Natural Milieu Research Articles

Electrospun gelatin/poly(L-lactide-co-ε-caprolactone) nanofibers for mechanically functional tissue-engineering scaffolds

Recently, much attention has been given to the fabrication of tissue-engineering scaffolds with nano-scaled structure to stimulate cell adhesion and proliferation in a microenvironment similar to the natural extracellular matrix milieu. In the present study, blends of gelatin and poly(L-lactide-co-ε-caprolactone) (PLCL) (blending ratio: 0, 30, 70 and 100 wt% gelatin to PLCL) were electrospun to prepare nano-structured non-woven fibers for the development of mechanically functional engineered skin grafts. The resulting nanofibers demonstrated the uniform and smooth fibers with mean diameters ranging from approx. 50 to 500 nm with interconnected pores, regardless of the composition. The contact angle decreased with increasing amount of gelatin in the blend and the water content of the nanofibers increased concurrently. PLCL nanofibers retained significant levels of recovery following application of uniaxial stress; GP-3 with 70% PLCL blend returned to the original length within less than 10% of deformation following 200% of uniaxial elongation. The overall tensile strength was inversely affected by increase in the gelatin content and degradation rates of the nanofibers were accelerated as the gelatin concentration increased. When seeded with human primary dermal fibroblasts and keratinocytes on the nanofibers, both initial cell adhesion and proliferation rate increased as a function of the gelatin content in the blend. Additionally, the total cell number was significantly greater on the nanofiber scaffolds than on polymer-coated glasses, indicating that nanofibrous structure facilitates cell proliferation. Taken together, gelatin/PLCL blend nanofiber scaffolds may serve as a promising artificial extracellular matrix for regeneration of mechanically functional skin tissue.

Embryo implantation rates in natural and stimulated assisted reproduction treatment cycles in poor responders

Controlled ovarian stimulation with exogenous gonadotrophins and gonadotrophin-releasing hormone (GnRH) analogues enables the collection of multiple oocytes and subsequent development of multiple embryos. However, interfering with the natural hormonal milieu may decrease the probability of successful embryo implantation due to effects on oocytes and/or endometrium. In order to provide a fair comparison of embryo implantation rates between natural cycles and stimulated cycles, bias caused by the presence of multiple embryos available for transfer in stimulated cycles should be avoided. This retrospective study analysed embryo implantation rates in cycles in which only a single embryo was available for transfer in 304 women who had poorly responded to ovarian stimulation in the previous cycle. Embryo implantation rates with different stimulation protocols were as follows: natural cycle, 20% (6/30); gonadotrophin only, 5.6% (3/54); long GnRH protocol, 3.8% (2/52); co-flare protocol, 1.9% (1/52); microdose flare-up, 15.4% (4/26); GnRH antagonists, 14.4% (13/90). Although the difference was not statistically significant there was a trend towards higher implantation rates with natural cycles in this group of women. Natural cycle IVF may be a reasonable and patient-friendly treatment choice yielding an acceptable outcome for women who are known or anticipated poor responders to ovarian stimulation.

