bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of apoptosis‐like endonuclease activity in avian thymocytes
Authors: Machaca Khaled , Compton Mark M.
Published: September 2004

Summary— In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid‐treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence‐activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P‐DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibito aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.
Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro
Authors: Rakoczy Piroska , Kennedy Christopher , Thompson‐Wallis Dawn , Mann Krishna , Constable Ian
Published: September 2004

Summary— The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4‐week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7‐year‐old donor compared with those from a 47‐year‐old donor. Within both groups the BROs‐challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S‐labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS‐challenged RPE cells of both bovine and human origin that did not appear in control or microsphere‐phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.
Composition of nuclear dense bodies and nucleolus‐associated bodies in interphase nuclei of the unicellular green alga Chlamydomonas reinhardtii
Authors: Tremblay Stéphane D. , Lafontaine Jean G.
Published: September 2004

Summary— The interphase nucleus of the green alga, Chlamydomonas reinhardtii, displayed two types of bodies some of them, the dense bodies, lying apparently free in the nucleoplasm while the others were attached to the nucleolus and were, therefore, referred to as nucleolus‐associated bodies (NABs). The presence of DNA, RNA and histones in dense bodies was investigated by means of post‐embedding immunocytochemistry and cytochemistry using a monoclonal antibody to single and double stranded DNA, a polyclonal antibody to rye H3 histones and RNase A‐gold complexes. The dense bodies were shown to contain significant amounts of RNA but neither DNA nor histones were detected; their composition was thus similar to that of the dense bodies described in higher plant cells. We propose that dense bodies might be implicated in the assembly of the 25 to 45 nm granules observed throughout the nucleoplasm of Chalamydomonas interphase nuclei. The composition of NABs was found to be distinct from that of the dense bodies since they were labeled by the antibody to DNA, specially in cryofixed and cryosubstituted specimens. The presence of DNA in NABs together with their intimate association to the nucleolus suggest that they may correspond to specific segments of chromosomes.
Adhesive properties of sea cucumber coelomoyctes
Authors: Canicatti Calogero , Rizzo Antonella , Montinari Maria Rosa
Published: September 2004

Summary— The adhesive properties of the coelomocytes of the sea cucumber, Holothuria polii, have been investigated by studying their ability to attach to glass coverslips in vitro, and their morphology examined by scanning electron microscopy. Both amoeboyctes and spherule cells in cell suspensions attached themselves to glass coverslips, but spreading activity was restricted to an amoebocyte subset which assumed an extremely flattened morphology. Coelomocyte adhesion was a time‐ and temperature‐dependent phenomenon and required cations for attachment to the glass surface. Mg2+ ions were more effective than Ca2+ in facilitating cell binding. The addition of potassium cyanide or sodium azide to the cell suspension did not inhibit amoebocyte attachment but vinblastine did. Cytochalasin B had no effect. Cell adhesiveness was greatly enhanced with both coelomic fluid and purified 200‐kDa coelomocyte‐aggregating factor.
Identification of novel adhesion proteins in mouse peritoneal macrophages
Authors: Tomita Mitsuko , Ishikawa Harunori
Published: September 2004

Summary— When mouse peritoneal macrophages were made to adhere firmly on glass surface and then removed by sequential treatment with hypotonic triethanolamine and Nonide P‐40, a set of proteins were found to be left behind at the sites of adherent cells. Such glass‐adherent proteins were detected as round or ellipsoidal patches of autofluorescence under a confocal laser microscope, and visualized ultrastructurally as aggregates to narrow threads of unique loop structures which were composed of linearly aligned particles of 22 ± 2 nm in diameter. Lithium dodecylsulfate‐polyacrylamide gel electrophoresis of the glass‐adherent proteins showed two major bands, 12 kDa and 14 kDa, which always co‐existed in any different sample. The polyclonal antibody raised against these two proteins specifically stained the glass‐adherent proteins in situ. The adhesion of macrophages to glass was significantly blocked with Fab fragments of the antibody. The in situ cross‐linking experiment suggested that these two proteins might be closely associated with each other to form complexes. Hence, these proteins can be reasonably considered to be responsible for non‐specific adhesion of macrophages to glass.
Immunodetection and localization of protein(s) related to retinal S‐antigen (arrestin) in kidney
Authors: Mirshahi Massoud , Razaghi Ahmad , Vandewalle Alain , Cluzeaud Françoise , Tarraf Mohammed , Faure Jean‐Pierre
Published: September 2004

