Glutamine transport at the blood-brain and blood-cerebrospinal fluid barriers

Glutamine has multiple physiological and pathophysiological roles in the brain. Because of their position at the interface between blood and brain, the cerebral capillaries and the choroid plexuses that form the blood-brain barriers (BBB) and blood-cerebrospinal fluid (CSF) barriers, have the potential to influence brain glutamine concentrations. Despite this, there has been a paucity of data on the mechanisms and polarity of glutamine transport at these barrier tissues. In situ brain perfusion in the rat, indicates that blood to brain L-[14C]glutamine transport at the blood-brain barrier is primarily mediated by a pH-dependent, Na(+)-dependent, System N transporter, but that blood to choroid plexus transport is primarily via a pH-independent System N transporter and a Na(+)-independent carrier that is not System L. Transport studies in isolated rat choroid plexuses and primary cultures of choroid plexus epithelial cells indicate that epithelial L-[14C]glutamine transport is polarized (apical uptake>basolateral) and that uptake at the apical membrane is mediated by pH dependent System N transporters (identified as SN1 and SN2 by polymerase chain reaction) and the Na(+)-independent System L. Blood-brain barrier System N transport is markedly effected by cerebral ischemia and may be a good marker of endothelial cell dysfunction. The multiple glutamine transporters at the blood-brain and blood-CSF barriers may have role in meeting the metabolic needs of the brain and the barrier tissues themselves. However, it is likely that the main role of these transporters is removing glutamine, and thus nitrogen, from the brain.     

How to Connect Cardiac Excitation to the Atomic Interactions of Ion Channels

Many have worked to create cardiac action potential models that explicitly represent atomic-level details of ion channel structure. Such models have the potential to define new therapeutic directions and to show how nanoscale perturbations to channel function predispose patients to deadly cardiac arrhythmia. However, there have been significant experimental and theoretical barriers that have limited model usefulness. Recently, many of these barriers have come down, suggesting that considerable progress toward creating these long-sought models may be possible in the near term.     

Effect of buried potential barrier in label-less electrochemical immunodetection of glutathione and glutathione-capped gold nanoparticles

The influence of potential barriers, introduced to the immunoglobulin-based sensory films, on voltammetric signals of a redox ion probe has been investigated. Films with positive and negative barriers have been examined by depositing charged self-assembled thiol monolayers as the basal layers of a sensory film. The studies performed with monoclonal anti-glutathione antibody-based sensors using ferricyanide ion probe have shown stronger sensor response to the layer components, as well as to the glutathione-capped gold nanoparticles acting as the antigen, for films with positive potential barrier buried deep in the film than for negative barrier films. The larger changes in differential resistance, peak separation and peak heights observed for films with positive barrier have been attributed to different depth and width of the charge distributions in these films. A buried positive barrier with narrow charge distribution width provides the best conditions for film stability and prevents fouling (less ion-exchanges with the medium). This conclusion has been confirmed by calculations of the electric field distribution and potential profiles in immunosensing films performed by numerical integration of Poisson equation for Gaussian distributions of fixed charges of covalently bound components. The proposed fixed-charge model can aid in rapid evaluation of sensory films in sensor development work. The implications of potential barriers in sensory film design are discussed.     

Microfluidics-assisted rapid generation of tubular cell-laden microgel inside glass capillaries

Drug screening using engineered blood vessels (EBVs) faces considerable barriers in approximating the conditions of an in vivo environment. To address this issue, we have introduced a microfluidic system for cell-laden tubular microgels. N-Carboxyethyl chitosan crosslinked with oxidized dextran was used for in situ gelable tubular scaffolds. The microfluidic system consisted of four glass capillaries that generated a coaxial flow of pre-polymer and phosphate buffered solutions. It rapidly produced cell-laden tubular microgels inside glass capillaries. The mechanical strength of the tubular microgels was suitable for their application as EBVs, with a maximum Young’s modulus of 12.2 ± 1.9 kPa. In vitro cell studies using human umbilical vein endothelial cells verified the biocompatibility and non-cytotoxicity of the gelation and fabrication process. Thus, in situ gelable cell-laden tubular microgels can be a potential platform for screening drugs to treat blood vessel diseases.     

