bluuebunny/crossref_metadata_2025 huggingface dataset:
Selenium-deficient diet enhances protein oxidation and affects methionine sulfoxide reductase (MsrB) protein level in certain mouse tissues
Authors: Moskovitz Jackob , Stadtman Earl R.
Published: June 2003

Mammals contain two methionine sulfoxide (MetO) reductases, MsrA and MsrB, that catalyze the thioredoxin-dependent reduction of the
S
-MetO and
R
-MetO derivatives, respectively, to methionine. The major mammalian MsrB is a selenoprotein (except in the heart). Here, we show that there is a loss of MsrB activity in the
MsrA
–
/
–
mouse that correlates with parallel losses in the levels of MsrB mRNA and MsrB protein, suggesting that MsrA might have a role in MsrB transcription. Moreover, mice that were grown on a selenium-deficient (SD) diet showed a substantial decrease in the levels of MsrB-catalytic activity, MsrB protein, and MsrB mRNA in liver and kidney tissues of both WT and
MsrA
–
/
–
mouse strains. Whereas no significant protein-MetO could be detected in tissue proteins of young mature mice grown on a selenium-adequate diet, growth on the SD diet led to substantial accumulations of MetO in proteins and also of protein carbonyl derivatives in the liver, kidney, cerebrum, and cerebellum, respectively. In addition, accumulation of protein-MetO derivatives increased with age in tissues of mice fed with a selenium-adequate diet. It should be pointed out that even though the total Msr level is at least 2-fold higher in WT than in
MsrA
–
/
–
mice, SD diet causes an equal elevation of protein-MetO (except in brain cerebellum) and carbonyl levels in both strains, suggesting involvement of other selenoproteins in regulation of the level of cellular protein-MetO accumulation. Furthermore, the development of the ``tip-toe'' walking behavior previously observed in the
MsrA
–
/
–
mice occurred earlier when they were fed with the SD diet.
Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice
Authors: de Boer Ernie , Rodriguez Patrick , Bonte Edgar , Krijgsveld Jeroen , Katsantoni Eleni , Heck Albert , Grosveld Frank , Strouboulis John
Published: June 2003

Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific
in vivo
biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties
in vivo
nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.
Prediction and measurement of an autoregulatory genetic module
Authors: Isaacs Farren J. , Hasty Jeff , Cantor Charles R. , Collins J. J.
Published: June 2003

The deduction of phenotypic cellular responses from the structure and behavior of complex gene regulatory networks is one of the defining challenges of systems biology. This goal will require a quantitative understanding of the modular components that constitute such networks. We pursued an integrated approach, combining theory and experiment, to analyze and describe the dynamics of an isolated genetic module, an
in vivo
autoregulatory gene network. As predicted by the model, temperature-induced protein destabilization led to the existence of two expression states, thus elucidating the trademark bistability of the positive feedback-network architecture. After sweeping the temperature, observed population distributions and coefficients of variation were in quantitative agreement with those predicted by a stochastic version of the model. Because model fluctuations originated from small molecule-number effects, the experimental validation underscores the importance of internal noise in gene expression. This work demonstrates that isolated gene networks, coupled with proper quantitative descriptions, can elucidate key properties of functional genetic modules. Such an approach could lead to the modular dissection of naturally occurring gene regulatory networks, the deduction of cellular processes such as differentiation, and the development of engineered cellular control.
Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing
Authors: Jiang Zhen Y. , Zhou Qiong L. , Coleman Kerri A. , Chouinard My , Boese Queta , Czech Michael P.
Published: June 2003

Glucose homeostasis is controlled by insulin in part through the translocation of intracellular glucose transporter 4 to the plasma membrane in muscle and fat cells. Akt/protein kinase B downstream of phosphatidylinositol 3-kinase has been implicated in this insulin-signaling pathway, but results with a variety of reagents including Akt1
–
/
–
and Akt2
–
/
–
mice have been equivocal. Here we report the application of small interfering RNA-directed gene silencing to deplete both Akt1 and Akt2 in cultured 3T3-L1 adipocytes. Loss of Akt1 alone slightly impaired insulin-mediated hexose transport activity but had no detectable effect on glycogen synthase kinase (GSK)-3 phosphorylation. In contrast, depletion of Akt2 alone by 70% inhibited approximately half of the insulin responsiveness. Combined depletions of Akt1 plus Akt2 in these cells even more markedly attenuated insulin action on glucose transporter 4 movements, hexose transport activity, and GSK-3 phosphorylation. These data demonstrate a primary role of Akt2 in insulin signaling, significant functional redundancy of Akt1 and Akt2 isoforms in this pathway, and an absolute requirement of Akt protein kinases for regulation of glucose transport and GSK-3 in cultured adipocytes.
Control of
in vitro
organogenesis by cyclin-dependent kinase activities in plants
Authors: Yamaguchi Masatoshi , Kato Hisashi , Yoshida Shigeo , Yamamura Saburo , Uchimiya Hirofumi , Umeda Masaaki
Published: June 2003

