bluuebunny/crossref_metadata_2025 huggingface dataset:
Asef controls vascular endothelial permeability and barrier recovery in the lung
Authors: Tian Xinyong , Tian Yufeng , Gawlak Grzegorz , Meng Fanyong , Kawasaki Yoshihiro , Akiyama Tetsu , Birukova Anna A.
Published: December 2014

Increased levels of hepatocyte growth factor (HGF) in injured lungs may reflect a compensatory response to diminish acute lung injury (ALI). HGF-induced activation of Rac1 GTPase stimulates endothelial barrier protective mechanisms. This study tested the involvement of Rac-specific guanine nucleotide exchange factor Asef in HGF-induced endothelial cell (EC) cytoskeletal dynamics and barrier protection in vitro and in a two-hit model of ALI. HGF induced membrane translocation of Asef and stimulated Asef Rac1-specific nucleotide exchange activity. Expression of constitutively activated Asef mutant mimicked HGF-induced peripheral actin cytoskeleton enhancement. In contrast, siRNA-induced Asef knockdown or expression of dominant-negative Asef attenuated HGF-induced Rac1 activation evaluated by Rac-GTP pull down and FRET assay with Rac1 biosensor. Molecular inhibition of Asef attenuated HGF-induced peripheral accumulation of cortactin, formation of lamellipodia-like structures, and enhancement of VE-cadherin adherens junctions and compromised HGF-protective effect against thrombin-induced RhoA GTPase activation, Rho-dependent cytoskeleton remodeling, and EC permeability. Intravenous HGF injection attenuated lung inflammation and vascular leak in the two-hit model of ALI induced by excessive mechanical ventilation and thrombin signaling peptide TRAP6. This effect was lost in Asef− /−mice. This study shows for the first time the role of Asef in HGF-mediated protection against endothelial hyperpermeability and lung injury.
Cytoskeletal forces during signaling activation in Jurkat T-cells
Authors: Hui King Lam , Balagopalan Lakshmi , Samelson Lawrence E. , Upadhyaya Arpita
Published: December 2014

T-cells are critical for the adaptive immune response in the body. The binding of the T-cell receptor (TCR) with antigen on the surface of antigen-presenting cells leads to cell spreading and signaling activation. The underlying mechanism of signaling activation is not completely understood. Although cytoskeletal forces have been implicated in this process, the contribution of different cytoskeletal components and their spatial organization are unknown. Here we use traction force microscopy to measure the forces exerted by Jurkat T-cells during TCR activation. Perturbation experiments reveal that these forces are largely due to actin assembly and dynamics, with myosin contractility contributing to the development of force but not its maintenance. We find that Jurkat T-cells are mechanosensitive, with cytoskeletal forces and signaling dynamics both sensitive to the stiffness of the substrate. Our results delineate the cytoskeletal contributions to interfacial forces exerted by T-cells during activation.
Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress
Authors: Guiney Evan L. , Goldman Aaron R. , Elias Joshua E. , Cyert Martha S.
Published: December 2014

During hyperosmotic shock, Saccharomyces cerevisiae adjusts to physiological challenges, including large plasma membrane invaginations generated by rapid cell shrinkage. Calcineurin, the Ca2+/calmodulin–dependent phosphatase, is normally cytosolic but concentrates in puncta and at sites of polarized growth during intense osmotic stress; inhibition of calcineurin-activated gene expression suggests that restricting its access to substrates tunes calcineurin signaling specificity. Hyperosmotic shock promotes calcineurin binding to and dephosphorylation of the PI(4,5)P2phosphatase synaptojanin/Inp53/Sjl3 and causes dramatic calcineurin-dependent reorganization of PI(4,5)P2-enriched membrane domains. Inp53 normally promotes sorting at the trans-Golgi network but localizes to cortical actin patches in osmotically stressed cells. By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells. In response to hyperosmotic shock and calcineurin-dependent regulation, Inp53 shifts from associating predominantly with clathrin to interacting with endocytic proteins Sla1, Bzz1, and Bsp1, suggesting that Inp53 mediates stress-specific endocytic events. This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion. We propose that activation of Ca2+/calcineurin and PI(4,5)P2signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.
Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100
Authors: Wang Shiyu , Park Shuin , Kodali Vamsi K. , Han Jaeseok , Yip Theresa , Chen Zhouji , Davidson Nicholas O. , Kaufman Randal J.
Published: December 2014

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding–impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.
c-di-GMP induction ofDictyosteliumcell death requires the polyketide DIF-1
Authors: Song Yu , Luciani Marie-Françoise , Giusti Corinne , Golstein Pierre
Published: December 2014

Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP–induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.
Insights into the molecular basis of the NOD2 signalling pathway
Authors: Boyle Joseph P. , Parkhouse Rhiannon , Monie Tom P.
Published: December 2014