Summary— S‐antigen (arrestin) is a cytosolic protein which regulates phototransduction in retinal rods. A protein immunologically related to S‐antigen was identified in fractions from soluble extract of bovine kidney enriched by gel filtration or by immunoaffinity chromatography using a polyclonal antibody to retinal S‐antigen. On immunoblots, this protein was recognized by a panel of monoclonal antibodies (mAbs S2D2, S1A3 and S9E2) directed against different S‐antigen epitopes and displayed the same apparent molecular mass (48 kDa) as retinal S‐antigen. All three mAbs revealed a specific immunoreactivity by indirect immunocytochemical technique on rat kidney sections. The three mAbs recognized some but not all glomerular cells, identified as epithelial cells by immunoelectron microscopy using the mAb S9E2. Both mAbs S2D2 and S1A3 gave a diffuse cytoplasmic staining in all tubule cells. Proximal tubule cells exhibited a weak immunoreactivity, whereas distal and collecting tubule cells were strongly labeled. In contrast, the mAb S9E2 immunoreaction was restricted to a cell subpopulation from distal and collecting tubules corresponding to intercalated cells identified by immunoelectron microscopy. With the mAb S9E2, the labeling of proximal tubule cells was localized in the apical region of the cytoplasm. These results suggest that two or more 48‐kDa proteins immunologically cross‐reactive with retinal S‐antigen are present in kidney. The observed pattern of distribution is in keeping with the hypothesis that such proteins could play a role in the regulation of G‐protein‐related receptors present in renal glomerules and tubule epithelial cells.
The chitin secreting system from deep sea hydrothermal vent worms
Authors: Gaill Françoise , Shillito Bruce , Lechaire Jean Pierre , Chanzy Henri , Goffinet Gérard
Published: September 2004

Summary— Deep‐sea hydrothermal vent worms livé in tubes made of giant β‐chitin crystallites (50 nm in diameter, several μm in length) embedded in a protein matrix. These chitin cyrstallites form a liquid‐crystal‐like structure differing from the wellknown cholesteric arrangement of classical chitin‐protein systems. Furthermore, and in contrast with the latter systems, the vestimentiferan chitin‐protein systems are produced by goatskin‐shaped glands. Rod‐shaped elements in the lumen of these glands were identified by DCTEM and Au‐WGA labeling and freeze fracture as chitin crystallites. The main characteristics of these “chitin secreting glands” are described.
Directed movements of ciliary and flagellar membrane components: A review
Authors: Bloodgood Robert A.
Published: September 2004

Summary— The ability to rapidly translocate polystyrene microspheres attached to the surface of a plasma membrane domain reflects a unique form of cellular force transduction occurring in association with the plasma membrane of microtubule based cell extensions. This unusual form of cell motility can be utilized by protistan organisms for whole cell locomotion, the early events in mating, and transport of food organisms along the cell surface, and possibly intracellular transport of certain organelles. Since surface motility is observed in association with cilia and flagella of algae, sea urchin embryos and cultured mammalian cells, it is likely that it serves an additional role beyond those already cited; this is likely to be the transport of precursors for the assembly and turnover of ciliary and flagellar membranes and axonemes. In the case of the Chalmyodomonas flagellum, where surface motility has been most extensively studied, it appears that cross‐linking of flagellar surface exposed proteins induces a transmembrane signaling pathway that activates machinery for moving flagellar membrane proteins in the plane of the flagellar membrane. This signaling pathway in vegetative Chlamydomonas reinhardtii appears to involve an influx of calcium, a rise in intraflagellar free calcium concentration and a change in the level of phosphorylation of specific membrane‐matrix proteins. It is hypothesized that flagellar surface contact with a solid substrate (during gliding), a polystyrene microsphere or another flagellum (during mating) will all activate a signaling pathway similar to the one artificially activated by the use of monoclonal antibodies to flagellar membrane glycoproteins. A somewhat different signaling pathway, involving a transient rise in intracellular cAMP level, may be associated with the mating of Clamydomonas gametes, which is initiated by flagellum‐flagellum contact. The hypothesis that the widespread observation of microsphere movements on various ciliary and flagellar surfaces may reflect a mechanism normally utilized to transport axonemal and membrane subunits along the internal surface of the organelle membrane presents a paradox in that one would expect this to be a constitutive mechanism, not one necessarily activated by a signaling pathway.
Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells
Authors: Clary‐Meinesz CF , Cosson J. , Huitorel P. , Blaive B.
Published: September 2004

Summary— Even though all human respiratory cilia are similar in structure, they experience a wide range of temperatures between the initial part of the nasal fossae which behave as heat exchangers and the inferior part of the trachea, particularly when we inhale exceedingly cold or hot air. The ciliary beat frequency of ciliated cells from human nasal mucosa and from bronchial mucosa averages 8 Hz when measured at room temperature. In the present study we compared the ciliary beat frequency of human cells from nasal and tracheal mucosa brushings at different temperatures from 5°C to 50°C using two different techniques, ex vivo and in vitro: ex vivo in culture medium less than 24 h after sampling and in vitro after demembranation and reactivation according to a standard procedure developed in our laboratory. Measuring the ATP‐reactivated ciliary beat frequency allowed us to check the thermal parameters of the dynein ATPase and all the axonemal machinery. No significant difference in frequency was observed between nasal fossae cilia and tracheal cilia when comparing extreme temperatures in both experimental procedures.
Rapid backward movement of anaphase chromosome whose kinetochore fibers were cut by ultraviolet microbeam irradiation
Authors: Izutsu Kosaku , Sato Hidemi
Published: September 2004

