bluuebunny/crossref_metadata_2025 huggingface dataset:
Vascular endothelial growth factor (VEGF) stimulates neurogenesis
in vitro
and
in vivo
Authors: Jin Kunlin , Zhu Yonghua , Sun Yunjuan , Mao Xiao Ou , Xie Lin , Greenberg David A.
Published: September 2002

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Because VEGF promotes the proliferation of vascular endothelial cells, we examined the possibility that it also stimulates the proliferation of neuronal precursors in murine cerebral cortical cultures and in adult rat brain
in vivo
. VEGF (>10 ng/ml) stimulated 5-bromo-2′-deoxyuridine (BrdUrd) incorporation into cells that expressed immature neuronal marker proteins and increased cell number in cultures by 20–30%. Cultured cells labeled by BrdUrd expressed VEGFR2/Flk-1, but not VEGFR1/Flt-1 receptors, and the effect of VEGF was blocked by the VEGFR2/Flk-1 receptor tyrosine kinase inhibitor SU1498. Intracerebroventricular administration of VEGF into rat brain increased BrdUrd labeling of cells in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), where VEGFR2/Flk-1 was colocalized with the immature neuronal marker, doublecortin (Dcx). The increase in BrdUrd labeling after the administration of VEGF was caused by an increase in cell proliferation, rather than a decrease in cell death, because VEGF did not reduce caspase-3 cleavage in SVZ or SGZ. Cells labeled with BrdUrd after VEGF treatment
in vivo
include immature and mature neurons, astroglia, and endothelial cells. These findings implicate the angiogenesis factor VEGF in neurogenesis as well.
Probing the transition state via photoelectron and photodetachment spectroscopy of H
3
O
−
Authors: Zhang Dong H. , Yang Minghui , Collins Michael A. , Lee Soo-Y.
Published: September 2002

The H
3
O
−
anion has stable and metastable structures that resemble configurations in the vicinity of the transition state for the neutral reactions OH + H
2
↔ H
2
O + H. Photoelectron spectroscopy of this anion probes the neutral reaction dynamics in the critical transition-state region. Accurate quantum dynamics calculations of the photoelectron intensity and photodissociation product energies are shown to provide a quantitatively reliable means of interpreting such experimental observations and reveal a detailed picture of the reaction dynamics.
A duplicated copy of
DMRT1
in the sex-determining region of the Y chromosome of the medaka,
Oryzias latipes
Authors: Nanda Indrajit , Kondo Mariko , Hornung Ute , Asakawa Shuichi , Winkler Christoph , Shimizu Atsushi , Shan Zhihong , Haaf Thomas , Shimizu Nobuyoshi , Shima Akihiro , Schmid Michael , Schartl Manfred
Published: September 2002

The genes that determine the development of the male or female sex are known in
Caenorhabditis elegans
,
Drosophila
, and most mammals. In many other organisms the existence of sex-determining factors has been shown by genetic evidence but the genes are unknown. We have found that in the fish medaka the Y chromosome-specific region spans only about 280 kb. It contains a duplicated copy of the autosomal
DMRT1
gene, named
DMRT1Y
. This is the only functional gene in this chromosome segment and maps precisely to the male sex-determining locus. The gene is expressed during male embryonic and larval development and in the Sertoli cells of the adult testes. These features make
DMRT1Y
a candidate for the medaka male sex-determining gene.
The histone deacetylase inhibitor SAHA arrests cancer cell growth, up-regulates thioredoxin-binding protein-2, and down-regulates thioredoxin
Authors: Butler Lisa M. , Zhou Xianbo , Xu Wei-Sheng , Scher Howard I. , Rifkind Richard A. , Marks Paul A. , Richon Victoria M.
Published: September 2002