The cytosolic pattern recognition receptor NOD2 is activated by the peptidoglycan fragment muramyl dipeptide to generate a proinflammatory immune response. Downstream effects include the secretion of cytokines such as interleukin 8, the upregulation of pro-interleukin 1β, the induction of autophagy, the production of antimicrobial peptides and defensins, and contributions to the maintenance of the composition of the intestinal microbiota. Polymorphisms in NOD2 are the cause of the inflammatory disorder Blau syndrome and act as susceptibility factors for the inflammatory bowel condition Crohn's disease. The complexity of NOD2 signalling is highlighted by the observation that over 30 cellular proteins interact with NOD2 directly and influence or regulate its functional activity. Previously, the majority of reviews on NOD2 function have focused upon the role of NOD2 in inflammatory disease or in its interaction with and response to microbes. However, the functionality of NOD2 is underpinned by its biochemical interactions. Consequently, in this review, we have taken the opportunity to address the more ‘basic’ elements of NOD2 signalling. In particular, we have focused upon the core interactions of NOD2 with protein factors that influence and modulate the signal transduction pathways involved in NOD2 signalling. Further, where information exists, such as in relation to the role of RIP2, we have drawn comparison with the closely related, but functionally discrete, pattern recognition receptor NOD1. Overall, we provide a comprehensive resource targeted at understanding the complexities of NOD2 signalling.
ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding
Authors: Akai Yuko , Kanai Ryuta , Nakazawa Norihiko , Ebe Masahiro , Toyoshima Chikashi , Yanagida Mitsuhiro
Published: December 2014

Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast
Schizosaccharomyces pombe
is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.
The low-frequency spectrum of small Helmholtz resonators
Authors: Schweizer B.
Published: December 2014

We analyse the spectrum of the Laplace operator in a complex geometry, representing a small Helmholtz resonator. The domain is obtained from a bounded set

Ω
⊂

R

n

by removing a small obstacle
Σ

ε

⊂
Ω
of size
ε
>0. The set
Σ

ε

essentially separates an interior domain

Ω
ε

inn

(the resonator volume) from an exterior domain

Ω
ε

out

, but the two domains are connected by a thin channel. For an appropriate choice of the geometry, we identify the spectrum of the Laplace operator: it coincides with the spectrum of the Laplace operator on
Ω
, but contains an additional eigenvalue

μ
ε

−
1

. We prove that this eigenvalue has the behaviour
μ

ε

≈
V

ε

L

ε

/
A

ε

, where
V

ε

is the volume of the resonator,
L

ε

is the length of the channel and
A

ε

is the area of the cross section of the channel. This justifies the well-known frequency formula

ω

HR

=

c
0

A

/

(
L
V
)

for Helmholtz resonators, where
c
0
is the speed of sound.
Murimonas intestini gen. nov., sp. nov., an acetate-producing bacterium of the family Lachnospiraceae isolated from the mouse gut
Authors: Kläring Karoline , Just Sarah , Lagkouvardos Ilias , Hanske Laura , Haller Dirk , Blaut Michael , Wenning Mareike , Clavel Thomas
Published: December 2014

Three strains of an anaerobic, Gram-stain-positive coccobacillus were isolated from the intestines of mice. These strains shared 100 % similarity in their 16S rRNA gene sequences, but were distantly related to any described members of the family

Lachnospiraceae

(<94 %). The most closely related species with names that have standing in nomenclature were

Robinsoniella peoriensis

,

Ruminococcus gnavus

,

Blautia producta

and

Clostridium xylanolyticum

. Phylogenetic relationships based on 16S rRNA gene sequence analysis were confirmed by partial sequencing of hsp60 genes. The use of an in-house database search pipeline revealed that the new isolates are most prevalent in bovine gut samples when compared with human and mouse samples for

Ruminococcus gnavus

and

B. producta

. All three isolated strains shared similar cellular fatty acid patterns dominated by C16 : 0 methyl ester. Differences in the proportions of C12 : 0 methyl ester, C14 : 0 methyl ester and C18 : 1
cis-11 dimethyl acetal were observed when compared with phylogenetically neighbouring species. The major short-chain fatty acid produced by strain SRB-530-5-HT was acetic acid. This strain tested positive for utilization of d-fructose, d-galacturonic acid, d-malic acid, l-alanyl l-threonine and l-glutamic acid but was negative for utilization of amygdalin, arbutin, α-d-glucose, 3-methyl d-glucose and salicin, in contrast to the type strain of the closest related species

Robinsoniella peoriensis

. The isolates were not able to use mannitol for growth. Based on genotypic, phenotypic and chemotaxonomic characteristics, we propose to create the new genus and species Murimonas intestini gen. nov., sp. nov. to accommodate the three strains SRB-530-5-HT ( = DSM 26524T = CCUG 63391T) (the type strain of Murimonas intestini), SRB-509-4-S-H ( = DSM 27577 = CCUG 64595) and SRB-524-4-S-H ( = DSM 27578 = CCUG 64594).
Actinokineospora spheciospongiae sp. nov., isolated from the marine sponge Spheciospongia vagabunda
Authors: Kämpfer Peter , Glaeser Stefanie P. , Busse Hans-Jürgen , Abdelmohsen Usama Ramadan , Ahmed Safwat , Hentschel Ute
Published: December 2014