Free energy analysis of conductivity and charge selectivity of M2GlyR-derived synthetic channels

Significant progresses have been made in the design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the second transmembrane domain of the α-subunit of the glycine receptor (GlyR). The latest designs, including p22 (KKKKP ARVGL GITTV LTMTT QS), are highly soluble in water with minimal aggregation propensity and insert efficiently into cell membranes to form highly conductive ion channels. The last obstacle to a potential lead sequence for channel replacement treatment of CF patients is achieving adequate chloride selectivity. We have performed free energy simulation to analyze the conductance and charge selectivity of M2GlyR-derived synthetic channels. The results reveal that the pentameric p22 pore is non-selective. Moderate barriers for permeation of both K⁺ and Cl⁻ are dominated by the desolvation cost. Despite previous evidence suggesting a potential role of threonine side chains in anion selectivity, the hydroxyl group is not a good surrogate of water for coordinating these ions. We have also tested initial ideas of introducing additional rings of positive changes to various positions along the pore to increase anion selectivity. The results support the feasibility of achieving anion selectivity by modifying the electrostatic properties of the pore, but at the same time suggest that the peptide assembly and pore topology may also be dramatically modified, which could abolish the effects of modified electrostatics on anion selectivity. This was confirmed by subsequent two-electrode voltage clamp measurements showing that none of the tested mono-, di- and tri-Dap substituted sequences was selective. The current study thus highlights the importance of controlling channel topology besides modifying pore electrostatics for achieving anion selectivity. Several strategies are now being explored in our continued efforts to design an anion selective peptide channel with suitable biophysical, physiological and pharmacological properties as a potential treatment modality for channel replacement therapy. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Ultramicroscopic organization of the vascular plexus in the rabbit cerebral lateral ventricles and its changes under the effect of pyrogens

By means of electron microscopical methods organization of various components of the vascular plexus in the rabbit cerebral lateral ventricles--ependymal and supraependymal cells, as well as capillaries, making the base of the hemato-encephalic and hemato-liquor barriers of the plexus have been studied. Injection of bacterial pyrogen (pyrogenal) to the animals is accompanied with an increasing permeability in the barriers of the vascular plexus for certain blood cells (lymphocytes, monocytes), their activation and transformation into plasma cells and macrophages. Under pyrogenal effect in the ventricle cavities activation of the supraependymal cells and intensification of their interaction with the underlying ependyma take place. In the ependymal layer local dilatation of intercellular spaces and intensification of exocytic processes are observed. The changes revealed demonstrate an active reaction of some elements of the vascular plexus to fever, dependent on injection of the bacterial pyrogens into the organism.     

Potentials of mean force in acidic proton transfer reactions in constrained geometries

Free energy barriers associated with the transfer of an excess proton in water and related to the potentials of mean force in proton transfer episodes have been computed in a wide range of thermodynamic states, from low-density amorphous ices to high-temperature liquids under the critical point for unconstrained and constrained systems. The latter were represented by set-ups placed inside hydrophobic graphene slabs at the nanometric scale allocating a few water layers, namely one or two in the narrowest case. Water–proton and carbon–proton forces were modelled with a Multi-State Empirical Valence Bond method. As a general trend, a competition between the effects of confinement and temperature is observed on the local hydrogen-bonded structures around the lone proton and, consequently, on the mean force exerted by its environment on the water molecule carrying the proton. Free energy barriers estimated from the computed potentials of mean force tend to rise with the combined effect of increasing temperatures and the packing effect due to a larger extent of hydrophobic confinement. The main reason observed for such enhancement of the free energy barriers was the breaking of the second coordination shell around the lone proton.     

Identification of lymph and blood capillaries by immunohistochemical staining for various basement membrane components.

In the present report we analyzed the presence and distribution of various basement membrane (BM) proteins in normal blood and lymph vessels with special emphasis on BM-associated heparan sulfate proteoglycan (HSPG) when compared to the BM-components collagen IV, laminin and fibronectin. We found that normal lymph capillaries have a BM that contains only collagen IV and small amounts of laminin, but almost no BM-associated HSPG and fibronectin, while blood capillaries showed a BM comprising of all components tested for. Larger lymphatics, however, were indistinguishable from blood vessels on the basis of BM staining. Lymphangiomas showed a BM pattern similar to that of lymph capillaries. Our findings provide evidence that the differential staining of BM-components may represent a reliable method for morphological distinction between blood and lymph capillaries. A comparison of these results with the BM-pattern in other functionally specialized blood vessels (glomerulus, sinusoids) provides evidence that the BM-composition may have some major impact on the functional properties. Thus, it is conceivable that the lack of HSPG in lymph capillaries may be essential for a free influx of fluid and proteins into these capillaries, which may have been extravasated into the interstitium.     

Identification of lymph and blood capillaries by immunohistochemical staining for various basement membrane components

Summary In the present report we analyzed the presence and distribution of various basement membrane (BM) proteins in normal blood and lymph vessels with special emphasis on BM-associated heparan sulfate proteoglycan (HSPG) when compared to the BM-components collagen IV, laminin and fibronectin. We found that normal lymph capillaries have a BM that contains only collagen IV and small amounts of laminin, but almost no BM-associated HSPG and fibronectin, while blood capillaries showed a BM comprising of all components tested for. Larger lymphatics, however, were indistinguishable from blood vessels on the basis of BM staining. Lymphangiomas showed a BM pattern similar to that of lymph capillaries. Our findings provide evidence that the differential staining of BM-components may represent a reliable method for morphological distinction between blood and lymph capillaries. A comparison of these results with the BM-pattern in other functionally specialized blood vessels (glomerulus, sinusoids) provides evidence that the BM-composition may have some major impact on the functional properties. Thus, it is conceivable that the lack of HSPG in lymph capillaries may be essential for a free influx of fluid and proteins into these capillaries, which may have been extravasated into the interstitium.     