Suberoylanilide hydroxamic acid (SAHA) is a potent inhibitor of histone deacetylases (HDACs) that causes growth arrest, differentiation, and/or apoptosis of many tumor types
in vitro
and
in vivo
. SAHA is in clinical trials for the treatment of cancer. HDAC inhibitors induce the expression of less than 2% of genes in cultured cells. In this study we show that SAHA induces the expression of vitamin D-up-regulated protein 1/thioredoxin-binding protein-2 (TBP-2) in transformed cells. As the expression of TBP-2 mRNA is increased, the expression of a second gene, thioredoxin, is decreased. In transient transfection assays, HDAC inhibitors induce TBP-2 promoter constructs, and this induction requires an NF-Y binding site. We report here that TBP-2 expression is reduced in human primary breast and colon tumors compared with adjacent tissue. These results support a model in which the expression of a subset of genes (i.e., including TBP-2) is repressed in transformed cells, leading to a block in differentiation, and culture of transformed cells with SAHA causes re-expression of these genes, leading to induction of growth arrest, differentiation, and/or apoptosis.
Essential myosin light chain as a target for caspase-3 in failing myocardium
Authors: Moretti Alessandra , Weig Hans-Jörg , Ott Thomas , Seyfarth Melchior , Holthoff Hans-Peter , Grewe Diana , Gillitzer Angelika , Bott-Flügel Lorenz , Schömig Albert , Ungerer Martin , Laugwitz Karl-Ludwig
Published: September 2002

Programmed cell death involves the activation of caspase proteases that can mediate the cleavage of vital cytoskeletal proteins. We have recently reported that, in failing cardiac myocytes, caspase-3 activation is associated with a reduction in contractile performance. In this study we used a modified yeast two-hybrid system to screen for caspase-3 interacting proteins of the cardiac cytoskeleton. We identified ventricular essential myosin light chain (vMLC1) as a target for caspase-3. By sequencing and site-directed mutagenesis, a noncanonical cleavage site for caspase-3 was mapped to the C-terminal DFVE
135
G motif. We demonstrated that vMLC1 cleavage in failing myocardium
in vivo
is associated with a morphological disruption of the organized vMLC1 staining of sarcomeres, and with a reduction in myocyte contractile performance. Adenoviral gene transfer of the caspase inhibitor p35
in vivo
prevented caspase-3 activation and vMLC1 cleavage, with positive impact on contractility. These data suggest that direct cleavage of vMLC1 by activated caspase-3 may contribute to depression of myocyte function by altering cross-bridge interaction between myosin and actin molecules. Therefore, activation of apoptotic pathways in the heart may lead to contractile dysfunction before cell death.
The program of androgen-responsive genes in neoplastic prostate epithelium
Authors: Nelson Peter S. , Clegg Nigel , Arnold Hugh , Ferguson Camari , Bonham Michael , White James , Hood Leroy , Lin Biaoyang
Published: September 2002

The human prostate gland is an important target organ of androgenic hormones. Testosterone and dihydrotestosterone interact with the androgen receptor to regulate vital aspects of prostate growth and function including cellular proliferation, differentiation, apoptosis, metabolism, and secretory activity. Our objective in this study was to characterize the temporal program of transcription that reflects the cellular response to androgens and to identify specific androgen-regulated genes (ARGs) or gene networks that participate in these responses. We used cDNA microarrays representing about 20,000 distinct human genes to profile androgen-responsive transcripts in the LNCaP adenocarcinoma cell line and identified 146 genes with transcript alterations more than 3-fold. Of these, 103 encode proteins with described functional roles, and 43 represent transcripts that have yet to be characterized. Temporal gene expression profiles grouped the ARGs into four distinct cohorts. Five uncharacterized ARGs demonstrated exclusive or high expression levels in the prostate relative to other tissues studied. A search of available DNA sequence upstream of 28 ARGs identified 25 with homology to the androgen response-element consensus-binding motif. These results identify previously uncharacterized and unsuspected genes whose expression levels are directly or indirectly regulated by androgens; further, they provide a comprehensive temporal view of the transcriptional program of human androgen-responsive cells.
1-Cys peroxiredoxin overexpression protects cells against phospholipid peroxidation-mediated membrane damage
Authors: Manevich Yefim , Sweitzer Tom , Pak Jhang Ho , Feinstein Sheldon I. , Muzykantov Vladimir , Fisher Aron B.
Published: September 2002