Little is known concerning the mechanisms by which auxin and cytokinin exert their effects on proliferation and differentiation. Cyclin-dependent kinases (CDKs) are major regulators of the eukaryotic cell cycle, thus they are assumed to control cell differentiation as well as proliferation in response to phytohormone signals. Here, we overexpressed rice
R2
cDNA, which encodes a CDK-activating kinase, in tobacco leaf explants by using the glucocorticoid-mediated transcriptional induction system. Transient expression of
R2
during the first 7 days of culture triggered callus formation in the absence of cytokinin. This phenotype was enhanced by higher expression of
R2
or coexpression of cyclin H, and suppressed by treatment with roscovitine, a CDK inhibitor. R2 expression at a later stage did not prevent cells from differentiation into roots, suggesting a restricted period for sensing CDK activities that control differentiation fate of cells during organogenesis.
Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum
Authors: Ghoumari A. M. , Dusart I. , El-Etr M. , Tronche F. , Sotelo C. , Schumacher M. , Baulieu E.-E.
Published: June 2003

Mifepristone (RU486), which binds with high affinity to both progesterone and glucocorticosteroid receptors (PR and GR), is well known for its use in the termination of unwanted pregnancy, but other activities including neuroprotection have been suggested. Cerebellar organotypic cultures from 3 to 7 postnatal day rat (P3—P7) were studied to examine the neuroprotective potential of RU486. In such cultures, Purkinje cells enter a process of apoptosis with a maximum at P3. This study shows that RU486 (20 μM) can protect Purkinje cells from this apoptotic process. The neuroprotective effect did involve neither PR nor GR, because it could not be mimicked or inhibited by other ligands of these receptors, and because it still took place in PR mutant (PR-KO) mice and in brain-specific GR mutant mice (GR
Nes/Cre
). Potent antioxidant agents did not prevent Purkinje cells from this developmental cell death. The neuroprotective effect of RU486 could also be observed in pathological Purkinje cell death. Indeed, this steroid is able to prevent Purkinje cells from death in organotypic cultures of cerebellar slices from
Purkinje cell degeneration
(
pcd
) mutant mice, a murine model of hereditary neurodegenerative ataxia. In P0 cerebellar slices treated with RU486 for 6 days and further kept in culture up to 21 days, the synthetic steroid increased by 16.2-fold the survival of
pcd
/
pcd
Purkinje cells. Our results show that RU486 may act through a new mechanism, not yet elucidated, to protect Purkinje cells from death.
Human acetyl-CoA carboxylase 1 gene: Presence of three promoters and heterogeneity at the 5′-untranslated mRNA region
Authors: Mao Jianqiang , Chirala Subrahmanyam S. , Wakil Salih J.
Published: June 2003

Acetyl-CoA carboxylase 1 (ACC1) catalyzes the formation of malonyl-CoA, the C
2
donor for
de novo
synthesis of long-chain fatty acids. We have identified 64 exons, including 7 alternatively spliced minor exons (1A, 1B, 1C, 3, 5A′, 5A, and 5B) in human ACC1 gene (≈330 kb). The gene is regulated by three promoters (PI, PII, and PIII), which are located upstream of exons 1, 2, and 5A, respectively. PI is a constitutive promoter and has no homology with the PI sequences of other mammalian ACC1. PII is regulated by various hormones. PIII is expressed in a tissue-specific manner. The presence of several alternatively spliced exons does not alter the translation of the 265-kDa ACC1 protein starting from an ATG present in exon 5. Translation of PIII transcripts from exon 5A generates a 259-kDa isoform in which the N-terminal 75 aa of 265-kDa ACC1 are replaced with a new sequence of 17 aa. Interestingly, the inclusion of exon 5B between 5A and 6 in PIII transcripts would yield a third 257-kDa isoform, which is translated from an ATG in exon 6. However, the presence of exon 5B in PI and PII transcripts leads to an in-frame stop codon that results in an ACC1-related 77-aa peptide. The presence of alternatively spliced exons and three isoforms of ACC1 could contribute to overall ACC1 activity either by influencing the mRNA stability and translational efficiency or by increasing the stability and specific activity of the ACC1 protein, respectively.
Targeted cytosine methylation for
in vivo
detection of protein–DNA interactions
Authors: Carvin Christopher D. , Dhasarathy Archana , Friesenhahn Laurie B. , Jessen Walter J. , Kladde Michael P.
Published: June 2003