A Gram-staining-positive, aerobic organism, isolated from the Red Sea sponge Spheciospongia vagabunda was investigated to determine its taxonomic position. On the basis of results of 16S rRNA gene sequence analysis strain EG49T was most closely related to

Actinokineospora cibodasensis

and

Actinokineospora baliensis

(both 97.3 % similarity) and

Actinokineospora diospyrosa

and

Actinokineospora auranticolor

(both 97.0 % similarity). The 16S rRNA gene sequence similarity to all other species of the genus

Actinokineospora

was <97.0 %. The quinone system of strain EG49T contained the menaquinones MK-9(H4) (47 %), MK-9(H6) (27 %) and MK-9(H2) (15 %) in major amounts. Minor amounts of MK-7(H4) (2 %), MK-9(H0) (1 %), MK-9(H8) (3 %) and MK-10(H4) (3 %) were detected as well in addition to MK-8(H4), MK-8(H6), MK-10(H2) and MK-10(H6) (all <1 %). The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. In the polar lipid profile, diphosphatidylglycerol, phosphatidylethanolamine and hydroxyphosphatidylethanolamine were predominant. Phosphatidylinositol-mannoside, two unidentified phospholipids and two glycoglipids as well as one aminoglycolipid, one aminolipid and one unidentified lipid were found in addition. The fatty acid profile was composed of mainly iso-branched fatty acids: iso-C16 : 0, iso-C14 : 0, iso-C15 : 0 and iso-C16 : 1H. All these findings clearly supported the classification of the strain as representing a member of the genus

Actinokineospora

. In addition, the results of physiological and biochemical tests also allowed phenotypic differentiation of strain EG49T from the most closely related species of the genus

Actinokineospora

. Strain EG49T represents a novel species of the genus

Actinokineospora

, for which we propose the name Actinokineospora spheciospongiae sp. nov., with strain EG49T ( = DSM 45935T = CCM 8480T = LMG 27700T) as the type strain.
A biophysical approach to predicting protein-DNA binding energetics
Authors: Locke George , Morozov Alexandre V.
Published: December 2014

Sequence-specific interactions between proteins and DNA play a central role in DNA replication, repair, recombination, and control of gene expression. These interactions can be studiedin vitrousing microfluidics, protein-binding microarrays (PBMs), and other high-throughput techniques. Here we develop a biophysical approach to predicting protein-DNA binding specificities from high-throughputin vitrodata. Our algorithm, called BindSter, accommodates multiple protein species competing for access to DNA and alternative binding modes of the same protein, while rigorously taking into account all sterically allowed configurations of DNA-bound particles. BindSter can be used with a hierarchy of protein-DNA interaction models of increasing complexity. We observe that the quality of BindSter predictions does not change significantly as some of the energy parameters vary over a sizable range. To take this degeneracy into account, we have developed a graphical representation of parameter uncertainties, called IntervalLogo. We find that our simplest model, in which each nucleotide in the binding site is treated independently, performs better than previous biophysical approaches. The extensions of this model, in which contributions of longer words are also considered, result in further improvements, underscoring the importance of higherorder effects in protein-DNA energetics. In contrast, we find little evidence for multiple binding modes for the transcription factors (TFs) in our dataset. Furthermore, there is limited consistency in predictions for the same TF utilizing microfluidics and PBM experimental platforms.
Common binding by redundant group B Sox proteins is evolutionarily conserved inDrosophila
Authors: Carl Sarah H. , Russell Steven
Published: December 2014

Background

Group B Sox proteins are a highly conserved group of transcription factors that act extensively to coordinate nervous system development in higher metazoans while showing both co-expression and functional redundancy across a broad group of taxa. InDrosophila melanogaster, the two group B Sox proteins Dichaete and SoxNeuro show widespread common binding across the genome. While some instances of functional compensation have been observed inDrosophila, the function of common binding and the extent of its evolutionary conservation is not known.

Results

We used DamID-seq to examine the genome-wide binding patterns of Dichaete and SoxNeuro in four species ofDrosophila. Through a quantitative comparison of Dichaete binding, we evaluated the rate of binding site turnover across the genome as well as at specific functional sites. We also examined the presence of Sox motifs within binding intervals and the correlation between sequence conservation and binding conservation. To determine whether common binding between Dichaete and SoxNeuro is conserved, we performed a detailed analysis of the binding patterns of both factors in two species.

