bluuebunny/crossref_metadata_2025 huggingface dataset:
Operant behavior can be triggered by the position of the rat relative to objects rotating on an inaccessible platform
Authors: Pastalkova E. , Kelemen E. , Bureš J.
Published: February 2003

The present study describes a task testing the ability of rats to trigger operant behavior by their relative spatial position to inaccessible rotating objects. Rats were placed in a Skinner box with a transparent front wall through which they could observe one or two adjacent objects fixed on a slowly rotating arena (d = 1 m) surrounded by an immobile black cylinder. The direction of arena rotation was alternated at a sequence of different time intervals. Rats were reinforced for the first bar-press that was emitted when a radius separating the two adjacent objects or dividing a single object into two halves (pointing radius) entered a 60° sector of its circular trajectory defined with respect to the stationary Skinner box (reward sector). Well trained rats emitted 62.1 ± 3.6% of responses in a 60° sector preceding the reward sector and in the first 30° of the reward sector. Response rate increased only when the pointing radius was approaching the reward sector, regardless of the time elapsed from the last reward. In the extinction session, when no reward was delivered, rats responded during the whole passage of the pointing radius through the former reward sector and spontaneously decreased responding after the pointing radius left this area. This finding suggests that rats perceived the reward sector as a continuous single region. The same results were obtained when the Skinner box with the rat was orbiting around the immobile scene. It is concluded that rats can recognize and anticipate their position relative to movable objects.
A mechanism converting psychosocial stress into mononuclear cell activation
Authors: Bierhaus Angelika , Wolf Jutta , Andrassy Martin , Rohleder Nicolas , Humpert Per M. , Petrov Dimitri , Ferstl Roman , von Eynatten Maximilian , Wendt Thoralf , Rudofsky Gottfried , Joswig Martina , Morcos Michael , Schwaninger Markus , McEwen Bruce , Kirschbaum Clemens , Nawroth Peter P.
Published: February 2003

Little is known about the mechanisms converting psychosocial stress into cellular dysfunction. Various genes, up-regulated in atherosclerosis but also by psychosocial stress, are controlled by the transcription factor nuclear factor κB (NF-κB). Therefore, NF-κB is a good candidate to convert psychosocial stress into cellular activation. Volunteers were subjected to a brief laboratory stress test and NF-κB activity was determined in peripheral blood mononuclear cells (PBMC), as a window into the body and because PBMC play a role in diseases such as atherosclerosis. In 17 of 19 volunteers, NF-κB was rapidly induced during stress exposure, in parallel with elevated levels of catecholamines and cortisol, and returned to basal levels within 60 min. To model this response, mice transgenic for a strictly NF-κB-controlled β-globin transgene were stressed by immobilization. Immobilization resulted in increased β-globin expression, which could be reduced in the presence of the α1-adrenergic inhibitor prazosin. To define the role of adrenergic stimulation in the up-regulation of NF-κB, THP-1 cells were induced with physiological amounts of catecholamines for 10 min. Only noradrenaline resulted in a dose- and time-dependent induction of NF-κB and NF-κB-dependent gene expression, which depended on pertussis-toxin-sensitive G protein-mediated phosphophatidylinositol 3-kinase, Ras/Raf, and mitogen-activated protein kinase activation. Induction was reduced by α
1
- and β-adrenergic inhibitors. Thus, noradrenaline-dependent adrenergic stimulation results in activation of NF-κB
in vitro
and
in vivo
. Activation of NF-κB represents a downstream effector for the neuroendocrine response to stressful psychosocial events and links changes in the activity of the neuroendocrine axis to the cellular response.
Ecological community description using the food web, species abundance, and body size
Authors: Cohen Joel E. , Jonsson Tomas , Carpenter Stephen R.
Published: February 2003

Measuring the numerical abundance and average body size of individuals of each species in an ecological community's food web reveals new patterns and illuminates old ones. This approach is illustrated using data from the pelagic community of a small lake: Tuesday Lake, Michigan, United States. Body mass varies almost 12 orders of magnitude. Numerical abundance varies almost 10 orders of magnitude. Biomass abundance (average body mass times numerical abundance) varies only 5 orders of magnitude. A new food web graph, which plots species and trophic links in the plane spanned by body mass and numerical abundance, illustrates the nearly inverse relationship between body mass and numerical abundance, as well as the pattern of energy flow in the community. Species with small average body mass occur low in the food web of Tuesday Lake and are numerically abundant. Larger-bodied species occur higher in the food web and are numerically rarer. Average body size explains more of the variation in numerical abundance than does trophic height. The trivariate description of an ecological community by using the food web, average body sizes, and numerical abundance includes many well studied bivariate and univariate relationships based on subsets of these three variables. We are not aware of any single community for which all of these relationships have been analyzed simultaneously. Our approach demonstrates the connectedness of ecological patterns traditionally treated as independent. Moreover, knowing the food web gives new insight into the disputed form of the allometric relationship between body mass and abundance.
Patterns of DNA sequence polymorphism at
Sod
vicinities in
Drosophila melanogaster
: Unraveling the footprint of a recent selective sweep
Authors: Sáez Alberto G. , Tatarenkov Andrey , Barrio Eladio , Becerra Nelsson H. , Ayala Francisco J.
Published: February 2003