“Turnover Proteome” of Human Atrial Trabeculae

Most of the biologically relevant data on cardiomyocytes are derived from isolated cells under conditions that are, to some extent, altered compared to the natural milieu of the functional heart. The handling procedure of the dissection, isolation, and short-term culturing induces changes in the cells such that the subsequently measured parameters (among others, the protein synthesis) reflect the actual experimental conduct rather than the intrinsic properties of these terminally differentiated cells. Although it is known that the protein synthetic machinery of isolated cardiomyocytes is operational and functional, the biosynthetic yield of human cardiomyocytes in the natural milieu of the trabeculae remains to be established, with a special emphasis to clarify whether the protein synthesis includes just a limited set of polypeptides or it encompasses all cellular constituents. Knowledge on this issue is a prerequisite for achieving further advances in our understanding of heart remodeling related to hypertrophy in particular, and for attempting interventions leading to repair of damaged heart in general. The experimental system of "organ bath" enables simultaneous registration of contractile forces of portions of cardiac muscle tissue (and other myocyte-containing tissues) and biosynthetic labeling of newly synthesized cellular constituents. The organ bath methodology was adapted for this project such as enabling to measure molecular changes in response to in vitro applied stimuli. Instead of Krebs-Henseleit-solution, as used in classical protocols of organ bath studies, we utilized cell culture media suitable to experimental conditions related to metabolic labeling. Proteome patterns established by performing two-dimensional gel electrophoresis of the extracts from biosynthetically labeled trabeculae revealed that cardiomyocytes synthesize the full spectrum of cellular proteins. Proteomic silver-stain readout was used to obtain samples for spot excisions, as material suitable for mass spectrometric analysis. Protein spots were identified both from the excised spots and also by matching with the in-house- and www-databases (Swiss-Prot/High-Performance Heart). From our findings that protein synthesis in terminally differentiated cardiomyocytes is not confined just to the synthesis of those structures needed for the post-mitotic house-keeping functions, we conclude that this model might serve as a valid experimental system to study and elucidate the effects of various pharmacological compounds under conditions where physiology (contractile forces) and biochemistry (protein synthesis) of intact human heart tissue are monitored simultaneously.

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity

Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease’s natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the β-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Aβ peptide, a key event that leads to Alzheimer’s disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.

In Defense of Praying with Images

The paper argues for a notion of religion that is based on a strong human sense for symbols. Symbols are the natural milieu for religion. I distinguish symbols from signs through the fact that the symbol brings together the elements kept separate in the sign. A symbol does contain something of the force of the reality which it represents. With this approach we can look at fides quaerens intellectum in a new light. Moreover, religious images and icons can gain from understanding religion as a symbolic practice. The paper argues that the theological debate on the religious value of icons should not be focused on the tension between the visibility and the invisibility of the divine. Against Marion 1 argue that touching, rather than seeing, is the core of religious images. People kiss and caress icons. Examples from ordinary life are adduced to illustrate this understanding.

Estructuras culturales y accesibilidad para la isla de San Erasmo en Venecia

<p>La puesta en valor de una antigua estructura defensiva de la Laguna de Venecia ha conllevado no sólo su conservación material sino también una intervención en su entorno inmediato y el paisaje circundante. Este artículo desvela la necesidad de concebir la restauración de un monumento en su medio natural como un conjunto indisoluble que requiere tanto de restauración física como de dotación de las infraestructuras necesarias para su disfrute, de la manera más discreta y silenciosa posible para evitar tergiversar su ambiente.</p>

De novo infection and propagation of wild-type Hepatitis C virus in human T lymphocytes in vitro

While exploring previous findings that ex vivo treatment of lymphoid cells from Hepatitis C virus (HCV)-infected individuals with T cell-stimulating mitogens augments detection of the residing virus, an in vitro HCV replication system was established, in which mitogen-induced T cell-enriched cultures served as HCV targets and the derived T cells multiplied virus during repeated serial passage. HCV replication was ascertained by detecting HCV RNA positive and negative strands, HCV NS5a and E2 proteins, release of HCV virions and nucleocapsids (confirmed by immunoelectron microscopy) and de novo infection of mitogen-induced T cells prepared from healthy donors. Further, affinity-purified normal human T lymphocytes were also susceptible to HCV infection in vitro and HCV replication was detected in pure T cells isolated from a patient with chronic hepatitis C. These results document that T cells can support propagation of HCV both in vivo and in vitro. The infection system established offers a valuable tool for in vitro studies on the entire cycle of HCV replication, virus cytopathogenicity and evaluation of antiviral agents against wild-type HCV in the natural host-cell milieu.