We report a technique, named targeted gene methylation (TAGM), for identifying
in vivo
protein-binding sites in chromatin. M.
Cvi
PI, a cytosine-5 DNA methyltransferase recognizing GC sites, is fused to a DNA-binding factor enabling simultaneous detection of targeted methylation, factor footprints, and chromatin structural changes by bisulfite genomic sequencing. Using TAGM with the yeast transactivator Pho4, methylation enrichments of up to 34- fold occur proximal to native Pho4-binding sites. Additionally, significant selective targeting of methylation is observed several hundred nucleotides away, suggesting the detection of long-range interactions due to higher-order chromatin structure. In contrast, at an extragenic locus lacking Pho4-binding sites, methylation levels are at the detection limit at early times after Pho4 transactivation. Notably, substantial amounts of methylation are targeted by Pho4-M.
Cvi
PI under repressive conditions when most of the transactivator is excluded from the nucleus. Thus, TAGM enables rapid detection of DNA–protein interactions even at low occupancies and has potential for identifying factor targets at the genome-wide level. Extension of TAGM from yeast to vertebrates, which use methylation to initiate and propagate repressed chromatin, could also provide a valuable strategy for heritable inactivation of gene expression.
Recombinant lymphocytic choriomeningitis virus expressing vesicular stomatitis virus glycoprotein
Authors: Pinschewer Daniel D. , Perez Mar , Sanchez Ana B. , de la Torre Juan Carlos
Published: June 2003

A recombinant S segment RNA (Sr) of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) where the glycoprotein of vesicular stomatitis virus (VSVG) was substituted for the glycoprotein of LCMV (LCMV-GP) was produced intracellularly from cDNA under the control of a polymerase I promoter. Coexpression of the LCMV proteins NP and L allowed expression of VSVG from Sr. Infection of transfected cells with WT LCMV (LCMVwt) resulted in reassortment of the L segment of LCMVwt with the Sr at low frequency. Isolation of recombinant LCMV (rLCMV) expressing VSVG (rLCMV/VSVG) was achieved by selection against LCMVwt by using a cell line deficient in the cellular protease S1P. This approach was based on the finding that processing of LCMV-GP by S1P was required for virus infectivity. Characterization of protein and RNA expression of rLCMV/VSVG in infected cells confirmed the expected virus genome organization. rLCMV/VSVG caused syncytium formation in cultured cells and grew to ≈100-fold lower titers than WT virus but, like the parent virus, it persisted in neonatally infected mice without clinical signs of disease.
Determination of the membrane topology of Ost4p and its subunit interactions in the oligosaccharyltransferase complex in
Saccharomyces cerevisiae
Authors: Kim Hyun , Yan Qi , von Heijne Gunnar , Caputo Gregory A. , Lennarz William J.
Published: June 2003

Ost4p is a minimembrane protein containing only 36 amino acids and is a subunit of oligosaccharyltransferase (OT) in
Saccharomyces cerevisiae
. It was found previously when amino acid residues 18–25 of Ost4p were mutated to ionizable amino acids and defects were observed in the interaction between Ost4p and either Stt3p or Ost3p, two other components of OT. The transmembrane segment of Ost4p is likely to extend from residues 10–25. This is consistent with the finding that α-helicity is estimated to be 36% by CD analysis of synthetic Ost4p in liposomes. This value is in reasonable agreement with the assumption that amino acids 10–25 (16 of 36 or 44%) are transmembrane. Therefore, the mutation-sensitive region (residues 18–25) is localized to only one half of the putative transmembrane domain of Ost4p. To learn where this region of Ost4p is situated in relation to the faces of endoplasmic reticulum (ER) membrane, we determined the membrane topology of Ost4p using an
in vivo
method and established that it is an N
lumen
-C
cyto
, type I membrane protein. These results indicate that the mutation-sensitive region of Ost4p is localized in the cytoplasmic leaflet of the ER membrane. In the current study, we also observed a loss of direct interaction between Ost3p and Stt3p in the presence of
ost4
temperature-sensitive mutants, which indicates Ost4p, via interactions with amino acid residues in the cytosolic leaflet of the ER membrane, functions to bind these two proteins together in a subcomplex of OT.
T-bet is required for optimal production of IFN-γ and antigen-specific T cell activation by dendritic cells
Authors: Lugo-Villarino Geanncarlo , Maldonado-López Roberto , Possemato Richard , Peñaranda Cristina , Glimcher Laurie H.
Published: June 2003