Summary— kinetochore spindle fibers in meiosis I and II grasshopper spermatocytes were cut with a heterochromatic ultraviolet (UV) microbeam converging on the specimen to form a slit‐shaped microspot 1.5 × 8 μm or 3 × 8 μm. A total exposure of 3 × 10−8 joules per μm2 was administered within 0.8–2.4 s, which was sufficient for severing. The cells were observed with a high extinction polarizing microscope or phase contrast optics and a record made by time‐lapse video microscopy, continuously before, during and after the irradiation. When kinetochore fibers were irradiated i anaphase with UV, an area of reduced birefringence (ARB) was produced at the exposed site. The newly created + ends of the microtubules rapidly disassembled poleward, at a constant speed of 17 μm/min. The — ends at the edge of ARB also depolymerized at a slower rate. When a kinetochore fiber was cut with UV in early anaphase at which time its associated chromosome had not disjoined from the partner chromosome, the chromosome of the irradiated kinetochore fiber moved rapidly back to its partner. The speed during this movement was faster than the normal poleward chromosome movement in anaphase by an order to magnitude or more. When a kinetochore and its associated kinetochore fiber were included in the irradiation are, the effects were more pronounced than the effects of irradiation on a kinetochore fiber alone; the direction of the line connecting the irradiated half‐bivalent with the partner half‐bivalent deviated so much from the longitudinal axis of the original spindle with time that the division assumed a tripolar figure.
Real‐time observation of microtubules attached to microtubule organizing centers in vitro*
Authors: Cibert Christian , Huitorel Philippe
Published: September 2004

Summary— The dynamics and organization of microtubules associated with axonemes and kinetochores in vitro were visualized using video microscopy techniques. Microtubules attached either at the ends of axonemes or to mitotic chromosomes behave accordining to dynamic instability in our conditions. Microtubules attached to kinetochores showed lower rates of elongation and shortening than those nucleated by axonemes in the same conditions. In addition, elementary bundles of microtubules appeared spontaneously in association with kinetochores, with microtubules elongating along previously attached microtubules at even lower rates. Such side interactions, either spontaneous or stabilized by factors such as MAPs, might affect microtubule dynamics directly.
Cytoskeletal elements in arthorpod sensilla and mammalian photoreceptors*
Authors: Wolfrum Uwe
Published: September 2004

Summary— Ciliary receptor cells, typified by cilia or modified cilia, are very common in the animal kingdom. In addition to the cytoskeleton of their ciliary processes these receptors possess other specific prominent cytoskeletal elements. Two representative systems are presented: i) scolopidia, mechanosensitive sensilla of various arthropod species; and ii) photoreceptor cells of the retina of the bovine eye. Two cytoskeletal structures are characteristic for arthropod scolopidia: a scolopale typifies the innermost auxiliary cell, and long ciliary rootlets are extending well into the sensory cells. The latter element is also charactristics for the inner segment of the photoreceptor cells in bovine. The scolopale of scolopidia is mainly composed of actin filaments. In the absence of myosin, the uniform polarity of the actin filaments and their association with tropomyosin all indicate a stabilizing role of the filament bundles within the scolopale. This function and a certina elasticity of actin filament bundles may be important during stimulation of the sensilla. The ciliary rootlets of both systems originate at the basal bodies at the ciliary base of the sensory cells and project proximally. These rootlets are composed of longitudinally oriented, fine filaments forming a characteristic regular cross‐striation. An α‐actinin immunoreactivity was detected within the ciliary rootlets of scolopidia. In addition, antibodies to centrin react with the rootlets of both types of receptors. Since centrin is largely responsible for the contraction of the flagellar rootlets in green algae, contraction may also occur in the ciliary rootlets of insect sensilla and vertebrate photoreceptors. In both system, contraction or relaxation of the ciliary rootlets could serve in sensory transduction or adaptation.
Centrin is a component of the periocentriola lattice
Authors: Baron Andre T. , Greenwood Tammy M. , Bazinet Christopher W. , Salisbury Jeffrey L.
Published: September 2004

Summary— Here, we use three polyclonal anticentrin antisera designated 08/28, 26/14‐1, and 26/14‐2 to further characterize the pericentriolar lattice of metazoan cells. all of these antibodies give an indistinguishable localization pattern that consists of a constellation of pericentrosomal spots. In QT6 cells these spots are few in number and closely associated with the centriolar region, whereas in PtK2 cells they are more numerous and distributed further from the point of microtubule focus. In mitotic cells, centrin is localized to the spindle poles and spindle apparatus. We demonstrate here that the pericentriolar lattice of PtK2 and QT6 cells is, in part, composed of proteins characterized by acidic pIs (4.4 to 5.4), low molecular mass (Mr 18 500–21000), and calcium‐binding; these attributes and the immunoreactivity of these proteins to anticentrin antibodies indicate that they are centrin isoforms of metazoan cells. Finally, we confirm our earlier observation that PtK2 cells contain a centrin‐related protein of Mr 165000; QT6 cells also contain centrin‐related proteins (Mr 64000–165000). We conclude that centrin is a component of the pericentriolar lattice of higher eukaryotic centrosomes.
Carpal tunnel pressure alters median nerve function in a dose‐dependent manner: A rabbit model for carpal tunnel syndrome
Authors: Diao Edward , Shao Fang , Liebenberg Ellen , Rempel David , Lotz Jeffrey C.
Published: September 2004

Background: An in vivo animal model for carpal tunnel syndrome (CTS) is presented which allows for graded application of pressure to the median nerve within the carpal canal. We hypothesized that such pressure would cause electrophysiologic changes in the median nerve in a dose‐related manner, with NCS/EMG changes consistent with CTS in humans.