Electrophoretic separation and characterization of urinary glycosaminoglycans and their roles in urolithiasis

Urinary polyanions recovered from the urine samples of kidney stone-formers and normal controls were subjected to preparative agarose gel electrophoresis, which yielded fractions 1–5 in a decreasing order of mobility. In both groups, chondroitin sulfates were identified in the fast-moving fractions and heparan sulfates in the slow-moving fractions. Furthermore, two types of heparan sulfates were identified based on their electrophoretic mobility: slow-moving and fast-moving. The fractionated urinary polyanions were then tested in an in vitro calcium oxalate crystallization assay and compared at the same uronic acid concentration, whereby, the chondroitin sulfates of stone-formers and heparan sulfates of normals enhanced crystal nucleation. Fraction 5 of the normals, containing glycoproteins (14–97 kDa) and associated glycosaminoglycans, were found to effectively inhibit crystallization. Papainization of this fraction in stone-formers revealed crystal-suppressive effects of glycoproteins, which was not seen in similar fractions of normals. It was concluded that glycoproteins could modulate the crystal-enhancing glycosaminoglycans such as chondroitin sulfates of stone-formers but not in normals. The differing crystallization activities of electrophoretic fraction 1 of normals and stone-formers revealed the presence of another class of glycosaminoglycan–hyaluronan. Hence, in the natural milieu, different macromolecules combine to have an overall outcome in the crystallization of calcium oxalate.

Can the Therapeutic Advantages of Allogenic Umbilical Cord Blood???Derived Stem Cells and Autologous Bone Marrow???Derived Mesenchymal Stem Cells Be Combined and Synergized?

Can the Therapeutic Advantages of Allogenic Umbilical Cord Blood???Derived Stem Cells and Autologous Bone Marrow???Derived Mesenchymal Stem Cells Be Combined and Synergized?

FROM A CENTRED TO A DECENTRED TROPICALITY: FRANCOPHONE COLONIAL AND POSTCOLONIAL GEOGRAPHY IN MONSOON ASIA

ABSTRACTThis paper discusses four key phases in the study of the tropical milieu of monsoon Asia by French geographers and colonial actors over the last 150 years. First, it sees how the natural milieu initially occupied a central and determining place in French colonial and scientific assessments of Indochina, and how the notion of monsoon Asia appeared within the framework of an academic geography (and formatively in the two regional theses produced by Charles Robequain and Pierre Gourou). It then shows how, after the Second World War, francophone geographers developed a “tropicalist” approach to monsoon Asia, one that was inextricably linked with the culturalist approach that was pioneered by Gourou, which later evolved into new, model‐building and interdisciplinary approaches to the study of tropical systems. It argues that these successive phases have marked a shift from a centred tropicality ‐ a system of knowledge anchored in French colonialism and centring on debate about the determinist influence of the natural milieu ‐ to a decentred tropicality that rejects environmental determinism, questions the ethnocentric character of tropical geography and integrates the study of tropical geography into the wider social sciences. The paper concludes with the suggestion that tropicality has had an epistemologically stronger and more institutionalised relationship with francophone geography than was the case in anglophone geography.

Indirect CB2Receptor and Mediator-Dependent Stimulation of Human Whole-Blood Neutrophils by Exogenous and Endogenous Cannabinoids

Immunomodulatory effects of endogenous and exogenous cannabinoids have been investigated in numerous studies, mostly performed with isolated cells or transformed cell lines, but only sparse data exist on human polymorphonuclear neutrophils (PMNs). We therefore investigated the respiratory burst reaction of human whole-blood PMNs under the influence of cannabinoids using flow cytometry. In their natural whole-blood milieu, a CB(2) receptor-dependent stimulation of the PMN respiratory burst was found at nanomolar concentrations of CP55 940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol] and methanandamide after a 3-h incubation period, whereas the short-living and rapidly hydrolyzed endogenous ligand anandamide did not alter the burst reaction of whole-blood PMNs under the same experimental conditions. The stimulatory cannabinoid effect was totally absent in isolated PMNs but could be transferred onto isolated PMNs by adding the cell-free low-molecular mass plasma fraction (<5000 Da) of cannabinoid-incubated blood, indicating an indirect mechanism depending on humoral products or mediators. Results of our further experiments suggest that products of the arachidonic acid metabolism are mediators of the cannabinoid-induced enhancement of the respiratory burst reaction of whole-blood PMNs.