IFN-γ is well known as the signature cytokine of CD4+T helper 1, CD8+, and natural killer cells, but recent studies demonstrate that antigen-presenting cells, in particular dendritic cells (DCs), are another potent source for this proinflammatory cytokine. T-bet, a transcription factor that controls IFN-γ expression in CD4+T cells, was reported recently to be expressed in human monocytes and myeloid DCs. In this study we investigate the role of T-bet in this important cell type. The development, differentiation, and activation of bone marrow and splenic DCs were unimpaired in mice lacking T-bet. However, T-bet was essential for the optimal production of IFN-γ by both CD8α+and CD8α-DCs. T-bet-deficient DCs were significantly impaired in their capacity to secrete IFN-γ after both stimulation with IL-12 alone or in combination with IL-18. Further, T-bet-/-DCs were impaired in their ability to activate the T helper 1 program of adoptively transferred antigen-specific T cellsin vivo. The rapid up-regulation of T-bet by IFN-γ in DCs coupled with a function for DC-derived IFN-γ in T cell activation may constitute a positive feedback loop to maximize type 1 immunity.
RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase
Authors: Frugier Magali , Giegé Richard , Schimmel Paul
Published: June 2003

The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs. The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3′ end of bound tRNA. Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain. Here we take the domain for synthesis of alanyl adenylate and fuse it to ``artificial'' peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNA
Ala
. Certain fusions confer aminoacylation activity on tRNA
Ala
and on hairpin microhelices modeled after its acceptor stem. Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNA
Ala
identity. Aminoacylation efficiency and specificity also depended on the specific peptide sequence. The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions. They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.
Mechanotransduction and flow across the endothelial glycocalyx
Authors: Weinbaum Sheldon , Zhang Xiaobing , Han Yuefeng , Vink Hans , Cowin Stephen C.
Published: June 2003

In this inaugural paper, we shall provide an overview of the endothelial surface layer or glycocalyx in several roles: as a transport barrier, as a porous hydrodynamic interface in the motion of red and white cells in microvessels, and as a mechanotransducer of fluid shearing stresses to the actin cortical cytoskeleton of the endothelial cell. These functions will be examined from a new perspective, the quasiperiodic ultrastructural model proposed in Squireet al. [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001)J. Struct. Biol.136, 239–255] for the 3D organization of the endothelial surface layer and its linkage to the submembranous scaffold. We shall show that the core proteins in the bush-like structures comprising the matrix have a flexural rigidity,EI, that is sufficiently stiff to serve as a molecular filter for plasma proteins and as an exquisitely designed transducer of fluid shearing stresses. However,EIis inadequate to prevent the buckling of these protein structures during the intermittent motion of red cells or the penetration of white cell microvilli. In these cellular interactions, the viscous draining resistance of the matrix is essential for preventing adhesive molecular interactions between proteins in the endothelial membrane and circulating cellular components.
Male Japanese quails with female brains do not show male sexual behaviors
Authors: Gahr Manfred
Published: June 2003

During embryonic development, gonadal steroid hormones (androgens and estrogens) are thought to organize the sexual differentiation of the brain in the heterogametic sexes of higher vertebrates (males in mammals, females in birds). Brain differentiation of the homogametic sexes is thought to proceed by default, not requiring sex hormones for sex-specific organization. In gallinaceous birds such as the Japanese quail, female brain organization is thought to develop via estrogen-dependent demasculinization of a default male brain phenotype. We performed male donor-to-female host (MF), female-to-male (FM), male-to-male (MM), and female-to-female (FF) isotopic, isochronic transplantation of the forebrain primordium in Japanese quail embryos before gonadal differentiation had occurred; brain chimeras had a forebrain (including the hypothalamus) originating exclusively from donor cells. MM, FF, and MF chimeras all showed sexual behavior governed by the genetic sex of the host. In contrast, FM chimeras (genetically female forebrain, all other tissues genetically male) showed no mounting and only rudimentary crowing behavior. Although MM, FF, MF, and FM chimeras all showed host-typical production of steroid hormones during embryonic life, only FM chimeras were hypogonadal, had atypical low levels of circulating testosterone in adulthood, and showed reduction (crowing) or absence (mounting) of reproductive behaviors. Morphological features of the medial preoptic nucleus (a sexually dimorphic brain area) also were not male-like in FM males. These data demonstrate a brain-intrinsic, genetically determined component that organizes the sex-typical production of gonadal hormones in adulthood and call for a reevaluation of the mechanisms underlying brain sexual differentiation in other higher-vertebrate species.
Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing
Authors: Wülfing Christoph , Purtic Bozidar , Klem Jennifer , Schatzle John D.
Published: June 2003

Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.
Epigenetic inactivation of CHFR in human tumors
Authors: Toyota Minoru , Sasaki Yasushi , Satoh Ayumi , Ogi Kazuhiro , Kikuchi Takefumi , Suzuki Hiromu , Mita Hiroaki , Tanaka Nobuyuki , Itoh Fumio , Issa Jean-Pierre J. , Jair Kam-Wing , Schuebel Kornel E. , Imai Kohzoh , Tokino Takashi
Published: June 2003

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the
CHFR
gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin–ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of
CHFR
expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of
CHFR
expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of
CHFR
was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of
CHFR
depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored
CHFR
expression. Finally, cells with
CHFR
methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.
Systematic discovery of multicomponent therapeutics
Authors: Borisy Alexis A. , Elliott Peter J. , Hurst Nicole W. , Lee Margaret S. , Lehár Joseph , Price E. Roydon , Serbedzija George , Zimmermann Grant R. , Foley Michael A. , Stockwell Brent R. , Keith Curtis T.
Published: June 2003

Multicomponent therapies, originating through deliberate mixing of drugs in a clinical setting, through happenstance, and through rational design, have a successful history in a number of areas of medicine, including cancer, infectious diseases, and CNS disorders. We have developed a high-throughput screening method for identifying effective combinations of therapeutic compounds. We report here that systematic screening of combinations of small molecules reveals unexpected interactions between compounds, presumably due to interactions between the pathways on which they act. Through systematic screening of ≈120,000 different two-component combinations of reference-listed drugs, we identified potential multicomponent therapeutics, including (
i
) fungistatic and analgesic agents that together generate fungicidal activity in drug-resistant
Candida albicans
, yet do not significantly affect human cells, (
ii
) glucocorticoid and antiplatelet agents that together suppress the production of tumor necrosis factor-α in human primary peripheral blood mononu-clear cells, and (
iii
) antipsychotic and antiprotozoal agents that do not exhibit significant antitumor activity alone, yet together prevent the growth of tumors in mice. Systematic combination screening may ultimately be useful for exploring the connectivity of biological pathways and, when performed with reference-listed drugs, may result in the discovery of new combination drug regimens.
Inducible nitric oxide synthase expression inhibition by adenovirus E1A
Authors: Cao Wangsen , Bao Clare , Lowenstein Charles J.
Published: June 2003

Nitric oxide (NO) is an antiviral effector of the innate immune system. Viruses that can interfere with NO synthesis may be able to replicate more rapidly than viruses that cannot limit NO synthesis. We show that the adenovirus E1A protein inhibits NO production by decreasing expression of the inducible NO synthase (NOS2). The amino-terminal portion of E1A decreases transactivation of the NOS2 5′-flanking region, limiting the DNA binding activity of NF-κB and inhibiting NOS2 expression. E1A is thus able to deactivate a critical component of the host defense against viral infection. Viral inhibition of NO production is a mechanism that may enable certain viruses to evade the host innate immune system.
Single-molecule detection of DNA hybridization
Authors: Singh-Zocchi Mukta , Dixit Sanhita , Ivanov Vassili , Zocchi Giovanni
Published: June 2003

We demonstrate the detection of nanometer-scale conformational changes of single DNA oligomers through a micromechanical technique. The quantity monitored is the displacement of a micrometer-size bead tethered to a surface by the probe molecule undergoing the conformational change. This technique allows probing of conformational changes within distances beyond the range of fluorescence resonance energy transfer. We apply the method to detect single hybridization events of label-free target oligomers. Hybridization of the target is detected through the conformational change of the probe.
Structural determinant of TRPV1 desensitization interacts with calmodulin
Authors: Numazaki Mitsuko , Tominaga Tomoko , Takeuchi Kumiko , Murayama Namie , Toyooka Hidenori , Tominaga Makoto
Published: June 2003

The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca
2+
-dependent desensitization of TRPV1 observed in patch–clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. Here, we show that the Ca
2+
-binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and that disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain.
Tetraspanin CD151 regulates α6β1 integrin adhesion strengthening
Authors: Lammerding Jan , Kazarov Alexander R. , Huang Hayden , Lee Richard T. , Hemler Martin E.
Published: June 2003