We survey DNA sequence polymorphisms at the
Sod
locus and four neighboring regions of
Drosophila melanogaster
, spanning 55,513 base pairs (bp), in 15 strains from a natural population, plus one reference laboratory strain and one strain of
Drosophila simulans
. Our objective is to characterize a proposed selective sweep that occurred at a locus close to
Sod
in
D. melanogaster
and to characterize the strength of the selection event, its time, and the size of the hitchhiked region. Two regions,
1819
and
6kbr3r
, show a pattern of polymorphism very similar to the one of
Sod
, implying that they have been affected by the same evolutionary process that impacted
Sod
. A third fragment,
2021
seems unaffected by the event. A fourth one,
4039
, on the opposite flank of
Sod
in relation to
2021
, is only partially affected. We estimate that the length of the chromosomal segment impacted by the selective sweep is 41–54 kb, the age of the selective sweep is 2,600–22,000 years, and the selective advantage is 0.020 <
s
< 0.103.
Efficient T cell repertoire selection in tetraparental chimeric mice independent of thymic epithelial MHC
Authors: Martinic Marianne M. , Rülicke Thomas , Althage Alana , Odermatt Bernhard , Höchli Matthias , Lamarre Alain , Dumrese Tilman , Speiser Daniel E. , Kyburz Diego , Hengartner Hans , Zinkernagel Rolf M.
Published: February 2003

Nonthymic epithelial cells were compared with thymic epithelial cells for their role in T cell repertoire selection. Tetraparental aggregation chimeras were generated from T and B cell-deficient mice (H-2dSCID or H-2bRag−/−) and thymus-deficient nude mice (H-2bor H-2d). These tetraparental mice showed primary protective CD8+T cell responses, after lymphocytic choriomeningitis virus infection, that were peptide-specifically restricted to either thymic or nonthymic epithelial MHC at comparable levels. These chimeras also mounted neutralizing IgG responses dependent on cognate CD4+T helper cell activity restricted to nonthymic epithelial MHC. Therefore, in contrast to earlier results with irradiation or thymus chimeras, these relatively undisturbed tetraparental mice reveal that the MHC of nonthymic epithelial cells efficiently selects a functional T cell repertoire.
Packing helices in proteins by global optimization of a potential energy function
Authors: Nanias Marian , Chinchio Maurizio , Pillardy Jarosław , Ripoll Daniel R. , Scheraga Harold A.
Published: February 2003

An efficient method has been developed for packing α-helices in proteins. It treats α-helices as rigid bodies and uses a simplified Lennard–Jones potential with Miyazawa–Jernigan contact-energy parameters to describe the interactions between the α-helical elements in this coarse-grained system. Global conformational searches to generate packing arrangements rapidly are carried out with a Monte Carlo-with-minimization type of approach. The results for 42 proteins show that the approach reproduces native-like folds of α-helical proteins as low-energy local minima of this highly simplified potential function.
Impacts of anthropogenic and natural NO
x
sources over the U.S. on tropospheric chemistry
Authors: Zhang Renyi , Tie Xuexi , Bond Donald W.
Published: February 2003

We evaluate the impact of anthropogenic and natural NO
x
sources over the contiguous United States on tropospheric NO
x
and O
3
levels by using a global 3D chemical transport model. The effects of major U.S. surface NO
x
emission sources (including anthropogenic, biomass burning, and soil emissions) are compared with that of lightning-produced NO
x
. Summer lightning is shown to play a dominant role in controlling NO
x
and O
3
concentrations in the middle and upper troposphere, despite the fact that fossil-fuel burning represents the largest source of NO
x
over the U.S. Furthermore, the effect of regional U.S. lightning is propagated through large areas of the Northern Hemisphere by atmospheric circulation. The results reveal that a thorough assessment of atmospheric NO
x
emission sources and their impact is required to devise control strategies for regional and global air pollution.
The strategy for coupling the RanGTP gradient to nuclear protein export
Authors: Becskei Attila , Mattaj Iain W.
Published: February 2003