176 IMPROVED GROWTH OF HUMAN EMBRYONIC STEM CELLS IN A REDUCED OXYGEN ATMOSPHERE

Traditional cell culture conditions entail the use of gas atmosphere consisting of 5–6% CO2 in air. These same conditions have also been used universally for the culture of human embryonic stem cells (hES), despite the natural milieu of the embryos, from which these cells are derived, being slightly hypoxic. The aim of this work was to examine if human embryonic stem cells would benefit from the reduced oxygen culture environment, as used for human pre-implantation embryos. In Experiment 1, the relative growth in high and low oxygen atmosphere was compared by cutting undifferentiared hES-colonies into equal-size pieces and transferring them to two dishes, one into the conventional incubator in 6% CO2 in air (HIGH), the other into K-MINC-1000 (Eight Mile Plains, Queensland) mini-incubator in 5% O2, 6% CO2 and 89% N2 (LOW). After 8 days the colony sizes and differentiation status were measured. In Experiment 2, the absolute growth in high and low oxygen atmosphere was compared by cutting exactly same size fragments from undifferentiated hES-colonies and distributing them to two treatments as described. All colonies were measured and passaged at Day 7 and final measurements taken at Day 14. In Experiment 1, improved growth was observed in reduced oxygen, mean percentage of undifferentiated growth from original colony (assigned as 100%) being 217% for HIGH and 482% for LOW over three replicates. In Experiment 2, the total of 16.9 mm2 of undifferentiated colonies in both treatment groups (24 colonies per group over 3 replicates) had by Day 7 grown to total of 51.1 and 79.5 mm2 (P &lt; 0.001), and by Day 14 to 216.8 and 373.3 mm2 (P &lt; 0.0001) in HIGH and LOW, respectively (see Table 1). In neither experiment were there differences in the differentiation status of the colonies between the treatments (mean 6% and 5% at Day 7, and 9% and 9% at Day 14 in HIGH and LOW, respectively). We conclude that culture in reduced oxygen improves growth of human embryonic stem cells. Table 1. Growth of hES colonies on high and low oxygen atmosphere (data from 3 replicates) We like to acknowledge Bresagen Ltd. for the supply of hesBGN-01 cell line.

Method for in situ detection of the mitochondrial function in neurons

Conventional studies of neuronal mitochondria have been limited to the use of purified preparations of isolated mitochondria, neural cell homogenates, living neurons, or brain slices. However, each technique has several drawbacks. Here, we demonstrate that the neuronal cell’s membrane can be effectively permeabilized by saponin-treatment and that these permeabilized neurons can be used for qualitative and quantitative assessments of oxygen consumption in combination with registration of mitochondrial membrane potential and free [Ca 2+] in the matrix. Under these conditions, the mitochondrial function can be studied without removing the mitochondria from their natural milieu thus avoiding the damage of the associated cytoskeleton and outer membrane. At the same time, the method allows the estimation of the mitochondrial function independently of other processes in the cell, and the easy manipulation of the milieu surrounding the mitochondria. Thus, the presented method offers the opportunity to study the neuronal mitochondrial function in situ and can also be applied to examine the mitochondrial function by other commonly used methods.

Observations on parasitism in deep-sea hydrothermal vent and seep limpets

Parasite burdens of shallow-water molluscs have been well documented, but little is known about parasite burdens of molluscs from deep-sea chemosynthetic environments (e.g. hydrothermal vents and seeps). Chemosynthetic habitats are characterized by high concentrations of reduced sulfur and, in the case of vents, high heavy metal concentrations. These compounds are noxious and even stress-inducing in some environments, but are part of the natural chemical milieu of vents and seeps. To examine parasite types and infection intensities in limpets from vents and seeps we documented parasite burdens in 4 limpet species from 4 hydrothermal vent fields (3 on the East Pacific Rise, 1 on the Mid-Atlantic Ridge) and 1 seep site (Florida Escarpment). Approximately 50 % of all limpets examined were infected with 1 or more types of parasites. Limpet parasites were predominantly rickettsia-like inclusions in the digestive and gill epithelia. Limpets collected from the vent field on the Mid-Atlantic Ridge were free of parasites. We detected no histopathological effects that we could attribute to parasites.