Conclusion

We find that, while the regulatory networks driven by Dichaete and SoxNeuro are largely conserved across the drosophilids studied, binding site turnover is widespread and correlated with phylogenetic distance. Nonetheless, binding is preferentially conserved at known cis-regulatory modules and core, independently verified binding sites. We observed the strongest binding conservation at sites that are commonly bound by Dichaete and SoxNeuro, suggesting that these sites are functionally important. Our analysis provides insights into the evolution of group B Sox function, highlighting the specific conservation of shared binding sites and suggesting alternative sources of neofunctionalisation between paralogous family members.
Ca3ZnGeO2[Ge4O12]: a Ca–Zn germanate related to the mineral taikanite
Authors: Redhammer Günther J.
Published: December 2014

The title compound, Ca3ZnGeO2[Ge4O12] (tricalcium zinc germanium dioxide dodecaoxidotetragermanate), adopts a taikanite-type structure. The tetrahedral [Ge4O12] chain geometry is very similar to that of the silicate chain of taikanite,i.e.BaSr2Mn3+2O2[Si4O12], while the major difference is found parallel to thecaxis. In taikanite, Mn3+octahedra form an infinite zigzag chain, whereas the title compound has a chain of distorted ZnO6octahedra, which alternates with distorted GeO4tetrahedra connected to each otherviatwo common edges. Eightfold-coordinated Ca2+polyhedra and ZnO6octahedra form a slab parallel to (001) which alternates with another slab containing the tetrahedrally coordinated Ge sites along thecaxis.
Symmetry groups associated with tilings on a flat torus
Authors: Loyola Mark L. , De Las Peñas Ma. Louise Antonette N. , Estrada Grace M. , Santoso Eko Budi
Published: December 2014

This work investigates symmetry and color symmetry properties of Kepler, Heesch and Laves tilings embedded on a flat torus and their geometric realizations as tilings on a round torus in Euclidean 3-space. The symmetry group of the tiling on the round torus is determined by analyzing relevant symmetries of the planar tiling that are transformed to axial symmetries of the three-dimensional tiling. The focus on studying tilings on a round torus is motivated by applications in the geometric modeling of nanotori and the determination of their symmetry groups.
Agriculture, health, and wealth convergence: bridging traditional food systems and modern agribusiness solutions
Authors: Dubé Laurette , Webb Patrick , Arora Narendra K. , Pingali Prabhu
Published: December 2014

The causes of many vexing challenges facing 21st‐century society are at the nexus of systems involved in agriculture, health and wealth production, consumption, and distribution. Using food as a test bed, and on the basis of emerging roadmaps that set achievable objectives over a 1‐ to 3‐year horizon, we introduce this special feature with convergence thinking and practice at its core. Specifically, we discuss academic papers structured around four themes: (1) evidence for a need for convergence and underlying mechanisms at the individual and societal levels; (2) strategy for mainstreaming convergence as a driver of business engagement and innovation; (3) convergence in policy and governance; (4) convergence in metrics and methods. Academic papers under each theme are accompanied by a roadmap paper reporting on the current status of concrete transformative convergence‐building projects associated with that theme. We believe that the insights provided by these papers have the potential to enable all actors throughout society to singly and collectively work to build supply and demand for nutritious food, in both traditional and modern food systems, while placing the burdens of malnutrition and ill health on their core strategic agendas.
Influence of GABA‐gated bicarbonate conductance on potential, current and intracellular chloride in crayfish muscle fibres.
Authors: Kaila K , Pasternack M , Saarikoski J , Voipio J
Published: December 2014

1. The effects of gamma‐aminobutyric acid (GABA) on membrane potential and conductance as well as on the intracellular Cl‐ activity (aiCl) and intracellular pH (pHi) were studied in crayfish muscle fibres using a three‐microelectrode voltage clamp and ion‐selective microelectrodes. In the presence of CO2‐HCO3‐, the intracellular HCO3‐ activity (aiHCO3) was estimated from pHi. 2. In a nominally HCO3(‐)‐free solution, a near‐saturating concentration of GABA (0.2 mM) produced a marked increase in membrane conductance but little change in potential. In a solution containing 30 mM‐HCO3‐ (equilibrated with 5% CO2 + 95% air; pH 7.4), the GABA‐induced increase in conductance was associated with a depolarization of about 15 mV, with an increase in aiCl and with a decrease in aiHCO3. All these effects were blocked by picrotoxin (PTX). The depolarizing action of GABA was augmented following depletion of extracellular and intracellular Cl‐. 3. The GABA‐induced increase in aiCl which took place in the presence of HCO3‐ was blocked by clamping the membrane potential at its resting level. This indicates that the increase in aiCl was due to passive redistribution of Cl‐. In both the presence and absence of HCO3‐, the GABA‐activated transmembrane flux of Cl‐ showed reversal at the level of the resting potential, which indicates that under steady‐state conditions the Cl‐ equilibrium potential (ECl) is identical to the resting potential. 4. In a Cl(‐)‐free, 30 mM‐HCO3(‐)‐containing solution, 0.5 mM‐GABA produced a PTX‐sensitive increase in conductance which amounted to 15% of the conductance activated in the presence of Cl‐. In the absence of both Cl‐ and HCO3‐, the respective figure was 2.8%. Assuming constant‐field conditions, the conductance data yielded a permeability ratio PHCO3/PCl of 0.42 for the GABA‐activated channels. 5. In a Cl(‐)‐containing, HCO3(‐)‐free solution, the reversal potential of the GABA‐activated current (EGABA) was, by about 1 mV, less negative than the resting membrane potential (RP). In a solution containing Cl‐ and 30 mM‐HCO3‐, EGABA‐RP was 12 mV. Simultaneous measurements of EGABA, aiCl and aiHCO3 (pHi) gave a PHCO3/PCl value of 0.33. 6. In a Cl(‐)‐free, HCO3(‐)‐containing solution EGABA was close to the HCO3‐ equilibrium potential (EHCO3) and an experimental acidosis which produced a negative shift in EHCO3 was associated with a similar shift in EGABA.(ABSTRACT TRUNCATED AT 400 WORDS)
Development of Y‐axon innervation of cortical area 18 in the cat.
Authors: Friedlander M J , Martin K A
Published: December 2014