The Ran GTPase plays critical roles in both providing energy for and determining the directionality of nucleocytoplasmic transport. The mechanism that couples the RanGTP gradient to nuclear protein export will determine the rate of and limits to accumulation of export cargoes in the cytoplasm, but is presently unknown. We reasoned that plausible coupling mechanisms could be distinguished by comparing the rates of reverse motion of export cargoes through the nuclear pore complex (NPC) with the predictions of a mathematical model. Measurement of reverse export rates in
Xenopus
oocytes revealed that nuclear export signals can facilitate RanGTP-dependent cargo movement into the nucleus against the RanGTP gradient at rates comparable to export rates. Although export cargoes with high affinity for their receptor are exported faster than those with low affinity, their reverse transport is also greater. The ratio of the rates of reverse and forward export of a cargo is proportional to its rate of diffusion through the NPC, i.e., to the ability of the cargo to penetrate the NPC permeability barrier. The data substantiate a diffusional mechanism of coupling and suggest the existence of a high concentration of RanGTP-receptor complexes within the NPC that decreases sharply at the cytoplasmic boundary of the NPC permeability barrier.
Multiple elements of the allergic arm of the immune response modulate autoimmune demyelination
Authors: Pedotti Rosetta , DeVoss Jason J. , Youssef Sawsan , Mitchell Dennis , Wedemeyer Jochen , Madanat Rami , Garren Hideki , Fontoura Paulo , Tsai Mindy , Galli Stephen J. , Sobel Raymond A. , Steinman Lawrence
Published: February 2003

Analysis of mRNA from multiple sclerosis lesions revealed increased amounts of transcripts for several genes encoding molecules traditionally associated with allergic responses, including prostaglandin D synthase, histamine receptor type 1 (H1R), platelet activating factor receptor, Ig Fc ɛ receptor 1 (FcɛRI), and tryptase. We now demonstrate that, in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), mediated by T helper 1 (Th1) T cells, histamine receptor 1 and 2 (H1R and H2R) are present on inflammatory cells in brain lesions. Th1 cells reactive to myelin proteolipid protein expressed more H1R and less H2R than Th2 cells. Pyrilamine, an H1R antagonist, blocked EAE, and the platelet activating factor receptor antagonist CV6209 reduced the severity of EAE. EAE severity was also decreased in mice with disruption of the genes encoding Ig FcγRIII or both FcγRIII and FcɛRI. Prostaglandin D synthase and tryptase transcripts were elevated in EAE brain. Taken together, these data reveal extensive involvement of elements of the immune response associated with allergy in autoimmune demyelination. The pathogenesis of demyelination must now be viewed as encompassing elements of both Th1 responses and “allergic” responses.
Exonucleolytic degradation of double-stranded RNA by an activity in
Xenopus laevis
germinal vesicles
Authors: Fruscoloni Paolo , Zamboni Michela , Baldi M. Irene , Tocchini-Valentini Glauco P.
Published: February 2003

We have identified, in extracts from
Xenopus laevis
germinal vesicles, a 5′ exonuclease activity that cleaves double-stranded RNA (dsRNA). Features of the 5′ ends of dsRNAs determine whether the strands are symmetrically or asymmetrically degraded. The activity hydrolyzes in the 5′ to 3′ direction, releasing 5′-mononucleotides processively, favoring strands with 5′-monophosphate termini; molecules with capped ends are resistant to digestion. Because of its ability to processively digest dsRNA to mononucleotides, we have named the exonuclease Chipper, which could cooperate or compete with Dicer (an endonuclease that produces molecules with a 5′-phosphate) in the processing of dsRNA.
Negative specific heat of a magnetically self-confined plasma torus
Authors: Kiessling Michael K.-H. , Neukirch Thomas
Published: February 2003

It is shown that the thermodynamic maximum-entropy principle predicts negative specific heat for a stationary, magnetically self-confined current-carrying plasma torus. Implications for the magnetic self-confinement of fusion plasma are considered.
cAMP-dependent protein kinase phosphorylation of the acid-sensing ion channel-1 regulates its binding to the protein interacting with C-kinase-1
Authors: Leonard A. Soren , Yermolaieva Olena , Hruska-Hageman Alesia , Askwith Candice C. , Price Margaret P. , Wemmie John A. , Welsh Michael J.
Published: February 2003

The acid-sensing ion channel-1 (ASIC1) contributes to synaptic plasticity and may influence the response to cerebral ischemia and acidosis. We found that cAMP-dependent protein kinase phosphorylated heterologously expressed ASIC1 and endogenous ASIC1 in brain slices. ASIC1 also showed significant phosphorylation under basal conditions. Previous studies showed that the extreme C-terminal residues of ASIC1 bind the PDZ domain of the protein interacting with C-kinase-1 (PICK1). We found that protein kinase A phosphorylation of Ser-479 in the ASIC1 C terminus interfered with PICK1 binding. In contrast, minimizing phosphorylation or mutating Ser-479 to Ala enhanced PICK1 binding. Phosphorylation-dependent disruption of PICK1 binding reduced the cellular colocalization of ASIC1 and PICK1. Thus, the ASIC1 C terminus contains two sites that influence its binding to PICK1. Regulation of this interaction by phosphorylation provides a mechanism to control the cellular localization of ASIC1.
Interference of hepatitis C virus RNA replication by short interfering RNAs
Authors: Kapadia Sharookh B. , Brideau-Andersen Amy , Chisari Francis V.
Published: February 2003