1-Cys peroxiredoxin (1-cysPrx) is a novel antioxidant enzyme able to reduce phospholipid hydroperoxides
in vitro
by using glutathione as a reductant. This enzyme is widely expressed and is enriched in lungs. A fusion protein of green fluorescent protein with 1-cysPrx was stably expressed in a lung-derived cell line (NCI-H441) lacking endogenous enzyme. Overexpressing cells (C17 or C48) degraded H
2
O
2
and
t
-butylhydroperoxide more rapidly and showed decreased sensitivity to oxidant stress as measured by
51
Cr release. On exposure to
•
OH generated by Cu
2+
-ascorbate (Asc), overexpressing cells compared with H441 showed less increase in thiobarbituric acid-reactive substance and phosphatidylcholine hydroperoxide content. This effect was reversed by depletion of cellular glutathione. Diphenyl-1-pyrenoylphosphonium fluorescence, used as a real-time probe of membrane phospholipid peroxidation, increased immediately on exposure to Cu
2+
-Asc and was abolished by preincubation of cells with Trolox (a soluble vitamin E) or Tempol (a radical scavenger). The rate of diphenyl-1-pyrenoylphosphonium fluorescence increase with Cu
2+
-Asc exposure was markedly attenuated in C17 and C48 cells as compared with H441. Annexin V-Cy3 was used to detect phosphatidylserine translocation from the inner to outer leaflet of the plasma membrane. Cu
2+
-Asc treatment induced phosphatidylserine translocation within 2 h in H441 cells but none was observed in C48 cells up to 24 h. These results indicate that 1-cysPrx can scavenge peroxides but in addition can reduce peroxidized membrane phospholipids. Thus, the enzyme can protect cells against oxidant-induced plasma membrane damage, thereby playing an important role in cellular defense against oxidant stress.
A linear lattice model for polyglutamine in CAG-expansion diseases
Authors: Bennett Melanie J. , Huey-Tubman Kathryn E. , Herr Andrew B. , West Anthony P. , Ross Scott A. , Bjorkman Pamela J.
Published: September 2002

Huntington's disease and several other neurological diseases are caused by expanded polyglutamine [poly(Gln)] tracts in different proteins. Mechanisms for expanded (>36 Gln residues) poly(Gln) toxicity include the formation of aggregates that recruit and sequester essential cellular proteins [Preisinger, E., Jordan, B. M., Kazantsev, A. & Housman, D. (1999)
Phil. Trans. R. Soc. London B
354, 1029–1034; Chen, S., Berthelier, V., Yang, W. & Wetzel, R. (2001)
J. Mol. Biol.
311, 173–182] and functional alterations, such as improper interactions with other proteins [Cummings, C. J. & Zoghbi, H. Y. (2000)
Hum. Mol. Genet.
9, 909–916]. Expansion above the “pathologic threshold” (≈36 Gln) has been proposed to induce a conformational transition in poly(Gln) tracts, which has been suggested as a target for therapeutic intervention. Here we show that structural analyses of soluble huntingtin exon 1 fusion proteins with 16 to 46 glutamine residues reveal extended structures with random coil characteristics and no evidence for a global conformational change above 36 glutamines. An antibody (MW1) Fab fragment, which recognizes full-length huntingtin in mouse brain sections, binds specifically to exon 1 constructs containing normal and expanded poly(Gln) tracts, with affinity and stoichiometry that increase with poly(Gln) length. These data support a “linear lattice” model for poly(Gln), in which expanded poly(Gln) tracts have an increased number of ligand-binding sites as compared with normal poly(Gln). The linear lattice model provides a rationale for pathogenicity of expanded poly(Gln) tracts and a structural framework for drug design.
Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes
Authors: Orwig Kyle E. , Ryu Buom-Yong , Avarbock Mary R. , Brinster Ralph L.
Published: September 2002