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including α6β1. To probe strength of α6β1-dependent adhesion to laminin-1, defined forces (0–1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-α6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening α6β1 integrin-mediated adhesion to laminin-1.
Regression of established tumors and metastases by potent vascular endothelial growth factor blockade
Authors: Huang Jianzhong , Frischer Jason S. , Serur Anna , Kadenhe Angela , Yokoi Akiko , McCrudden Kimberly W. , New Tamara , O'Toole Kathleen , Zabski Stephanie , Rudge John S. , Holash Jocelyn , Yancopoulos George D. , Yamashiro Darrell J. , Kandel Jessica J.
Published: June 2003

Vascular endothelial growth factor (VEGF) is a critical promoter of blood vessel growth during embryonic development and tumorigenesis. To date, studies of VEGF antagonists have primarily focused on halting progression in models of minimal residual cancer. Consistent with this focus, recent clinical trials suggest that blockade of VEGF may impede cancer progression, presumably by preventing neoangiogenesis. However, VEGF is also a key mediator of endothelial–vascular mural cell interactions, a role that may contribute to the integrity of mature vessels in advanced tumors. Here, we report that high-affinity blockade of VEGF, using the recently described VEGF-Trap, abolishes mature, preexisting vasculature in established xenografts. Eradication of vasculature is followed by marked tumor regression, including regression of lung micrometastases. Thus, the contribution of relatively low levels of VEGF to vessel integrity may be critical to maintenance of even very small tumor masses. Potent blockade of VEGF may provide a new therapeutic option for patients with bulky, metastatic cancers.
Mice with targeted disruption of the fatty acid transport protein 4 (Fatp 4, Slc27a4) gene show features of lethal restrictive dermopathy
Authors: Herrmann Thomas , van der Hoeven Frank , Gröne Hermann-Josef , Stewart Adrian Francis , Langbein Lutz , Kaiser Iris , Liebisch Gerhard , Gosch Isabella , Buchkremer Florian , Drobnik Wolfgang , Schmitz Gerd , Stremmel Wolfgang
Published: June 2003

The fatty acid transport protein family is a group of evolutionarily conserved proteins that are involved in the cellular uptake and metabolism of long and very long chain fatty acids. However, little is known about their respective physiological roles. To analyze the functional significance of fatty acid transport protein 4 (Fatp4, Slc27a4), we generated mice with a targeted disruption of the Fatp4 gene. Fatp4-null mice displayed features of a neonatally lethal restrictive dermopathy. Their skin was characterized by hyperproliferative hyperkeratosis with a disturbed epidermal barrier, a flat dermal–epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis. The rigid skin consistency resulted in an altered body shape with facial dysmorphia, generalized joint flexion contractures, and impaired movement including suckling and breathing deficiencies. Lipid analysis demonstrated a disturbed fatty acid composition of epidermal ceramides, in particular a decrease in the C26:0 and C26:0-OH fatty acid substitutes. These findings reveal a previously unknown, essential function of Fatp4 in the formation of the epidermal barrier.
Back signaling by the Nrg-1 intracellular domain
Authors: Bao Jianxin , Wolpowitz Deon , Role Lorna W. , Talmage David A.
Published: June 2003

Transmembrane isoforms of neuregulin-1 (Nrg-1), ligands for erbB receptors, include an extracellular domain with an EGF-like sequence and a highly conserved intracellular domain (ICD) of unknown function. In this paper, we demonstrate that transmembrane isoforms of Nrg-1 are bidirectional signaling molecules in neurons. The stimuli for Nrg-1 back signaling include binding of erbB receptor dimers to the extracellular domain of Nrg-1 and neuronal depolarization. These stimuli elicit proteolytic release and translocation of the ICD of Nrg-1 to the nucleus. Once in the nucleus, the Nrg-1 ICD represses expression of several regulators of apoptosis, resulting in decreased neuronal cell death in vitro. Thus, regulated proteolytic processing of Nrg-1 results in retrograde signaling that appears to mediate contact and activity-dependent survival of Nrg-1–expressing neurons.
Critical role for scaffolding adapter Gab2 in FcγR-mediated phagocytosis
Authors: Gu Haihua , Botelho Roberto J. , Yu Min , Grinstein Sergio , Neel Benjamin G.
Published: June 2003

Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding molecules, plays important roles in regulating the growth, differentiation, and function of many hematopoietic cell types. In this paper, we reveal a novel function of Gab2 in Fcγ receptor (FcγR)–initiated phagocytosis in macrophages. Upon FcγR activation, Gab2 becomes tyrosyl phosphorylated and associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and the protein–tyrosine phosphatidylinositol Shp-2. FcγR-mediated phagocytosis is severely impaired in bone marrow–derived macrophages from Gab2−/− mice. The defect in phagocytosis correlates with decreased FcγR-evoked activation of Akt, a downstream target of PI3K. Using confocal fluorescence microscopy, we find that Gab2 is recruited to the nascent phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires the pleckstrin homology domain of Gab2 and is sensitive to treatment with the PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary role in recruitment to the phagosome. Because PI3K activity is required for FcγR-mediated phagocytosis, our results indicate that Gab2 acts as a key component of FcγR-mediated phagocytosis, most likely by amplifying PI3K signaling in the nascent phagosome.
Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure
Authors: Reiser Vladimír , Raitt Desmond C. , Saito Haruo
Published: June 2003

Very little is known about how cellular osmosensors monitor changes in osmolarity of the environment. Here, we report that in yeast, Sln1 osmosensor histidine kinase monitors changes in turgor pressures. Reductions in turgor caused by either hyperosmotic stress, nystatin, or removal of cell wall activate MAPK Hog1 specifically through the SLN1 branch, but not through the SHO1 branch of the high osmolarity glycerol pathway. The integrity of the periplasmic region of Sln1 was essential for its sensor function. We found that activity of the plant histidine kinase cytokinin response 1 (Cre1) is also regulated by changes in turgor pressure, in a manner identical to that of Sln1, in the presence of cytokinin. We propose that Sln1 and Cre1 are turgor sensors, and that similar turgor-sensing mechanisms might regulate hyperosmotic stress responses both in yeast and plants.
Dynamic instability of microtubules is regulated by force
Authors: Janson Marcel E. , de Dood Mathilde E. , Dogterom Marileen
Published: June 2003

Microtubules are long filamentous protein structures that randomly alternate between periods of elongation and shortening in a process termed dynamic instability. The average time a microtubule spends in an elongation phase, known as the catastrophe time, is regulated by the biochemical machinery of the cell throughout the cell cycle. In this light, observed changes in the catastrophe time near cellular boundaries (Brunner, D., and P. Nurse. 2000. Cell. 102:695–704; Komarova, Y.A., I.A. Vorobjev, and G.G. Borisy. 2002. J. Cell Sci. 115:3527–3539) may be attributed to regulatory effects of localized proteins. Here, we argue that the pushing force generated by a microtubule when growing against a cellular object may itself provide a regulatory mechanism of the catastrophe time. We observed an up to 20-fold, force-dependent decrease in the catastrophe time when microtubules grown from purified tubulin were polymerizing against microfabricated barriers. Comparison with catastrophe times for microtubules growing freely at different tubulin concentrations leads us to conclude that force reduces the catastrophe time only by limiting the rate of tubulin addition.
Low density lipoprotein receptor–related protein is a calreticulin coreceptor that signals focal adhesion disassembly
Authors: Orr Anthony Wayne , Pedraza Claudio E. , Pallero Manuel Antonio , Elzie Carrie A. , Goicoechea Silvia , Strickland Dudley K. , Murphy-Ullrich Joanne E.
Published: June 2003

Thrombospondin (TSP) signals focal adhesion disassembly (the intermediate adhesive state) through interactions with cell surface calreticulin (CRT). TSP or a peptide (hep I) of the active site induces focal adhesion disassembly through binding to CRT, which activates phosphoinositide 3-kinase (PI3K) and extracellular signal–related kinase (ERK) through Gαi2 proteins. Because CRT is not a transmembrane protein, it is likely that CRT signals as part of a coreceptor complex. We now show that low density lipoprotein receptor–related protein (LRP) mediates focal adhesion disassembly initiated by TSP binding to CRT. LRP antagonists (antibodies, receptor-associated protein) block hep I/TSP-induced focal adhesion disassembly. LRP is necessary for TSP/hep I signaling because TSP/hep I is unable to stimulate focal adhesion disassembly or ERK or PI3K signaling in fibroblasts deficient in LRP. LRP is important in TSP–CRT signaling, as shown by the ability of hep I to stimulate association of Gαi2 with LRP. The isolated proteins LRP and CRT interact, and LRP and CRT are associated with hep I in molecular complexes extracted from cells. These data establish a mechanism of cell surface CRT signaling through its coreceptor, LRP, and suggest a novel function for LRP in regulating cell adhesion.
A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation
Authors: Dailey Lisa , Laplantine Emmanuel , Priore Riccardo , Basilico Claudio
Published: June 2003