Methods: In 40 New Zealand white rabbits, ranging from 2 to 2.5 kg, angioplasty catheters were placed in the carpal tunnel in the forepaws and pressures ranging from 50 to 80 mm Hg applied to one side while the contralateral side served as the control and remained uninflated. Pressure was applied until a 15% increase in distal motor latency was obtained for 2 consecutive weeks by nerve conduction studies.

Results: All the experimental limbs exhibited a 15% increase in distal motor latency. None of the control limbs showed a significant increase in distal motor latency. In the experimental animals the 15% delay was achieved in approximately 4–5 weeks in the 50–70 mm Hg groups and in approximately 1 week in the 80 mm Hg group.

Conclusion: This new animal model for CTS demonstrates a direct cause and effect relationship between carpal tunnel pressure and median nerve dysfunction. We anticipate that this in vivo model with clinically relevant outcomes will facilitate identification of injury mechanisms, and will serve as a basis for future development of novel interventions and treatments. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Asymmetric atrophy of the supraspinatus muscle following tendon tear
Authors: Meyer Dominik C. , Pirkl Christoph , Pfirrmann Christian W. A. , Zanetti Marco , Gerber Christian
Published: September 2004

Muscle atrophy is a known consequence of muscle disuse, muscle denervation and tendon tear. Whereas after nerve injury muscle atrophies in the denervated area, the distribution of muscle atrophy following tear of its tendon is not known.

Standardized MRI scans of 64 consecutive, painful shoulders were evaluated for supraspinatus tendon tearing, myotendinous retraction, supraspinatus muscle atrophy, fatty infiltration, ratio of the scapular (deep) and fascial (superficial) muscle area (“symmetry”) and position of the central tendon within the supraspinatus fossa.

There were thirteen shoulders with no and eleven shoulders with partial thickness supraspinatus tendon tears. In the forty cases with full thickness tendon tear, there was significant muscle atrophy and fatty infiltration. Atrophy of the fascial muscle portion was 43%, on the bony side it was 9% (p < 0.005). The position of the central tendon within the supraspinatus fossa, was unaltered.

Muscular changes following tendon tear occur highly asymmetrically: The muscle portion originating from the fascia primarily atrophies, the portion originating from the scapula primarily undergoes fatty infiltration. Muscular changes are not simply a consequence of muscle disuse, but dependent on architectural changes in the muscle. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Movement of the posterior cruciate ligament during knee flexion—MRI analysis
Authors: Komatsu Takeshi , Kadoya Yoshinori , Nakagawa Shigeru , Yoshida Gen , Takaoka Kunio
Published: September 2004

The movement of the posterior cruciate ligament (PCL) during flexion of the living knee is unknown. The purpose of the present study was to analyze the movement of the PCL using magnetic resonance imaging (MRI). The posterior cruciate ligaments in 20 normal knees were visualized using MRI from extension to deep flexion. Sagittal inclination relative to the longitudinal axis of the tibia was measured and analyzed with reference to the patellar tendon (PT) and the anterior cruciate ligament (ACL). Although the PCL was slack in extension, it straightened with anterior inclination (24.1 ± 5.1°) at 90° flexion. At active maximum flexion (129.2 ± 8.1°), the ligament was almost parallel (3.9 ± 7.4° inclination) to the longitudinal axis of the tibia. At passive maximum flexion (158.8 ± 5.8°), the inclination was reversed anteroposteriorly, measuring −23.0 ± 6.7°. The PCL and PT moved in a corresponding manner within 20° of discrepancy. The results of this in vivo study of the PCL have clinical relevance to conservative therapy for PCL knee injuries. The results of this study could also be useful in PCL reconstruction surgery to determine the optimum graft position to allow maximum postoperative motion. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
An experimentally determined protein folding energy landscape
Authors: Mello Cecilia C. , Barrick Doug
Published: September 2004

Energy landscapes have been used to conceptually describe and model protein folding but have been difficult to measure experimentally, in large part because of the myriad of partly folded protein conformations that cannot be isolated and thermodynamically characterized. Here we experimentally determine a detailed energy landscape for protein folding. We generated a series of overlapping constructs containing subsets of the seven ankyrin repeats of the
Drosophila
Notch receptor, a protein domain whose linear arrangement of modular structural units can be fragmented without disrupting structure. To a good approximation, stabilities of each construct can be described as a sum of energy terms associated with each repeat. The magnitude of each energy term indicates that each repeat is intrinsically unstable but is strongly stabilized by interactions with its nearest neighbors. These linear energy terms define an equilibrium free energy landscape, which shows an early free energy barrier and suggests preferred low-energy routes for folding.
Crystal structure of the clathrin adaptor protein 1 core
Authors: Heldwein Ekaterina E. , Macia Eric , Wang Jing , Yin Helen L. , Kirchhausen Tomas , Harrison Stephen C.
Published: September 2004