Gonocytes are a transient population of male germ-line stem cells that are derived from primordial germ cells in the embryo and give rise to spermatogonial stem cells, which establish and maintain spermatogenesis in the postnatal testis. In contrast to spermatogonial stem cells, gonocytes can be identified easily in neonatal rat testis cell suspensions based on their large size and distinct morphology. Furthermore, histological analysis of testes from neonatal transgenic rats demonstrated that gonocytes are the only cells that express alacZreporter transgene. Two gonocyte subpopulations, designated pseudopod and round, were identified and isolated from neonatal (0–4 days postpartum) rat testis cell suspensions. Male germ-line stem cells, identified by their ability to produce and maintain colonies of spermatogenesis upon transplantation into infertile recipient testes, were present almost exclusively in the pseudopod gonocyte subpopulation. In contrast, annexin V staining indicated that the majority of round gonocytes undergo apoptosis. These results indicate that a nearly pure population of male germ-line stem cells can be prospectively identified in neonatal rat testis cell suspensions by morphological criteria. Together, the pseudopod and round gonocyte populations will provide powerful tools for the study of cellular mechanisms that control cell fates and the establishment of spermatogenesis in the postnatal testis.
Crosslinked HIV-1 envelope–CD4 receptor complexes elicit broadly cross-reactive neutralizing antibodies in rhesus macaques
Authors: Fouts Timothy , Godfrey Karla , Bobb Kathryn , Montefiori David , Hanson Carl V. , Kalyanaraman V. S. , DeVico Anthony , Pal Ranajit
Published: September 2002

The identification of HIV envelope structures that generate broadly cross-reactive neutralizing antibodies is a major goal for HIV-vaccine development. In this study, we evaluated one such structure, expressed as either a gp120–CD4 or a gp140–CD4 complex, for its ability to elicit a neutralizing antibody response. In rhesus macaques, covalently crosslinked complexes of soluble human CD4 (shCD4) and HIV-1IIIBenvelope glycoproteins (gp120 or gp140) generated antibodies that neutralized a wide range of primary HIV-1 isolates regardless of the coreceptor usage or genetic subtype. Ig with cross-reactive neutralizing activity was recovered by affinity chromatography with a chimeric single-chain polypeptide containing sequences for HIVBaLgp120 and a mimetic peptide that induces a CD4-triggered envelope structure. These results suggest that covalently crosslinked complexes of the HIV-1 surface envelope glycoprotein and CD4 elicit broadly neutralizing humoral responses that, in part, may be directed against a novel epitope(s) found on the HIV-1 envelope.
Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy
Authors: Gao Guang-Ping , Alvira Mauricio R. , Wang Lili , Calcedo Roberto , Johnston Julie , Wilson James M.
Published: September 2002

Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1–6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.
TCL1 participates in early embryonic development and is overexpressed in human seminomas
Authors: Narducci Maria Grazia , Fiorenza Maria Teresa , Kang Sang-Moo , Bevilacqua Arturo , Di Giacomo Monica , Remotti Daniele , Picchio Maria Cristina , Fidanza Vincenzo , Cooper Max D. , Croce Carlo Maria , Mangia Franco , Russo Giandomenico
Published: September 2002

Overexpression of the
TCL1
oncogene has been shown to play a causative role in T cell leukemias of humans and mice. The characterization of
Tcl1
-deficient mice in these studies indicates an important developmental role for Tcl1 in early embryogenesis. In wild-type embryos, Tcl1 is abundant in the first three mitotic cycles, during which it shuttles between nuclei and the embryo cortical regions in a cell-cycle-dependent fashion. The absence of this protein in early embryogenesis results in reduced fertility of female mice. The present studies elucidate the mechanism responsible for the reduced female fertility through analysis of the oogenesis stages and early embryo development in
Tcl1
-deficient mice. Even though
Tcl1
−/−
females display normal oogenesis and rates of oocyte maturation/ovulation and fertilization, the lack of maternally derived Tcl1 impairs the embryo's ability to undergo normal cleavage and develop to the morula stage, especially under
in vitro
culture conditions. Beyond this crisis point, differentiative traits of zygotic genome activation and embryo compaction can take place normally. In contrast with this unanticipated role in early embryogenesis, we observed an overexpression of TCL1 in human seminomas. This finding suggests that
TCL1
dysregulation could contribute to the development of this germinal cell cancer as well as lymphoid malignancies.
PDZ-domain interactions and apical expression of type IIa Na/P
i
cotransporters
Authors: Hernando Nati , Déliot Nadine , Gisler Serge M. , Lederer Eleanor , Weinman Edward J. , Biber Jürg , Murer Heini
Published: September 2002