Activating mutations in FGF receptor 3 (FGFR3) cause several human dwarfism syndromes by affecting both chondrocyte proliferation and differentiation. Using microarray and biochemical analyses of FGF-treated rat chondrosarcoma chondrocytes, we show that FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E–Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3. Thus, FGF may coordinate the onset of differentiation with chondrocyte growth arrest in the developing growth plate.
The tale of tail-anchored proteins
Authors: Borgese Nica , Colombo Sara , Pedrazzini Emanuela
Published: June 2003

A group of integral membrane proteins, known as C-tail anchored, is defined by the presence of a cytosolic NH2-terminal domain that is anchored to the phospholipid bilayer by a single segment of hydrophobic amino acids close to the COOH terminus. The mode of insertion into membranes of these proteins, many of which play key roles in fundamental intracellular processes, is obligatorily posttranslational, is highly specific, and may be subject to regulatory processes that modulate the protein's function. Although recent work has elucidated structural features in the tail region that determine selection of the correct target membrane, the molecular machinery involved in interpreting this information, and in modulating tail-anchored protein localization, has not been identified yet.
The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts
Authors: Wignall Sarah M. , Deehan Renée , Maresca Thomas J. , Heald Rebecca
Published: June 2003

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.
A requirement for MAP kinase in the assembly and maintenance of the mitotic spindle
Authors: Horne Melinda M. , Guadagno Thomas M.
Published: June 2003

Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.
The conjugacy problem for groups, and Higman embeddings
Authors: Ol’shanskii A. , Sapir M.
Published: June 2003

For every finitely generated recursively presented group

G

{\mathcal G}

we construct a finitely presented group

H

{\mathcal H}

containing

G

{\mathcal G}

such that

G

{\mathcal G}

is (Frattini) embedded into

H

{\mathcal H}

and the group

H

{\mathcal H}

has solvable conjugacy problem if and only if

G

{\mathcal G}

has solvable conjugacy problem. Moreover,

G

{\mathcal G}

and

H

{\mathcal H}

have the same r.e. Turing degrees of the conjugacy problem. This solves a problem by D. Collins.
Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis
Authors: Holt Jason A. , Luo Guizhen , Billin Andrew N. , Bisi John , McNeill Y. Yvette , Kozarsky Karen F. , Donahee Mary , Wang Da Yuan , Mansfield Traci A. , Kliewer Steven A. , Goodwin Bryan , Jones Stacey A.
Published: June 2003

The nuclear bile acid receptor FXR has been proposed to play a central role in the feedback repression of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), the first and rate-limiting step in the biosynthesis of bile acids. We demonstrate that FXR directly regulates expression of fibroblast growth factor-19 (FGF-19), a secreted growth factor that signals through the FGFR4 cell-surface receptor tyrosine kinase. In turn, FGF-19 strongly suppresses expression ofCYP7A1in primary cultures of human hepatocytes and mouse liver through a c-Jun N-terminal kinase (JNK)-dependent pathway. This signaling cascade defines a novel mechanism for feedback repression of bile acid biosynthesis and underscores the vital role of FXR in the regulation of multiple pathways of cholesterol catabolism in the liver.
DrosophilaFACT contributes toHoxgene expression through physical and functional interactions with GAGA factor
Authors: Shimojima Tsukasa , Okada Masahiro , Nakayama Takahiro , Ueda Hitoshi , Okawa Katsuya , Iwamatsu Akihiro , Handa Hiroshi , Hirose Susumu
Published: June 2003

Chromatin structure plays a critical role in the regulation of transcription.DrosophilaGAGA factor directs chromatin remodeling to its binding sites. We show here thatDrosophilaFACT (facilitateschromatintranscription), a heterodimer of dSPT16 and dSSRP1, is associated with GAGA factor through its dSSRP1 subunit, binds to a nucleosome, and facilitates GAGA factor-directed chromatin remodeling. Moreover, genetic interactions betweenTrithorax-likeencoding GAGA factor andspt16implicate the GAGA factor–FACT complex in expression ofHoxgenesUltrabithorax, Sex combs reduced, andAbdominal-B. Chromatin immunoprecipitation experiments indicated the presence of the GAGA factor–FACT complex in the regulatory regions ofUltrabithoraxandAbdominal-B.These data illustrate a crucial role of FACT in the modulation of chromatin structure for the regulation of gene expression.