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFNα in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.
Individuality and variation in gene expression patterns in human blood
Authors: Whitney Adeline R. , Diehn Maximilian , Popper Stephen J. , Alizadeh Ash A. , Boldrick Jennifer C. , Relman David A. , Brown Patrick O.
Published: February 2003

The nature and extent of interindividual and temporal variation in gene expression patterns in specific cells and tissues is an important and relatively unexplored issue in human biology. We surveyed variation in gene expression patterns in peripheral blood from 75 healthy volunteers by using cDNA microarrays. Characterization of the variation in gene expression in healthy tissue is an essential foundation for the recognition and interpretation of the changes in these patterns associated with infections and other diseases, and peripheral blood was selected because it is a uniquely accessible tissue in which to examine this variation in patients or healthy volunteers in a clinical setting. Specific features of interindividual variation in gene expression patterns in peripheral blood could be traced to variation in the relative proportions of specific blood cell subsets; other features were correlated with gender, age, and the time of day at which the sample was taken. An analysis of multiple sequential samples from the same individuals allowed us to discern donor-specific patterns of gene expression. These data help to define human individuality and provide a database with which disease-associated gene expression patterns can be compared.
Activated microglia in cortex of mouse models of mucopolysaccharidoses I and IIIB
Authors: Ohmi Kazuhiro , Greenberg David S. , Rajavel Kavitha S. , Ryazantsev Sergey , Li Hong Hua , Neufeld Elizabeth F.
Published: February 2003

α-
N
-Acetylglucosaminidase deficiency (mucopolysaccharidosis IIIB, MPS IIIB) and α-
l
-iduronidase deficiency (MPS I) are heritable lysosomal storage diseases; neurodegeneration is prominent in MPS IIIB and in severe cases of MPS I. We have obtained morphologic and molecular evidence for the involvement of microglia in brain pathology of mouse models of the two diseases. In the cortex, a subset of microglia (sometimes perineuronal) consists of cells that are probably phagocytic; they have large storage vacuoles, react with MOMA-2 (monoclonal antibody against macrophages) and
Griffonia simplicifolia
isolectin IB
4
, and stain intensely for the lysosomal proteins Lamp-1, Lamp-2, and cathepsin D as well as for G
M3
ganglioside. MOMA-2-positive cells appear at 1 and 6 months in MPS IIIB and MPS I mice, respectively, but though their number increases with age, they remain sparse. However, a profusion of cells carrying the macrophage CD68/macrosialin antigen appear in the cortex of both mouse models at 1 month. mRNA encoding CD68/macrosialin also increases at that time, as shown by microarray and Northern blot analyses. Ten other transcripts elevated in both mouse models are associated with macrophage functions, including complement C4, the three subunits of complement C1q, lysozyme M, cathepsins S and Z, cytochrome b558 small subunit, macrophage-specific protein 1, and DAP12. An increase in IFN-γ and IFN-γ receptor was observed by immunohistochemistry. These functional increases may represent activation of resident microglia, an influx and activation of blood monocytes, or both. They show an inflammatory component of brain disease in the two MPS, as is known for many neurodegenerative disorders.
The origin of the boson peak and thermal conductivity plateau in low-temperature glasses
Authors: Lubchenko Vassiliy , Wolynes Peter G.
Published: February 2003

We argue that the intrinsic glassy degrees of freedom in amorphous solids giving rise to the thermal conductivity plateau and the “boson peak” in the heat capacity at moderately low temperatures are directly connected to those motions giving rise to the two-level-like excitations seen at still lower temperatures. These degrees of freedom can be thought of as strongly anharmonic transitions between the local minima of the glassy energy landscape that are accompanied by ripplon-like domain wall motions of the glassy mosaic structure predicted to occur atTgby the random first-order transition theory. The energy spectrum of the vibrations of the mosaic depends on the glass transition temperature, the Debye frequency, and the molecular length scale. The resulting spectrum reproduces the experimental low-temperature boson peak. The “nonuniversality” of the thermal conductivity plateau depends onkBTg/ℏωDand arises from calculable interactions with the phonons.
Desensitization, internalization, and signaling functions of β-arrestins demonstrated by RNA interference
Authors: Ahn Seungkirl , Nelson Christopher D. , Garrison Tiffany Runyan , Miller William E. , Lefkowitz Robert J.
Published: February 2003