Lipid Peroxidation in the Presence of Albumin, Inhibitory and Prooxidative Effects

Oxidative modifications of LDL are involved in atherogenesis. Previously we have developed a simple assay to evaluate the susceptibility of lipids to copper-induced peroxidation in the relatively natural milieu of unfractionated serum in the presence of excess citrate. Based on our previous results we have proposed that the inducer of peroxidation in our optimized assay is a copper-citrate complex. Recent investigations indicate that under certain conditions a copper-albumin complex may induce peroxidation of ascorbate. Two different complexes may be formed in albumin-containing systems (e.g. serum) namely 1:1 and 2:1 copper-albumin complexes. The aim of the present work was to evaluate the possibility that at least one of these complexes may be responsible for the induction of peroxidation of lipids in lipidic systems containing copper and albumin, including our optimized assay. Towards this end, we have investigated the dependence of copper-induced peroxidation on the concentration of added albumin in lipidic systems in the absence and presence of citrate. In all the systems investigated in this study (PLPC liposomes, LDL, HDL and mixtures of HDL and LDL) we found that at low concentrations of free copper (e.g. in the presence of excess citrate) the 2:1 copper-albumin complex is redox-active and that this complex is the major contributor to the initiation of lipid peroxidation in these systems and in our optimized assay. The possible relevance of the induction of peroxidation in vivo by the latter complex has yet to be studied. <footnote id="fn1"><no>*</no>This work was performed in partial fulfillment of the requirements for a Ph.D. degree of Dorit Samocha-Bonet, Sackler Faculty of Medicine, Tel-Aviv University, Israel. </footnote>

Urbanismo y naturaleza en América Latina: un matrimonio indisoluble

In many latinoamerican countries, the increase of urban population and the absence of planning have exposed an important fraction of the population to natural risks. People’s attitude toward their environment has varied with time, from fear and submission to active protection. In a country as Colombia, a theoretical equilibrium has been reached through environmental law: rational use of the natural milieu, using resources but respecting restrictions. However much remains to be done to obtain a truly “natural risk culture”, particularly with respect to a sufficient knowledge of landscape dynamics after human intervention. This knowledge is indispensable, since mankind. presently facing the possibility of a global change on a short basis. Systematic monitoring of changes in land use and natural or modified surface or subsurface processes is thus necessary to establish the presence of global change. Previous strategies and a serious effort to foster related educational programs should be organised at regional and national scales in order to face the problems that the planet is suffering.

INTRA-, EXTRA- AND INTERMUSCULAR MYOFASCIAL FORCE TRANSMISION OF SYNERGISTS AND ANTAGONISTS: EFFECTS OF MUSCLE LENGTH AS WELL AS RELATIVE POSITION

The concepts of intramuscular myofascial force transmission is reintroduced and reviewed on the basis of experiments involving tenotomy and aponeurotomy of dissected rat EDL muscle studied in situ. Results from experiments with measurements of force of EDL muscle, of which the muscle belly was not dissected (i.e. the muscle is surrounded by its natural connective tissue milieu) are discussed. In such experiments, force was measured at proximal as well as distal EDL tendons. Examples of experimental evidence for both extramuscular and intermuscular myofascial force transmission within the rat anterior crural compartment are presented. Evidence is presented also for differential effects of proximal and distal lengthening on myofascial force transmission from EDL, even for the case in which symmetric length changes were imposed on the muscle. It is shown that myofascial force transmission effects are not limited to synergists located within one compartment, but do also play a very substantial role in the interaction between antagonist muscles in neighbouring anterior crural and peroneal compartments.