1. Geniculocortical Y‐axons (n = 38) in the optic radiations of 4‐5‐week‐old kittens (n = 20) and adult cats (n = 18) were studied both physiologically and morphologically. Axons were recorded from intracellularly and subsequently filled ionophoretically with horseradish peroxidase (HRP). The HRP filled the axons' terminal arborizations in visual cortex (particularly well for those innervating area 18). Fourteen axons appeared to be completely filled with HRP (n = 8 in kitten, n = 6 in adult) and served as the basis for the quantitative analysis of the terminal arborizations reported in this study. 2. The distribution and correspondence of the axonal boutons to presynaptic elements in cortical layer 4A was analysed at both the light and electron microscope level using computerized three‐dimensional analysis and serial section reconstruction, respectively. Compared to adult axons, the boutons of the kitten axons were smaller (means = 0.75 vs. 1.75 microns length, P less than 0.001) and more densely spaced both along individual axon branches (means = 6.60 vs. 11.20 microns interbouton interval, P less than 0.001) and between neighbouring branches of the same axon (means = 4.7 vs. 6.4 microns nearest‐neighbour distance, P less than 0.01). 3. Most kitten boutons made a single Gray's type 1 synapse on a cortical neurone, unlike adult boutons which usually contacted two or more postsynaptic targets. Both kitten and adult axons had dendritic spines as their major target. Occasionally, HRP reaction‐product was observed in cortical neurones postsynaptic to the labelled geniculocortical axon, which gave some estimate of the number of synaptic contacts between a single geniculocortical axon and target cell (about five). 4. The kitten Y‐axons innervated the visual cortex in a pattern similar to that of the adult, with the richest terminal branching and bouton density in layer 4A with some additional boutons distributed in layers 3, 4B and 6. The extent of the terminal arborizations primarily in layer 4A (as measured in surface views) of kitten Y‐axons in area 18 was significantly less than that of adult Y‐axons in area 18 (means = 0.9 mm2 vs. means = 1.2 mm2, P = 0.04). 5. We conclude that between 4 and 5 postnatal weeks and 1 year, geniculocortical Y‐axons projecting to cortical area 18 undergo four major changes. These include a reduction in synaptic bouton density (both in three‐dimensional space and along individual branches), a concomitant moderate expansion in the surface area of cortex innervated, an increase in bouton size and an increase in the number of synaptic contacts made by each bouton. A general proportional growth of the individual axons' terminal arborization together with fusion and/or separation of neighbouring boutons is sufficient to explain this development.
Desensitization of swine arterial smooth muscle to transplasmalemmal Ca2+ influx.
Authors: Rembold C M
Published: December 2014

1. The effect of transplasmalemmal Ca2+ influx on the [Ca2+]i dependence of smooth muscle contraction was evaluated by measuring intracellular [Ca2+] (as estimated by aequorin), myosin phosphorylation, and isometric stress in swine carotid media. 2. Extracellular Ca2+ was removed by incubation in physiological saline with 1 mM‐EGTA and no added CaCl2 for 20 min (termed EGTA treatment). In some preparations, intracellular Ca2+ was released by a brief (5 min) histamine stimulation while in this Ca2(+)‐free EGTA solution (termed histamine treatment). 3. Restoration of extracellular CaCl2 to EGTA and histamine‐treated preparations in the continued presence of histamine was associated with an initial large aequorin light transient. However, this light transient was not initially associated with an increase in myosin phosphorylation or rapid stress development, suggesting that the contractile apparatus was desensitized to aequorin‐estimated myoplasmic [Ca2+]. The desensitization was temporary, and resolved by 10 min after restoration of extracellular CaCl2. 4. The light transient observed upon restoration of extracellular CaCl2 was smaller in preparations only EGTA treated when compared to preparations treated with both EGTA and histamine, suggesting that histamine treatment further desensitized the contractile apparatus. 5. The stress development rate was not slowed when histamine and extracellular CaCl2 were simultaneously added to EGTA‐treated preparations, suggesting that the desensitization was only to transplasmalemmal Ca2+ influx (from extracellular CaCl2 readdition), and not intracellular Ca2+ release (from the histamine stimulation). 6. In EGTA and histamine‐treated preparations, restoration of extracellular CaCl2 in the presence of 109 mM‐KCl was associated with a larger aequorin light signal than was observed upon readdition of CaCl2 in the presence of histamine, suggesting that depolarization also further desensitized the contractile apparatus. 7. Depolarization of EGTA‐treated preparations did not increase [Ca2+] or stress, suggesting that depolarization did not release intracellular Ca2+ stores. 8. No significant light transient was observed upon addition of extracellular LaCl3, suggesting that tissue damage or leakage of aequorin into the extracellular space was not the cause of the Ca2(+)‐reintroduction light signal. 9. These data suggest that removal of extracellular CaCl2 desensitizes the contractile apparatus of smooth muscle to transplasmalemmal Ca2+ influx. This desensitization is only to readdition of extracellular Ca2+; the contractile apparatus still responds to intracellular Ca2+ release. The desensitization is increased by prior depolarization or brief histamine treatment (potentially by depleting intracellular Ca2+). The source of activator Ca2+ appears to affect the relationship between aequorin light and phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
Peripheral projections of nociceptive unmyelinated axons in the human peroneal nerve.
Authors: Jørum E , Lundberg L E , Torebjörk H E
Published: December 2014