β-Arrestins bind to activated G protein-coupled receptor kinase-phosphorylated receptors, which leads to their desensitization with respect to G proteins, internalization via clathrin-coated pits, and signaling via a growing list of “scaffolded” pathways. To facilitate the discovery of novel adaptor and signaling roles of β-arrestins, we have developed and validated a generally applicable interfering RNA approach for selectively suppressing β-arrestins 1 or 2 expression by up to 95%. β-Arrestin depletion in HEK293 cells leads to enhanced cAMP generation in response to β
2
-adrenergic receptor stimulation, markedly reduced β
2
-adrenergic receptor and angiotensin II receptor internalization and impaired activation of the MAP kinases ERK 1 and 2 by angiotensin II. This approach should allow discovery of novel signaling and regulatory roles for the β-arrestins in many seven-membrane-spanning receptor systems.
DNA unzipped under a constant force exhibits multiple metastable intermediates
Authors: Danilowicz Claudia , Coljee Vincent W. , Bouzigues Cedric , Lubensky David K. , Nelson David R. , Prentiss Mara
Published: February 2003

Single molecule studies, at constant force, of the separation of double-stranded DNA into two separated single strands may provide information relevant to the dynamics of DNA replication. At constant applied force, theory predicts that the unzipped length as a function of time is characterized by jumps during which the strands separate rapidly, followed by long pauses where the number of separated base pairs remains constant. Here, we report previously uncharacterized observations of this striking behavior carried out on a number of identical single molecules simultaneously. When several single λ phage molecules are subject to the same applied force, the pause positions are reproducible in each. This reproducibility shows that the positions and durations of the pauses in unzipping provide a sequence-dependent molecular fingerprint. For small forces, the DNA remains in a partially unzipped state for at least several hours. For larger forces, the separation is still characterized by jumps and pauses, but the double-stranded DNA will completely unzip in less than 30 min.
PUMA
mediates the apoptotic response to p53 in colorectal cancer cells
Authors: Yu Jian , Wang Zhenghe , Kinzler Kenneth W. , Vogelstein Bert , Zhang Lin
Published: February 2003

Although several genes that might mediate p53-induced apoptosis have been proposed, none have previously been shown to play an essential role in this process through a rigorous gene disruption approach. We used a gene-targeting approach to evaluate p53-mediated death in human colorectal cancer cells. Expression of p53 in these cells induces growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor
p21
. If
p21
is disrupted via gene targeting, the cells die through apoptosis. If the
PUMA
gene is also disrupted in such cells, apoptosis is prevented. The effects of PUMA on apoptosis were observed after exogenous overexpression of p53 as well as after exposure to hypoxia, a physiologic activator of p53, and DNA damage. The PUMA protein interacts with Bcl-X
L
and promotes mitochondrial translocation and multimerization of Bax. Accordingly, genetic disruption of
BAX
makes cells resistant to the apoptosis resulting from PUMA expression. These results suggest that the balance between PUMA and p21 is pivotal in determining the responses to p53 activation and provide a model for understanding the basis of p53 mutations in human cancer.
Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin
Authors: Wang Qiang , Xie Yi , Du Quan-Sheng , Wu Xiao-Jun , Feng Xu , Mei Lin , McDonald Jay M. , Xiong Wen-Cheng
Published: February 2003

Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion–activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion–targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2–gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.
Elevated glucose inhibits VEGF-A–mediated endocardial cushion formation
Authors: Enciso Josephine M. , Gratzinger Dita , Camenisch Todd D. , Canosa Sandra , Pinter Emese , Madri Joseph A.
Published: February 2003

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1–negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1–positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.
Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the β2-adrenergic receptor
Authors: Rangarajan Savithri , Enserink Jorrit M. , Kuiperij H. Bea , de Rooij Johan , Price Leo S. , Schwede Frank , Bos Johannes L.
Published: February 2003

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1–GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the Gαs-coupled β2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.
Amassin, an olfactomedin protein, mediates the massive intercellular adhesion of sea urchin coelomocytes
Authors: Hillier Brian J. , Vacquier Victor D.
Published: February 2003