The heterotetrameric adaptor proteins (AP complexes) link the outer lattice of clathrin-coated vesicles with membrane-anchored cargo molecules. We report the crystal structure of the core of the AP-1 complex, which functions in the trans-Golgi network (TGN). Packing of complexes in the crystal generates an exceptionally long (1,135-Å) unit-cell axis, but the 6-fold noncrystallographic redundancy yields an excellent map at 4-Å resolution. The AP-1 core comprises N-terminal fragments of the two large chains, β1 and γ, and the intact medium and small chains, μ1 and σ1. Its molecular architecture closely resembles that of the core of AP-2, the plasma-membrane-specific adaptor, for which a structure has been determined. Both structures represent an “inactive” conformation with respect to binding of cargo with a tyrosine-based sorting signal. TGN localization of AP-1 depends on the small GTPase, Arf1, and the phosphoinositide, PI-4-P. We show that directed mutations of residues at a particular corner of the γ chain prevent recruitment to the TGN in cells and diminish PI-4-P-dependent, but not Arf1-dependent, liposome binding
in vitro
.
Superiority of network motifs over optimal networks and an application to the revelation of gene network evolution
Authors: Ott S. , Hansen A. , Kim S.-Y. , Miyano S.
Published: September 2004

Motivation: Estimating the network of regulative interactions between genes from gene expression measurements is a major challenge. Recently, we have shown that for gene networks of up to around 35 genes, optimal network models can be computed. However, even optimal gene network models will in general contain false edges, since the expression data will not unambiguously point to a single network.

Results: In order to overcome this problem, we present a computational method to enumerate the most likely m networks and to extract a widely common subgraph (denoted as gene network motif) from these. We apply the method to bacterial gene expression data and extensively compare estimation results to knowledge. Our results reveal that gene network motifs are in significantly better agreement to biological knowledge than optimal network models. We also confirm this observation in a series of estimations using synthetic microarray data and compare estimations by our method with previous estimations for yeast. Furthermore, we use our method to estimate similarities and differences of the gene networks that regulate tryptophan metabolism in two related species and thereby demonstrate the analysis of gene network evolution.

Availability: Commercial license negotiable with Gene Networks Inc. (cherkis@gene-networks.com)

Contact:  sascha-ott@gmx.net
Adenovirus stimulates choline efflux by increasing expression of organic cation transporter-2
Authors: Miakotina Olga L. , Agassandian Marianna , Shi Lei , Look Dwight C. , Mallampalli Rama K.
Published: September 2004

We examined the effect of wild-type human adenovirus (Ad5) on choline transport in murine lung epithelia (MLE) and in rodent primary alveolar type II cells. Cells were active in pH-sensitive, reversible transport of choline, a process blocked pharmacologically with phenoxybenzamine, an inhibitor of organic cation transporters (OCT). PCR products for the choline transporters, OCT-1 and OCT-2, were detected, but only OCT-2 protein was robustly expressed within MLE and primary alveolar epithelial cells. Ad5 produced a two- to threefold increase in choline efflux from cells, resulting in a significant reduction in intracellular choline content and its major product, phosphatidylcholine. Effects of Ad5 on choline efflux were inhibited with phenoxybenzamine, and choline efflux was attenuated by OCT-2 small interfering RNA. Adenovirus also produced a dose-dependent increase in immunoreactive OCT-2 levels concomitant with increased cellular OCT-2 steady-state mRNA. These results indicate that adenoviruses can significantly disrupt choline trafficking in lung epithelia by upregulating expression of an alveolar protein involved in organic cation transport.
Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung
Authors: van Tuyl Minke , Liu Jason , Wang Jinxia , Kuliszewski Maciek , Tibboel Dick , Post Martin
Published: September 2004

Recent investigations have suggested an active role for endothelial cells in organ development, including the lung. Herein, we investigated some of the molecular mechanisms underlying normal pulmonary vascular development and their influence on epithelial branching morphogenesis. Because the lung in utero develops in a relative hypoxic environment, we first investigated the influence of low oxygen on epithelial and vascular branching morphogenesis. Two transgenic mouse models, the C101-LacZ (epithelial-LacZ marker) and the Tie2-LacZ (endothelial-LacZ marker), were used. At embryonic day 11.5, primitive lung buds were dissected and cultured at either 20 or 3% oxygen. At 24-h intervals, epithelial and endothelial LacZ gene expression was visualized by X-galactosidase staining. The rate of branching of both tissue elements was increased in explants cultured at 3% oxygen compared with 20% oxygen. Low oxygen increased expression of VEGF, but not that of the VEGF receptor (Flk-1). Expression of two crucial epithelial branching factors, fibroblast growth factor-10 and bone morphogenetic protein-4, were not affected by low oxygen. Epithelial differentiation was maintained at low oxygen as shown by surfactant protein C in situ hybridization. To explore epithelial-vascular interactions, we inhibited vascular development with antisense oligonucleotides targeted against either hypoxia inducible factor-1α or VEGF. Epithelial branching morphogenesis in vitro was dramatically abrogated when pulmonary vascular development was inhibited. Collectively, the in vitro data show that a low-oxygen environment enhances branching of both distal lung epithelium and vascular tissue and that pulmonary vascular development appears to be rate limiting for epithelial branching morphogenesis.
LTD4induces hyperresponsiveness to histamine in bovine airway smooth muscle: role of SR-ATPase Ca2+pump and tyrosine kinase
Authors: Carbajal Verónica , Vargas Mario H. , Flores-Soto Edgar , Martínez-Cordero Erasmo , Bazán-Perkins Blanca , Montaño Luis M.
Published: September 2004