Ultrastructure of the capillaries of nerve tissue transplants growing in the anterior chamber of the eye

Blood capillaries have been studied electron microscopically in the areas of grafts (rat embryonal hippocamp and septal cerebral parts transplanted to mature rats) containing mainly nervous, glial or connective tissue cells. Certain differences in the capillary wall structure have been revealed. In areas with a great concentration of nervous cells, the blood capillaries are characterized by a dense arrangement of cellular elements in their walls, a continuous layer of the glial end-feet, this is specific for the CNS capillaries providing the blood--brain barrier. In peripheral area of the grafts, where glial elements predominate, the capillaries have loose arrangement of the mural cellular elements, great endotheliocyte activity, thick connective tissue tunic, lack of a dense glial surrounding. These characteristics make dubious the statement whether these capillaries possess the blood--brain barrier function. In places where connective tissue cells make aggregates, the capillaries do not possess the barrier properties because of perforations and fenestrae in endothelium and interruptions of the basal membrane, absence of pericapillary glial elements. All types of the capillaries demonstrate certain signs of a high functional activity. Formation of the capillary structure depends on the surrounding tissue.     

The ultrastructure of frog mesenteric capillaries of known filtration coefficient.

The capillaries of the frog mesentery were investigated by transmission electronmicroscopy. The mean diameter of these vessels was 20 micrometers and a single layer of five to six endothelial cells (on average) contributed to each circumference. Their ultrastructure was very similar to that of mammalian capillaries of the continuous type [Bennett, Luft and Hampton, 1959]. The endotherlial cells, which contained many vesicles, were joined by specialized junctions and surrounded by a continuous basement membrane. Eleven capillaries were examined after the filtration coefficient of their walls had been investigated by the microperfusion micro-occlusion technique of Michel, Mason, Curry and Tooke [1974]. No abnormal appearances were observed even after the vessels had been perfused with protein free solutions which have been shown to increase filtration coefficient five fold. No morphological differences between capillaries were seen to accompany large differences in the filtration coefficients.     

Climatic and geographic barriers drive distributional patterns of bird phenotypes within peninsular India

Modern phylogenetic data provide unparalleled ability to test biogeographic paradigms, often suggested by differences in species distribution patterns. However, such approaches have been applied less at regional scales, particularly in Asia. In the absence of such data, we examine if concordance of distributional patterns for phenotypes (subspecies) suggest potential biogeographic barriers for birds in peninsular India. Specifically, we examine climatic and physical factors that might limit phenotype distributions in this region. Various physical, vegetation and climatic barriers were demarcated to identify potential biogeographic units within peninsular India. We then collated occurrence of endemic or disjunctly distributed species and subspecies within these units using published range maps. We also quantified turnover between potential units, allowing us to identify significant biogeographic barriers. Three time‐step climate data (Last Glacial Maxima, mid‐Holocene and present) enabled us to examine differences between these potential biogeographic regions through time. The Palk Straits, followed by the Goa Gap (～ 16°N) and the Godavari River emerged as the major barriers in this region. The Palk Straits and Godavari are physical barriers while Goa Gap appears to be a climate‐mediated ecological divide. Mountain barriers like the Palghat Gap are not the most significant barriers as previously thought. Climatically intermediate regions appeared unstable in the past and showed inconsistent affinities to different geographic units across families. We suggest that relative climatic stability of the wet regions of the southern western Ghats could be responsible for high subspecies endemism here. Our approach provides hypotheses that can be tested with comparative multi‐species phylogeographic data in the future.     

Plant-derived antifungal proteins and peptides

Plants produce potent constitutive and induced antifungal compounds to complement the structural barriers to microbial infection. Approximately 250,000-500,000 plant species exist, but only a few of these have been investigated for antimicrobial activity. Nevertheless, a wide spectrum of compound classes have been purified and found to have antifungal properties. The commercial potential of effective plant-produced antifungal compounds remains largely unexplored. This review article presents examples of these compounds and discusses their properties.     