Sea urchins have a fluid-filled body cavity, the coelom, containing four types of immunocytes called coelomocytes. Within minutes after coelomic fluid is removed from the body cavity, a massive cell–cell adhesion of coelomocytes occurs. This event is referred to as clotting. Clotting is thought to be a defense mechanism against loss of coelomic fluid if the body wall is punctured, and it may also function in the cellular encapsulation of foreign material and microbes. Here we show that this intercoelomocyte adhesion is mediated by amassin, a coelomic plasma protein with a relative molecular mass (Mr) of 75 kD. Amassin forms large disulfide-bonded aggregates that adhere coelomocytes to each other. One half of the amassin protein comprises an olfactomedin (OLF) domain. Structural predictions show that amassin and other OLF domain-containing vertebrate proteins share a common architecture. This suggests that other proteins of the OLF family may function in intercellular adhesion. These findings are the first to demonstrate a function for a protein of the OLF family.
Characterization of the translocon of the outer envelope of chloroplasts
Authors: Schleiff Enrico , Soll Jürgen , Küchler Michael , Kühlbrandt Werner , Harrer Roswitha
Published: February 2003

The protein translocon of the outer envelope of chloroplasts (Toc) consists of the core subunits Toc159, Toc75, and Toc34. To investigate the molecular structure, the core complex was purified. This core complex has an apparent molecular mass of ∼500 kD and a molecular stoichiometry of 1:4:4–5 between Toc159, Toc75, and Toc34. The isolated translocon recognizes both transit sequences and precursor proteins in a GTP-dependent manner, suggesting its functional integrity. The complex is embedded by the lipids phosphatidylcholine and digalactosyldiacylglyceride. Two-dimensional structural analysis by EM revealed roughly circular particles consistent with the formation of a stable core complex. The particles show a diameter of ∼130 Å with a solid ring and a less dense interior structure. A three-dimensional map obtained by random conical tilt reconstruction of electron micrographs suggests that a “finger”-like central region separates four curved translocation channels within one complex.
Targeting of U4/U6 small nuclear RNP assembly factor SART3/p110 to Cajal bodies
Authors: Stanĕk David , Rader Stephen D. , Klingauf Mirko , Neugebauer Karla M.
Published: February 2003

The spliceosomal small nuclear RNAs (snRNAs) are distributed throughout the nucleoplasm and concentrated in nuclear inclusions termed Cajal bodies (CBs). A role for CBs in the metabolism of snRNPs has been proposed but is not well understood. The SART3/p110 protein interacts transiently with the U6 and U4/U6 snRNPs and promotes the reassembly of U4/U6 snRNPs after splicing in vitro. Here we report that SART3/p110 is enriched in CBs but not in gems or residual CBs lacking coilin. The U6 snRNP Sm-like (LSm) proteins, also involved in U4/U6 snRNP assembly, were localized to CBs as well. The levels of SART3/p110 and LSm proteins in CBs were reduced upon treatment with the transcription inhibitor α-amanitin, suggesting that CB localization reflects active processes dependent on transcription/splicing. The NH2-terminal HAT domain of SART3/p110 was necessary and sufficient for specific protein targeting to CBs. Overexpression of truncation mutants containing the HAT domain had dominant negative effects on U6 snRNP localization to CBs, indicating that endogenous SART3/p110 plays a role in targeting the U6 snRNP to CBs. We propose that U4 and U6 snRNPs accumulate in CBs for the purpose of assembly into U4/U6 snRNPs by SART3/p110.
Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane
Authors: Fons Ryen D. , Bogert Brigitte A. , Hegde Ramanujan S.
Published: February 2003

Although the transport of model proteins across the mammalian ER can be reconstituted with purified Sec61p complex, TRAM, and signal recognition particle receptor, some substrates, such as the prion protein (PrP), are inefficiently or improperly translocated using only these components. Here, we purify a factor needed for proper translocation of PrP and identify it as the translocon-associated protein (TRAP) complex. Surprisingly, TRAP also stimulates vectorial transport of many, but not all, other substrates in a manner influenced by their signal sequences. Comparative analyses of several natural signal sequences suggest that a dependence on TRAP for translocation is not due to any single physical parameter, such as hydrophobicity of the signal sequence. Instead, a functional property of the signal, efficiency of its post-targeting role in initiating substrate translocation, correlates inversely with TRAP dependence. Thus, maximal translocation independent of TRAP can only be achieved with a signal sequence, such as the one from prolactin, whose strong interaction with the translocon mediates translocon gating shortly after targeting. These results identify the TRAP complex as a functional component of the translocon and demonstrate that it acts in a substrate-specific manner to facilitate the initiation of protein translocation.
Compartmentalized megakaryocyte death generates functional platelets committed to caspase-independent death
Authors: Clarke Murray C.H. , Savill John , Jones David B. , Noble Brendon S. , Brown Simon B.
Published: February 2003