1. Previous knowledge of the anatomical course of unmyelinated (C) axons along a peripheral nerve has been scarce and has led to the concept of the axons in a constantly interchanging position. 2. Results obtained by microneurography in the peroneal nerve at knee or ankle levels in awake humans demonstrated that the receptive fields of neighbouring C units in the nerve cluster in close vicinity on the skin of the foot or the ankle. These findings indicate that C afferents run closely together throughout large portions of the peripheral nerve. 3. Intraneural microstimulation performed at neural sites where nociceptive C units were recorded induced painful sensations projected to the skin. When the stimulus intensity was increased, there was typically a concentric increase in the area of projected pain, rather than recruitment of several scattered pain projections. This finding further supports the hypothesis of a neighbouring relation of nociceptive C axons within nerve fascicles, implying spatial recruitment of adjacent axons in the nerve with adjacent peripheral projections. 4. A pain locognosia test performed during ischaemic block of impulse conduction in myelinated fibres demonstrated a fairly precise cerebral localization of noxious events on the foot from the input of C afferent fibres alone.
Electromyographic cross‐talk within a compartmentalized muscle of the cat.
Authors: English A W , Weeks O I
Published: December 2014

1. Experiments were conducted to test the extent to which the electromyographic (EMG) activity generated by the activation of single motor units is conducted from one neuromuscular compartment of the cat lateral gastrocnemius (LG) muscle into adjacent compartments. 2. Potentials produced by stimulation of forty‐five single motor units were monitored from bipolar fine‐wire EMG electrodes which had been implanted either into the centres of each of the four neuromuscular compartments of LG or into regions of the muscle known to lie on the border of contiguous compartments. 3. In all cases single unit potentials could be recorded from the electrodes in the centre of the compartments which clearly identified the compartment of residence of the muscle unit. Regardless of unit type, the amplitude of the potential recorded from electrodes in one compartment was always greater than that recorded from any other compartment. 4. Smaller potentials could be recorded from electrodes in the centre of compartments adjacent to the compartment of residence of the muscle unit. For those motor units where the amplitude of the EMG potentials recorded from the compartment of residence was large, the amplitude of such ‘cross‐talk’ could be greater than the amplitude of potentials recorded from the compartment of residence of smaller motor units. 5. In the case of electrodes placed at compartment boundaries, no clear compartment selectivity of recording of motor unit potentials was evident. 6. These results indicate that great care must be taken in choosing sites of EMG electrode placement when performing kinesiological studies, especially when the amplitude of the EMG activity recorded is of consideration.
Acetazolamide and transient responses of basolateral membrane potential of rabbit kidney proximal tubules perfused in vitro.
Authors: Beck J S , Potts D J
Published: December 2014

1. A study was made of the partial recovery of basolateral membrane potential that follows some depolarizing manoeuvres in cells of isolated perfused segments of rabbit proximal convoluted tubules. 2. Peritubular application of 10(‐4) M‐acetazolamide (a known inhibitor of the basolateral sodium‐bicarbonate co‐transporter) caused a hyperpolarization of both the basolateral membrane potential (Vbl) and the transepithelial potential (Vte). 3. Activation of electrogenic apical sodium co‐transport caused a depolarization of the basolateral membrane followed by partial recovery of potential, and a sustained transepithelial hyperpolarization. The partial recovery of basolateral membrane potential was significantly smaller in the presence of 10(‐4) M‐acetazolamide applied to the peritubular fluid, although the magnitude of the initial depolarization was not significantly altered. 4. Addition to the bath of 0.5 mM‐barium, a potassium conductance blocker, caused a transepithelial and basolateral membrane depolarization followed by partial recovery of potential. The partial recovery of basolateral membrane potential was significantly smaller in the presence of 10(‐4) M‐acetazolamide applied to the peritubular fluid, although the magnitude of the initial depolarization was again not significantly altered. 5. Increases in bath potassium concentration from 5 to 20 mM led to transepithelial and basolateral membrane depolarization followed by partial recovery of potentials. In paired experiments the partial recovery of basolateral potential was significantly reduced when 10(‐4) M‐acetazolamide was present in the bath. 6. These observations are consistent with the hypothesis that the basolateral sodium‐bicarbonate co‐transporter plays a role in the recovery of Vbl following these depolarizing manoeuvres.
ATP‐sensitive K+ channels in rat pancreatic beta‐cells: modulation by ATP and Mg2+ ions.
Authors: Ashcroft F M , Kakei M
Published: December 2014