El anillo verde de Vitoria-Gasteiz

Basically, the purpose of this initiative was to resolve the environmental problems of a number of areas of high ecological value which, located on the outskirts of the city, and subjected to many forms of aggression, coexisted with other degraded and marginal spaces that had been formed as a result of urban and industrial expansion. The restoration of the ecology and landscaping of these areas and their preparation for use by the public, have allowed the formation of a multifunctional green around the city which is providing rnajor environmental, social and economic benefits. The green ring has allowed nature to integrate with the life of the city distributing the offer of green areas equally among al1 the districts of the city and favouring the connection between green urban spaces and the surrounding agricultura1 land. This has also favoured the development of new leisure and recreational activities as well as those associated with training, education and environmental awareness; activities which, on the one hand, have represented a salutary lesson for the environmental business community as well as the integration into society and the job market of certain social groups, and on the other, have contributed to the conservation of these spaces, as their users have become their most fervent defenders and guardians. The use of some of these spaces for storm water management purposes in times of heavy rainfall and in order to resolve the problem of flooding in industrial areas, is another example of the added value obtained by un overall approach to this problem. The excellent results obtained after 9 years of work show the technical and economic viability and the social benefits of this project - eloquent proof of the determination of this municipality to advance towards a sustainable model of a city fully integrated in the surrounding natural milieu.

In vivo behaviour of long-circulating liposomes in blood vessels in hamster inflammation and septic shock models-use of intravital fluorescence microscopy.

This study aimed to observe liposome uptake by leukocytes in vivo. The study was performed on skin by using a dorsal skin-fold chamber implanted in golden hamsters using intravital microscopy. 5 and 6-CF-encapsulated polyethylene glycolated liposomes were injected intravenously. The skin microcirculation was observed with an intravital Eclipse E800 Nikon microscope (using x40, x80 magnification) fitted with a Xenon light source and an epifluorescence assembly (excitation, 470 nm, FWHM 40 nm; emission, 540 nm, FWHM 40 nm). An ultra-high sensitivity videocamera mounted on the microscope projected the image onto a monitor, and the images (720 x 576 pixels) were recorded for playback analysis with a digital video cassette recorder. An acute inflammatory response was obtained by removing one complete layer of skin and the underlying fascia and avascular tissue on the opposing side of the flap corresponding to an area equivalent to the window aperture. Using this model and set-up, leukocyte rolling and adhesion were easily observed and the entry of PEGylated liposomes into hamster blood leukocytes was studied for a period of 6 h. PEGylated liposomes were clearly identified alone inside the blood flow and inside the leukocytes as soon as the inflammatory reaction appeared. This study shows for the first time that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up, liposomes. This observation is in accordance with previous in vitro studies.

Multiple Ligand and Cell-Dependent States of Lateral Mobility of Plasmalemmal G Protein-Coupled Cholecystokinin Receptors

Lateral movement of receptor molecules in the plane of the plasmalemma has important implications for signal transduction and receptor regulation, yet mechanisms affecting such movement are not well understood. We have studied the lateral mobility of the G protein-coupled cholecystokinin (CCK) receptor expressed in the natural milieu of the rat pancreatic acinar cell and in a model cell system, the CHO-CCKR cell, after occupation with fluorescent agonist and antagonist. Lateral diffusion characteristics were distinct in each type of cell and for receptors occupied by each type of ligand, fluorescent agonist, rhodamine-Gly-[(Nle28,31)CCK-26-33], and fluorescent antagonist, rhodamine-Gly-[(D-Trp30,Nle28,31)CCK-26-32]-phenethyl ester. Multiple states of mobility were detected for CCK receptors. The slowest population of mobile receptors on the CHO-CCKR cells moved at similar rates when occupied by both antagonist and agonist, while the faster-moving populations moved at a faster rate when occupied with antagonist than with agonist. The fastest component of mobile receptors may reflect unconstrained interactions of the antagonist-occupied receptors with signaling or anchoring structures, while the slowest component may represent the fraction of ligand-occupied receptors that ultimately undergo internalization. The intermediate mobility states may reflect receptor interactions with signal transduction and regulatory machinery. While only a single population of mobile receptors was demonstrable on the acinar cells, increased ligand concentrations (agonist and antagonist) resulted in increased percentages of mobile receptors, suggesting a stoichiometric limitation of immobilizing molecular constraints. Inhibition of protein kinase C had no significant effect on the lateral mobility of agonist-occupied CCK receptors.