Caspase-directed apoptosis usually fragments cells, releasing nonfunctional, prothrombogenic, membrane-bound apoptotic bodies marked for rapid engulfment by macrophages. Blood platelets are functional anucleate cells generated by specialized fragmentation of their progenitors, megakaryocytes (MKs), but committed to a constitutive caspase-independent death. Constitutive formation of the proplatelet-bearing MK was recently reported to be caspase-dependent, apparently involving mitochondrial release of cytochrome c, a known pro-apoptogenic factor. We extend those studies and report that activation of caspases in MKs, either constitutively or after Fas ligation, yields platelets that are functionally responsive and evade immediate phagocytic clearance, and retain mitochondrial transmembrane potential until constitutive platelet death ensues. Furthermore, the exclusion from the platelet progeny of caspase-9 present in the progenitor accounts for failure of mitochondrial release of cytochrome c to activate caspase-3 during platelet death. Thus, progenitor cell death by apoptosis can result in birth of multiple functional anucleate daughter cells.
The FGF receptor uses the endocannabinoid signaling system to couple to an axonal growth response
Authors: Williams Emma-Jane , Walsh Frank S. , Doherty Patrick
Published: February 2003

Akey role for DAG lipase activity in the control of axonal growth and guidance in vitro and in vivo has been established. For example, DAG lipase activity is required for FGF-stimulated calcium influx into neuronal growth cones, and this response is both necessary and sufficient for an axonal growth response. The mechanism that couples the hydrolysis of DAG to the calcium response is not known. The initial hydrolysis of DAG at the sn-1 position (by DAG lipase) will generate 2-arachidonylglycerol, and this molecule is well established as an endogenous cannabinoid receptor agonist in the brain. In the present paper, we show that in rat cerebellar granule neurons, CB1 cannabinoid receptor antagonists inhibit axonal growth responses stimulated by N-cadherin and FGF2. Furthermore, three CB1 receptor agonists mimic the N-cadherin/FGF2 response at a step downstream from FGF receptor activation, but upstream from calcium influx into cells. In contrast, we could find no evidence for the CB1 receptor coupling the TrkB neurotrophin receptor to an axonal growth response in the same neurons. The observation that the CB1 receptor can couple the activated FGF receptor to an axonal growth response raises novel therapeutic opportunities.
The inner membrane protein Mdm33 controls mitochondrial morphology in yeast
Authors: Messerschmitt Marlies , Jakobs Stefan , Vogel Frank , Fritz Stefan , Dimmer Kai Stefan , Neupert Walter , Westermann Benedikt
Published: February 2003

Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Δmdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein–protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.
Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle
Authors: Handwerger Korie E. , Murphy Christine , Gall Joseph G.
Published: February 2003

Cajal bodies (CBs) are evolutionarily conserved nuclear organelles that contain many factors involved in the transcription and processing of RNA. It has been suggested that macromolecular complexes preassemble or undergo maturation within CBs before they function elsewhere in the nucleus. Most such models of CB function predict a continuous flow of molecules between CBs and the nucleoplasm, but there are few data that directly support this view. We used fluorescence recovery after photobleaching (FRAP) on isolated Xenopus oocyte nuclei to measure the steady-state exchange rate between the nucleoplasm and CBs of three fluorescently tagged molecules: U7 small nuclear RNA, coilin, and TATA-binding protein (TBP). In the nucleoplasm, the apparent diffusion coefficients for the three molecules ranged from 0.26 to 0.40 μm2 s−1. However, in CBs, fluorescence recovery was markedly slower than in the nucleoplasm, and there were at least three kinetic components. The recovery rate within CBs was independent of bleach spot diameter and could not be attributed to high CB viscosity or density. We propose that binding to other molecules and possibly assembly into larger complexes are the rate-limiting steps for FRAP of U7, coilin, and TBP inside CBs.
Selective Activation of the c-Jun NH2-terminal Protein Kinase Signaling Pathway by Stimulatory KIR in the Absence of KARAP/DAP12 in CD4+ T Cells
Authors: Snyder Melissa R. , Lucas Mathias , Vivier Eric , Weyand Cornelia M. , Goronzy Jörg J.
Published: February 2003