1. The inside‐out configuration of the patch‐clamp method was used to study the effects of MgATP, free ATP and Mg2+ on single ATP‐sensitive K+ channel currents in rat pancreatic beta‐cells. 2. Magnesium ions caused a marked reduction of channel activity: 5 mM‐free Mg2+ produced a 50% reduction in the activity of inward currents recorded at ‐60 mV in symmetrical K+ concentrations. 3. Inhibition of channel activity by MgATP does not involve phosphorylation as both free ATP (i.e. ATP in the absence of divalent cations) and non‐hydrolysable ATP analogues were effective inhibitors. 4. Magnesium ions produced a striking reduction in the ability of ATP (total) to inhibit channel activity. When channel activity was plotted as a function of the total ATP concentration, the Ki for channel inhibition was 4 microM in Mg2(+)‐free solution, compared to a Ki of 26 microM in the presence of 2 mM‐Mg2+. The shape of the relationship between channel activity and the total ATP concentration was not changed by Mg2+. When channel activity was plotted as a function of the free ATP concentration, however, Mg2+ had little effect on Ki. This suggests that free ATP is the more potent inhibitor of channel activity and that MgATP has little inhibitory effect. 5. ATP analogues that dissociate only as far as the tribasic form were also able to inhibit channel activity. This suggests that both ATP4‐ and ATPH3‐ can block the channel. 6. Like ATP, ADP was more effective at inhibiting channel activity in the absence of Mg2+, that is as the free base. The non‐hydrolysable ATP analogues AMP‐PNP and AMP‐PCP, however, were more effective in the presence of Mg2+. 7. It is suggested that (1) the potency of inhibition is related to the amount of negative charge carried by the ion and (2) the intracellular concentration of free ATP will be an important modulator of channel activity in the intact beta‐cell.
Ionic basis of pacemaker generation in dog colonic smooth muscle.
Authors: Barajas-López C , Den Hertog A , Huizinga J D
Published: December 2014

1. The ionic basis of the slow waves in the circular muscle of the dog colon, in particular the ionic conductances involved in their initiation, were investigated by measuring intracellular electrical activity in the Abe‐Tomita‐type chamber for voltage control. 2. The depolarization that initiates the slow wave activity could be evoked by an increase in inward current and/or by a block of outward current. According to previous work, inward current could be carried by Na+, Cl‐, and Ca2+ ions; K+ ions would carry outward current. 3. The Na+ channel blocker tetrodotoxin (5 x 10(‐7) M) did not affect the slow wave amplitude nor its rate of rise. After omission of Na+, by replacing Na+ with N‐methyl‐D‐glucamine, large slow waves continued to develop although some changes in slow wave characteristics occurred. 4. Replacement of 91% of the Cl‐ by isethionate decreased the slow wave frequency and increased the slow wave amplitude. However, NaCl substitution by sucrose increased the slow wave frequency and decreased the slow wave amplitude. 5. Slow wave activity continued to develop after blockade of Ca2+ influx by D600 (10(‐6) M) or CoCl2 (1‐3 mM). D600 and Co2+ did not affect the membrane potential but reduced the slow wave amplitude and abolished the plateau potential. Slow waves were abolished after omission of extracellular Ca2+ (plus 1 mM‐EGTA). This suggests that Ca2+ influx is probably not necessary but extracellular presence of Ca2+ ions is indispensible for the slow wave generation. 6. The combination of 0 Na+, Li+ HEPES solution, by replacing Na+ with Li+, plus D600 depolarized the cells (up to approximately ‐40 mV) and abolished slow wave activity. This effect was voltage dependent since repolarization caused slow waves to return. 7. Abolition of the slow wave activity was also obtained by current‐induced depolarization to approximately ‐40 mV. However, during high‐K+‐induced depolarization (to approximately ‐40 mV) high amplitude (16 mV) slow waves were still present, slowing that the voltage dependence of the slow waves was shifted positively. This effect probably occurs due to modification by extracellular K+ of a voltage‐dependent K+ conductance, which would suggest that a K+ conductance is involved in slow wave generation. 8. In conclusion, slow waves are generated by cyclic membrane conductance changes, which are dependent on the presence of extracellular Ca2+ ions and on the membrane potential. Our data are consistent with the hypothesis that slow waves are initiated by the blockade of a K+ conductance.
Measurements of intracellular Ca2+ in dissociated type I cells of the rabbit carotid body.
Authors: Biscoe T J , Duchen M R , Eisner D A , O'Neill S C , Valdeolmillos M
Published: December 2014