Airway hyperresponsiveness is a key feature of asthma, but its mechanisms remain poorly understood. Leukotriene D4(LTD4) is one of the few molecules capable of producing airway hyperresponsiveness. In this study, LTD4, but not leukotriene C4(LTC4), produced a leftward displacement of the concentration-response curve to histamine in bovine airway smooth muscle strips. Neither LTC4nor LTD4modified the concentration-response curve to carbachol. In simultaneous measurements of intracellular Ca2+([Ca2+]i) and contraction, histamine or carbachol produced a transient Ca2+peak followed by a plateau, along with a contraction. LTD4increased the histamine-induced transient Ca2+peak and contraction but did not modify responses to carbachol. Enhanced responses to histamine induced by LTD4were not modified by staurosporine or chelerythrine but were abolished by genistein. Western blot showed that carbachol, but not histamine, caused intense phosphorylation of extracellular signal-regulated kinase 1/2 and that LTD4significantly enhanced the phosphorylation induced by histamine, but not by carbachol. L-type Ca2+channel participation in the hyperresponsiveness to histamine was discarded because LTD4did not modify the [Ca2+]ichanges induced by KCl. In tracheal myocytes, LTD4enhanced the transient Ca2+peak induced by histamine (but not by carbachol) and the sarcoplasmic reticulum (SR) Ca2+refilling. Genistein abolished this last LTD4effect. Partial blockade of the SR-ATPase Ca2+pump with cyclopiazonic acid reduced the Ca2+transient peak induced by histamine but not by carbachol. These results suggested that LTD4induces hyperresponsiveness to histamine through activation of the tyrosine kinase pathway and an increasing SR-ATPase Ca2+pump activity. L-type Ca2+channels seemed not to be involved in this phenomenon.
Metabolic activation of AMP kinase in vascular smooth muscle
Authors: Rubin L. J. , Magliola L. , Feng X. , Jones A. W. , Hale C. C.
Published: September 2004

AMP-activated kinase (AMPK) is a highly conserved heterotrimeric kinase that functions as a metabolic master switch to coordinate cellular enzymes involved in carbohydrate and fat metabolism that regulate ATP conservation and synthesis. AMPK is activated by conditions that increase AMP-to-ATP ratio, such as exercise and metabolic stress. In the present study, we probed whether AMPK was expressed in vascular smooth muscle and would be activated by metabolic stress. Endothelium-denuded porcine carotid artery segments were metabolically challenged with 2-deoxyglucose (10 mM) plus N2 (N2-2DG). These vessels exhibited a rapid increase in AMPK activity by 1 min that was near maximal by 20 min. AMPK inactivation on return to normal physiological saline was ∼50% in 1 min and fully recovered by 5 min. Immunoprecipitation of the α1- and α2-catalytic subunit followed by immunoblot analysis for [P]Thr172-AMPK indicates that α1-AMPK accounts for all activity. Little if any α2-AMPK was detected in carotid smooth muscle. AMPK activity was not increased by contractile agonist (endothelin-1) or by the reported AMPK activators 5-aminoimidazole-4-carboxamide ribofuranoside (2 mM), metformin (2 mM), or phenformin (0.2 mM). AMPK activation by N2-2DG was associated with a rapid and pronounced reduction in endothelin-induced force and reduced phosphorylation of Akt and Erk 1/2. These data demonstrate that AMPK expression differs in vascular smooth muscle compared with striated muscles and that activation and inactivation after metabolic stress occur rapidly and are associated with signaling pathways that may regulate smooth-muscle contraction
Elevated n-3 fatty acids in a high-fat diet attenuate the increase in PDH kinase activity but not PDH activity in human skeletal muscle
Authors: Turvey Erin A. , Heigenhauser George J. F. , Parolin Michelle , Peters Sandra J.
Published: September 2004

We tested the hypothesis that a high-fat diet (75% fat; 5% carbohydrates; 20% protein), for which 15% of the fat content was substituted with n-3 fatty acids, would not exhibit the diet-induced increase in pyruvate dehydrogenase kinase (PDK) activity, which is normally observed in human skeletal muscle. The fat content was the same in both the regular high-fat diet (HF) and in the n-3-substituted diet (N3). PDK activity increased after both high-fat diets, but the increase was attenuated after the N3 diet (0.051 ± 0.007 and 0.218 ± 0.047 min−1 for pre- and post-HF, respectively; vs. 0.073 ± 0.016 and 0.133 ± 0.032 min−1 for pre- and post-N3, respectively). However, the active form of pyruvate dehydrogenase (PDHa) activity decreased to a similar extent in both conditions (0.93 ± 0.17 and 0.43 ± 0.09 mmol/kg wet wt pre- and post-HF; vs. 0.87 ± 0.19 and 0.39 ± 0.05 mmol/kg wet wt pre- and post-N3, respectively). This suggested that the difference in PDK activity did not affect PDHa activation in the basal state, and it was regulated by intramitochondrial effectors, primarily muscle pyruvate concentration. Muscle glycogen content was consistent throughout the study, before and after both diet conditions, whereas muscle glucose-6-phosphate, glycerol-3-phosphate, lactate, and pyruvate were decreased after the high-fat diets. Plasma triglycerides decreased after both high-fat diets but decreased to a greater extent after the N3, whereas plasma free fatty acids increased after both diets, but to a lesser extent after the N3. In summary, PDK activity is decreased after a high-fat diet that is rich in n-3 fatty acids, although PDHa activity was unaltered. In addition, our data demonstrated that the hypolipidemic effect of n-3 fatty acids occurs earlier (3 days) than previously reported and is evident even when the diet has 75% of its total energy derived from fat.
Effects of hyperoxia on skeletal muscle carbohydrate metabolism during transient and steady-state exercise
Authors: Stellingwerff Trent , Glazier Lee , Watt Matthew J. , LeBlanc Paul J. , Heigenhauser George J. F. , Spriet Lawrence L.
Published: September 2004

