documented in 18% of the 88 patients who underwent follow-up angiography, including three total occlusions. The results, both with respect to QCA and the occurrence of MACE, compare favourably with studies using elective stenting in both stable and unstable angina patients. As a result of this pilot study, a large randomized trial comparing direct balloon angioplasty with direct stenting in 900 patients with AMI was initiated in December 1996.