1. The carotid body chemoreceptors are stimulated in situ by cyanide (CN‐), which mimics the effect of hypoxia. We have shown that CN‐ increases a calcium‐dependent potassium conductance (gK(Ca)) in single type I cells dissociated from the carotid body of the rabbit. We have now used the Ca2(+)‐sensitive fluorophore, Fura‐2, to measure intracellular Ca2+ directly in single type I cells. 2. CN‐ reversibly increased [Ca2+]i from approximately 90 nM to a mean of approximately 200 nM. Some of this Ca2+ originated from an intracellular store, which was depleted by exposure to Ca2(+)‐free solutions. Prolonged application of CN‐ caused a sustained increase in [Ca2+]i, suggesting that CN‐ impairs the removal or sequestration of Ca2+. 3. pHi measured with the dye BCECF (2,7‐bis(2‐carboxyethyl)‐5(and‐6)‐carboxyfluorescein) did not change consistently in response to CN‐, although pHi changed predictably in response to both ammonium chloride and to acidification of the superfusate with CO2. 4. Potassium‐induced depolarization (35 mM‐K+) caused a large, cadmium‐sensitive rise in [Ca2+]i. The K(+)‐induced Ca2+ load was used to study the regulation of [Ca2+]i. 5. The clearance of a Ca2+ load was slowed either by removal of [Na+]o or by application of CN‐. This shows that both a Na+‐Ca2+ exchange and an energy‐dependent process or processes contribute to the regulation of [Ca2+]i. 6. Carbachol (CCh, 10‐100 microM), which also hyperpolarizes type I cells, caused a small transient rise in [Ca2+]i, indicating release from an exhaustible intracellular pool. The response to CN‐ was unaffected by prior or continued exposure to CCh, suggesting that the two stimuli operate by distinct mechanisms. 7. The increased gK(Ca) seen in type I cells in response to CN‐ thus reflects a change in cellular Ca2+ homeostasis. The rise in [Ca2+]i presumably underlies the documented increase in transmitter release from the carotid body in response to CN‐. If chemotransduction is a consequence of the release of transmitters from the type I cell, the response of the carotid body to CN‐, and possibly also to hypoxia, is thus a direct consequence of the energy dependence of Ca2+ homeostasis in the type I cell.
Amino acid efflux in the isolated perfused rat pancreas: trans‐stimulation by extracellular amino acids.
Authors: Mann G E , Norman P S , Smith I C
Published: December 2014

1. Epithelial uptake and efflux of the non‐metabolized system A analogue 2‐methylaminoisobutyric acid (MeAIB) and L‐serine were studied in the isolated perfused rat pancreas using a dual tracer loading and wash‐out technique. Uptakes of 2‐[14C]MeAIB and L‐[3H]serine were measured relative to D‐[3H or 14C]mannitol (extracellular tracer) during a 20 min cell loading period. Maximal uptake for MeAIB (34 +/‐ 2%, n = 6) occurred within 2‐3 min and decreased to 14 +/‐ 2% after 20 min tracer loading. Uptake for L‐serine reached a maximum (62 +/‐ 4%, n = 7) within 1 min and decreased to 19 +/‐ 2% after 20 min tracer loading. 2. When tracer wash‐out was monitored during subsequent perfusion of the preloaded pancreas with an isotope‐free solution, D‐mannitol predominantly cleared from a fast exchanging compartment (0.54 +/‐ 0.05 ml g‐1, n = 9) with a time constant (Tfast) of 0.68 +/‐ 0.04 min. Although MeAIB and L‐serine exhibited similar fast phases of wash‐out, a much larger efflux occurred from a slowly exchanging pool with respective time constants (Tslow) of 15.47 +/‐ 0.45 min (n = 6) and 5.98 +/‐ 0.46 min (n = 7). 3. A rapid vascular challenge of the pancreas with 100 mM‐L‐serine transiently accelerated cellular efflux of 2‐[14C]MeAIB and L‐[3H]serine without affecting wash‐out of D‐[14C]mannitol. Tracer efflux following cell loading with 2‐[14C]MeAIB or L‐[3H]serine was not stimulated by a challenge with 100 mM‐MeAIB. 4. The time course of amino acid evoked 2‐[14C]MeAIB and L‐[3H]serine efflux paralleled the extracellular dilution profile of a vascular stimulus, suggesting that the acceleration of efflux was due to trans‐stimulation. 5. Trans‐stimulation of 2‐[14C]MeAIB and L‐[3H]serine efflux by a further twenty‐two naturally occurring and three synthetic amino acids was then examined. L‐Proline, N‐methyl‐DL‐alanine, L‐lysine and D‐lysine selectively stimulated MeAIB efflux. Efflux of both tracer amino acids was accelerated by aminoisobutyric acid (AIB), L‐serine, L‐alanine, L‐cysteine, L‐threonine, glycine, branched‐chain and large neutral amino acids, but affected negligibly by D‐alanine, beta‐alanine, MeAIB, L‐arginine, L‐aspartate, L‐glutamate, taurine or D‐mannitol. 6. In summary, we have characterized amino acid exchange mechanisms in the isolated pancreas and conclude that efflux of intracellular amino acids from pancreatic acinar cells may be mediated by at least two transporters with differing specificity for extracellular amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)