This study compared the effects of inspiring either a hyperoxic (60% O2) or normoxic gas (21% O2) while cycling at 70% peak O2uptake on 1) the ATP derived from substrate phosphorylation during the initial minute of exercise, as estimated from phosphocreatine degradation and lactate accumulation, and 2) the reliance on carbohydrate utilization and oxidation during steady-state cycling, as estimated from net muscle glycogen use and the activity of pyruvate dehydrogenase (PDH) in the active form (PDHa), respectively. We hypothesized that 60% O2would decrease substrate phosphorylation at the onset of exercise and that it would not affect steady-state exercise PDH activity, and therefore muscle carbohydrate oxidation would be unaltered. Ten active male subjects cycled for 15 min on two occasions while inspiring 21% or 60% O2, balance N2. Blood was obtained throughout and skeletal muscle biopsies were sampled at rest and 1 and 15 min of exercise in each trial. The ATP derived from substrate-level phosphorylation during the initial minute of exercise was unaffected by hyperoxia (21%: 52.2 ± 11.1; 60%: 54.0 ± 9.5 mmol ATP/kg dry wt). Net glycogen breakdown during 15 min of cycling was reduced during the 60% O2trial vs. 21% O2(192.7 ± 25.3 vs. 138.6 ± 16.8 mmol glycosyl units/kg dry wt). Hyperoxia had no effect on PDHa, because it was similar to the 21% O2trial at rest and during exercise (21%: 2.20 ± 0.26; 60%: 2.25 ± 0.30 mmol·kg wet wt−1·min−1). Blood lactate was lower (6.4 ± 1.0 vs. 8.9 ± 1.0 mM) at 15 min of exercise and net muscle lactate accumulation was reduced from 1 to 15 min of exercise in the 60% O2trial compared with 21% (8.6 ± 5.1 vs. 27.3 ± 5.8 mmol/kg dry wt). We concluded that O2availability did not limit oxidative phosphorylation in the initial minute of the normoxic trial, because substrate phosphorylation was unaffected by hyperoxia. Muscle glycogenolysis was reduced by hyperoxia during steady-state exercise, but carbohydrate oxidation (PDHa) was unaffected. This closer match between pyruvate production and oxidation during hyperoxia resulted in decreased muscle and blood lactate accumulation. The mechanism responsible for the decreased muscle glycogenolysis during hyperoxia in the present study is not clear.
Recombinant guinea pig CCL5 (RANTES) differentially modulates cytokine production in alveolar and peritoneal macrophages
Authors: Skwor Troy A , Cho Hyosun , Cassidy Craig , Yoshimura Teizo , McMurray David N
Published: September 2004

The CC chemokine ligand 5 (CCL5; regulated on activation, normal T expressed and secreted) is known to recruit and activate leukocytes; however, its role in altering the responses of host cells to a subsequent encounter with a microbial pathogen has rarely been studied. Recombinant guinea pig (rgp)CCL5 was prepared, and its influence on peritoneal and alveolar macrophage activation was examined by measuring cytokine and chemokine mRNA expression in cells stimulated with rgpCCL5 alone or exposed to rgpCCL5 prior to lipopolysaccharide (LPS) stimulation. Levels of mRNA for guinea pig tumor necrosis factor α (TNF-α), interleukin (IL)-1β, CCL2 (monocyte chemoattractant protein-1), and CXC chemokine ligand 8 (IL-8) were analyzed by reverse transcription followed by real-time polymerase chain reaction analysis using SYBR Green. Bioactive TNF-α protein concentration was measured using the L929 bioassay. Both macrophage populations displayed significant enhancement of all the genes and TNF-α protein levels when stimulated with rgpCCL5, except for CCL2 in alveolar macrophages. When peritoneal or alveolar macrophages were pretreated with rgpCCL5 for 2 h and then exposed to low concentrations of LPS, diminished cytokine and chemokine mRNA levels were apparent at 6 h compared with LPS alone. At the protein level, there was a reduction in TNF-α protein at 6 h in the CCL5-pretreated cells compared with LPS alone. These results further support a role for CCL5 in macrophage activation in addition to chemotactic properties and suggest a role in regulating the inflammatory response to LPS in the guinea pig by modulating the production of proinflammatory cytokines by macrophages.
Bounding the optimal burn‐in time for a system with two types of failure
Authors: Cha Ji Hwan , Lee Sangyeol , Mi Jie
Published: September 2004