Electrical resistance of a capillary endothelium

The electrical resistance of consecutive segments of capillaries has been determined by a method in which the microvessels were treated as a leaky, infinite cable. A two-dimensional analytical model to describe the potential field in response to intracapillary current injection was formulated. The model allowed determination of the electrical resistance from four sets of data: the capillary radius, the capillary length constant, the length constant in the mesentery perpendicular to the capillary, and the relative potential drop across the capillary wall. Of particular importance were the mesothelial membranes covering the mesenteric capillaries with resistances several times higher than that of the capillary endothelium. 27 frog mesenteric capillaries were characterized. The average resistance of the endothelium was 1.85 omega cm2, which compares well with earlier determinations of the ionic permeability of such capillaries. However, heterogeneity with respect to resistance was observed, that of 10 arterial capillaries being 3.0 omega cm2 as compared with 0.95 omega cm2 for 17 mid- and venous capillaries. The average in situ length constant was 99 micrometers for the arterial capillaries and 57 micrometers for the mid- and venous capillaries. It is likely that the ions that carry the current must move paracellularly, through junctions that are leaky to small solutes.     

Pathogenesis of chronic venous insufficiency and possible effects of compression and pentoxifylline.

It has been recognized for over 2000 years that ulceration of the leg may be associated with visible varices of the lower limb. More recent physiological investigation has shown that the pressure in the veins of the lower limb remains raised in patients with venous ulceration during ambulation, whereas in normal subjects the pressure in superficial veins falls to a low level. This elevated pressure appears to cause damage to the superficial capillaries in the skin culminating in the production of venous ulceration. Events in the dermal capillaries which result in skin destruction have yet to be fully defined. Pericapillary fibrin cuffs have been demonstrated histologically and suggested as a cause of diminished nutrition to the skin. White blood cells have been shown to accumulate in the lower limb of patients with venous disease and these accumulations are particularly located around the dermal capillaries. Activated white blood cells releasing free radicals and destructive enzymes may precipitate skin destruction. An understanding of these mechanisms may help to explain the efficacy of compression hosiery and bandaging as well as some of the new pharmacological agents which have been shown to influence venous ulcer healing.     

Mucin dynamics and enteric pathogens

The extracellular secreted mucus and the cell surface glycocalyx prevent infection by the vast numbers of microorganisms that live in the healthy gut. Mucin glycoproteins are the major component of these barriers. In this Review, we describe the components of the secreted and cell surface mucosal barriers and the evidence that they form an effective barricade against potential pathogens. However, successful enteric pathogens have evolved strategies to circumvent these barriers. We discuss the interactions between enteric pathogens and mucins, and the mechanisms that these pathogens use to disrupt and avoid mucosal barriers. In addition, we describe dynamic alterations in the mucin barrier that are driven by host innate and adaptive immune responses to infection.     

Multimeasurement analysis of the quantitative spatial organization of epicardial lymphatic capillaries under normal conditions

Principles of quantitative-spatial organization of the epicardial lymphatic capillaries have been studied in 26 normal dogs' hearts. Multimeasurable systemic models have been constructed to demonstrate how quantitative indices of metrical properties of the capillaries depend on the cardiac size. The possibility is discussed to apply the data obtained in the study of microcirculatory pathology of different genesis.     

Pulmonary gene delivery using polymeric nonviral vectors

Pulmonary delivery provides an easy and well tolerated means of access for the administration of biomacromolecules to the pulmonary epithelium and could therefore be an attractive approach for local and systemic therapies. A growing number of reports, which are summarized in this review, mirror the viability of pulmonary gene delivery. Special attention has been paid to the biological barriers in the lung that must be overcome for successful delivery, and which can be divided into anatomic, physical, immunologic, and metabolic barriers. In light of these barriers, successful nonviral polymer-based formulations of therapeutic genes are presented depending on the chemical nature of the polymer. In addition to polyethyleneimine-based nonviral vectors, which have been most intensively studied for pulmonary gene delivery in the past, other polymeric, dendritic, and targeted materials are also described here, including novel and biodegradable polymers. As new materials need in vitro or ex vivo testing before in vivo application, sophisticated models for all three approaches have been illustrated. Although pulmonary siRNA delivery enjoys popularity in clinical trials, pulmonary gene delivery has so far not been translated into clinical applications. With this review, potential hurdles are demonstrated, but novel approaches that may lead to optimized systems are described as well.     

Barriers To Biological Fieldwork: What Really Prevents Teaching Out of Doors?

This paper considers a range of factors that may contribute to an unwillingness or inability of teachers to participate in the teaching of biology through fieldwork. Through a synthesis of the views of both pre-service teachers in training and primary school teachers in practice we explore the relative importance of a wide range of potential barriers and potential responses to them in the context of the wider literature. We conclude that although fieldwork may be impeded by the interaction of a wide range of individual barriers, including an individual’s predisposition towards the outdoors, it is possible to group interacting barriers into two main areas: school culture and teacher confidence. It is also apparent that barriers may assume different levels of significance when considered in general terms rather than when applied to a particular context and that the significance of barriers may change through time. Encouragingly, we have also shown that in-service teachers have a willingness to overcome these barriers.