Activation of CD4+ T cells is governed by interplay between stimulatory and inhibitory receptors; predominance of stimulatory signals favors autoimmune reactions. In patients with rheumatoid arthritis, expression of the critical costimulatory molecule, CD28, is frequently lost. Instead, CD4+CD28null T cells express killer immunoglobulin-like receptors (KIRs) with a preferential expression of the stimulatory receptor, CD158j. The frequency of CD4+CD28null T cells in rheumatoid arthritis (RA) correlates with the risk for more severe disease. Moreover, the KIR2DS2 gene, which encodes for CD158j, is a genetic risk factor for rheumatoid vasculitis. CD158j signals through the adaptor molecule, KARAP/DAP12, to positively regulate cytotoxic activity in NK cells. However, the majority of CD4+CD28null T cell clones lacked the expression of KARAP/DAP12. Despite the absence of KARAP/DAP12, CD158j was functional and augmented interferon-γ production after T cell receptor stimulation. Cross-linking of CD158j resulted in selective phosphorylation of c-Jun NH2-terminal protein kinase (JNK) and its upstream kinase, MKK4 that led to the expression of ATF-2 and c-Jun, all in the absence of extracellular signal–regulated kinase (ERK)1/2 phosphorylation. Mutation of the lysine residue within the transmembrane domain of CD158j abolished JNK activation, suggesting that an alternate adaptor molecule was being used. CD4+CD28null T cells expressed DAP10 and inhibition of phosphatidylinositol 3-kinase, which acts downstream of DAP10, inhibited JNK activation; however, no interaction of DAP10 with CD158j could be detected. Our data suggest that CD158j in T cells functions as a costimulatory molecule through the JNK pathway independent of KARAP/DAP12 and DAP10. Costimulation by CD158j may contribute to the autoreactivity of CD4+CD28null T cells in RA.
A Role of Suppressor of Cytokine Signaling 3 (SOCS3/CIS3/SSI3) in CD28-mediated Interleukin 2 Production
Authors: Matsumoto Akira , Seki Yoh-ichi , Watanabe Ryosuke , Hayashi Katsuhiko , Johnston James A. , Harada Yohsuke , Abe Ryo , Yoshimura Akihiko , Kubo Masato
Published: February 2003

Suppressor of cytokine signaling (SOCS)3 has been characterized as a negative feedback regulator in cytokine-mediated Janus kinase signal transducer and activator of transcription signaling. However, this study shows that T cells from transgenic mice expressing SOCS3 exhibit a significant reduction in interleukin (IL)-2 production induced by T cell receptor cross-linking when T cells are costimulated with CD28. Decreased protein expression in SOCS3+/− mice enhanced CD28-mediated IL-2 production, clearly indicating the correlation between expression level of SOCS3 and IL-2 production ability. The SOCS3 protein interacted with phosphorylated CD28 through its SH2 domain but not the kinase inhibitory region. In addition, a point mutation in the SOCS3 SH2 domain attenuated the inhibition of CD28 function in IL-2 promoter activation. Committed T helper (Th)2 cells exclusively expressed SOCS3 and production of Th2 cytokines, such as IL-4 and IL-5, was much less dependent on CD28 costimulation compared with interferon γ and IL-2 production in Th1 cells. Consistent with this notion, the expression level of SOCS3 in early T cell activation influenced the ability of IL-2 production induced by CD28 costimulation. Therefore, the SOCS3 may play an alternative role in prohibiting excessive progression of CD28-mediated IL-2 production.
Prevention of Arthritis by Interleukin 10–producing B Cells
Authors: Mauri Claudia , Gray David , Mushtaq Naseem , Londei Marco
Published: February 2003

In this study we have shown that activation of arthritogenic splenocytes with antigen and agonistic anti-CD40 gives raise to a B cell population that produce high levels of interleukin (IL)-10 and low levels of interferon (IFN)-γ. Transfer of these B cells into DBA/1-TcR-β-Tg mice, immunized with bovine collagen (CII) emulsified in complete Freund's adjuvant inhibited T helper type 1 differentiation, prevented arthritis development, and was also effective in ameliorating established disease. IL-10 is essential for the regulatory function of this subset of B cells, as the B cells population isolated from IL-10 knockout mice failed to mediate this protective function. Furthermore, B cells isolated from arthritogenic splenocytes treated in vitro with anti–IL-10/anti–IL-10R were unable to protect recipient mice from developing arthritis. Our results suggest a new role of a subset of B cells in controlling T cell differentiation and autoimmune disorders.
T Cell Regulation as a Side Effect of Homeostasis and Competition
Authors: Barthlott Thomas , Kassiotis George , Stockinger Brigitta
Published: February 2003

We have previously hypothesized that maintaining a balanced peripheral immune system may not be the sole responsibility of a specialized subset of T cells dedicated to immune regulation, but also a side effect of normal competition for shared resources within an intact immune system. Here we show that regulatory activity is correlated with high homeostatic expansion potential, reflecting the avidity for self-peptide:MHC complexes. Monoclonal transgenic T cells with high homeostatic expansion potential and lacking characteristics previously associated with regulatory function were able to regulate wasting disease induced by transfer of a small number of naive CD45RBhi CD4 T cells into lymphopenic hosts. Self-regulatory function is also found in the naive polyclonal T cell repertoire depleted of CD25+ T cells. T cells capable of preventing immune pathology, like the transgenic T cells, express higher than average levels of CD5, an indicator of avidity for self:MHC peptide complexes. We therefore propose that dysregulated expansion of potentially pathogenic T cells in a lymphopenic environment can be prevented by members of the naive T cell repertoire, irrespective of their specificity, as a side effect of their response to homeostatic and antigenic stimulation.