Burn‐in is a widely used method to improve the quality of products or systems after they have been produced. In this paper, we consider the problem of determining bounds to the optimal burn‐in time and optimal replacement policy maximizing the steady state availability of a repairable system. It is assumed that two types of system failures may occur: One is Type I failure (minor failure), which can be removed by a minimal repair, and the other is Type II failure (catastrophic failure), which can be removed only by a complete repair. Assuming that the underlying lifetime distribution of the system has a bathtub‐shaped failure rate function, upper and lower bounds for the optimal burn‐in time are provided. Furthermore, some other applications of optimal burn‐in are also considered. © 2004 Wiley Periodicals, Inc. Naval Research Logistics, 2004
Host‐defence skin peptides of the Australian Common Froglet Crinia signifera: sequence determination using positive and negative ion electrospray mass spectra
Authors: Maselli Vita M. , Brinkworth Craig S. , Bowie John H. , Tyler Michael J.
Published: September 2004

The amino acid sequences of seven signiferin peptides are provided by consideration of tandem mass spectrometric (MS/MS) data for the respective MH+ and [MH]− precursor ions. These methods do not differentiate between isomeric residues Leu and Ile; these were identified using the Edman degradation technique. The sequence of signiferin 1, a new smooth muscle contracting neuropeptide (RLCIPYIIPC‐OH) containing a disulfide bridge, is best determined using the collision‐induced dissociation (CID) spectrum of the [MH]− anion. The initial fragmentation of this system involves loss of H2S2, which furnishes an open‐chain system that is readily sequenced using the α and β backbone cleavage anions. Copyright © 2004 John Wiley & Sons, Ltd.
Basic fibroblast growth factor high and low affinity binding sites in developing mouse brain, hippocampus and cerebellum
Authors: Fayein Nicole‐Adeline , Courtois Yves , Jeanny Jean‐Claude
Published: September 2004

Summary— Fibroblast growth factors (FGFs), first extracted from brain and retina, are potent neurotrophic factors. They stimulate neuroblast proliferation and neuron differentiation and survival. In order to study the spatial and temporal distribution of the target cells in the mouse brain we studied by autoradiography and quantified by image analysis 125I‐bFGF binding sites as a function of development. We have revealed the presence of two types of specific bFGF receptors. One is heparitinase sensitive and is co‐localized with heparan sulfate proteoglycans of the basement membranes (meninges, choroid plexus and blood vessels). It is not developmentally regulated and corresponds to the low affinity receptors. It may be a storage form. The second type is heparitinase resistant and is modified during development, matching, in the adult, layering of the hippocampus and cerebellum. At 13 days of embryonic development there is a preferential distribution of silver grains on the ecto‐ and neuroectodermal tissues. In the adult, the labeling is localized on the neural process layers. It likely corresponds to the specific binding to cell high affinity receptors. Binding patterns according to the developmental stages of the brain can be correlated with mitotic, migration and differentiation phases of the neuronal cells.
The role of calcium, polyamines and centrosomes in the formation and organization of cleavage furrows in amphibian eggs
Authors: Aimar Christian , Grant Nancy
Published: September 2004

Summary— Cleavage furrows of amphibian eggs exhibit characteristic morphological features; the presence of finger‐like microvilli (MV) along their outer edges, the formation of furrow walls from new plasma membrane lacking MV, and the subsequent retrieval of this membrane during the infolding of the furrow. A similar structure can be induced, specifically, by certain cytoplasmic components such as centrosomes, polyamines and calcium. Their respective roles in the events associated with the furrowing process have been investigated by injecting these agents into nucleated and enucleated Pleurodeles eggs and evaluating their effects using cytochemical labelling of the egg surface with a biotin‐streptavidin system. The injection of polyamines (spermine or spermidine) and in some cases, calcium into enucleated eggs provoked MV elongation and the appearance of newly formed, smooth plasma membrane. In these eggs, this membrane was not incorporated into the furrows, and as a consequence, the blastomeres did not actually separate. In contrast, the injection of centrosomes into enucleated eggs induced both the incorporation and internalization of new membrane, resulting in the formation of furrows and a true cellularization of the eggs, identical to the cleavage process observed in fertilized eggs. The present results provide further evidence that the establishment of the furrow depends on two complementary interacting systems: the contractile elements of the egg cortex which regulate the insertion of new membrane and the mitotic center which is essential for the invagination of the furrow.
Analysis of the mechanism of dinoflagellate flagella contraction‐relaxation cycle
Authors: Cachon Monique , Greuet Claude , Cosson Jacky , Huitorel Philippe
Published: September 2004

Summary— Dinoflagellates possess two flagella. One of them, the longitudinal flagellum, retracts from time to time in some species, such as Ceratium and Peridinium. Additional structures which run along the axoneme seem to be responsible for this particular behaviour. The retraction which is rapid (less than 60 ms) may be subdivided into several steps: i) the undulating movement stops; ii) the flagellum appears then as a jagged line during 20 ms; iii) finally a rapid retraction (20 ms) takes place, the flagellum being folded 20 times inside the cylindrical flagellar pocket. The measurements on video‐records suggest that the R‐fibre shortens to 30% of its original length. The contraction and relaxation mechanism of nanofilaments is proposed to be through coiling and uncoiling dependent on Ca2+ concentration.