Type IIa Na/P
i
cotransporters are expressed in renal proximal brush border and are the major determinants of inorganic phosphate (P
i
) reabsorption. Their carboxyl-terminal tail contains information for apical expression, and interacts by means of its three terminal amino acids with several PSD95/DglA/ZO-1-like domain (PDZ)-containing proteins. Two of these proteins, NaPi-Cap1 and Na/H exchanger-regulatory factor 1 (NHERF1), colocalize with the cotransporter in the proximal brush border. We used opossum kidney cells to test the hypothesis of a potential role of PDZ-interactions on the apical expression of the cotransporter. We found that opossum kidney cells contain NaPi-Cap1 and NHERF1 mRNAs. For NHERF1, an apical location of the protein could be documented; this location probably reflects interaction with the cytoskeleton by means of the MERM-binding domain. Overexpression of PDZ domains involved in interaction with the cotransporter (PDZ-1/NHERF1 and PDZ-3/NaPi-Cap1) had a dominant–negative effect, disturbing the apical expression of the cotransporter without affecting the actin cytoskeleton or the basolateral expression of Na/K-ATPase. These data suggest an involvement of PDZ-interactions on the apical expression of type IIa cotransporters.
Sterols block binding of COPII proteins to SCAP, thereby controlling SCAP sorting in ER
Authors: Espenshade Peter J. , Li Wei-Ping , Yabe Daisuke
Published: September 2002

Sterols inhibit their own synthesis in mammalian cells by blocking the vesicular endoplasmic reticulum-to-Golgi transport of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), a sterol-sensing protein that escorts SREBPs. Unable to reach the Golgi, SREBPs are not processed by Golgi-resident proteases, and they fail to activate genes required for cholesterol synthesis. The current studies were designed to reveal whether sterols block SCAP movement by inhibiting synthesis of special vesicles dedicated to SCAP, or whether sterols block SCAP incorporation into common coat protein (COP)II-coated vesicles. Through immunoisolation, we show that SCAP-containing vesicles, formed
in vitro
, also contain vesicular stomatitis virus glycoprotein (VSVG) protein, a classic marker of COPII-coated vesicles. Sterols selectively block incorporation of SCAP into these vesicles without blocking incorporation of VSVG protein. We show that the mammalian vesicular budding reaction can be reconstituted by recombinant yeast COPII proteins that support incorporation of SCAP as well as VSVG into vesicles. Sterols block SCAP incorporation into vesicles by blocking Sar1-dependent binding of the COPII proteins Sec 23/24 to SCAP. These studies demonstrate feedback control of a biosynthetic pathway by the regulated binding of COPII proteins to an endoplasmic reticulum-to-Golgi transport protein.
Specification of unique Pit-1 activity in the
hGH
locus control region
Authors: Shewchuk Brian M. , Liebhaber Stephen A. , Cooke Nancy E.
Published: September 2002

The human GH (
hGH
) gene cluster is regulated by a remote 5′ locus control region (LCR). HSI, an LCR component located 14.5 kb 5′ to the
hGH
-
N
promoter, constitutes the primary determinant of high-level
hGH
-
N
activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity
in vivo
. Furthermore, these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted
hGH
-
N
transgene activation. In contrast, the
hGH
-
N
transgene is not activated by Pit-1 sites native to either the
hGH
-
N
or rat (r)
GH
gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of
hGH
in the developing pituitary.
Evidence for an ancient selective sweep in the MHC class I gene repertoire of chimpanzees
Authors: de Groot Natasja G. , Otting Nel , Doxiadis Gaby G. M. , Balla-Jhagjhoorsingh Sunita S. , Heeney Jonathan L. , van Rood Jon J. , Gagneux Pascal , Bontrop Ronald E.
Published: September 2002

MHC class I molecules play an essential role in the immune defense against intracellular infections. The hallmark of the MHC is its extensive degree of polymorphism at the population level. However, the present comparison of MHC class I gene intron variation revealed that chimpanzees have experienced a severe repertoire reduction at the orthologues of theHLA-A,-B, and-Cloci. The loss of variability predates the (sub)speciation of chimpanzees and did not effect other known gene systems. Therefore the selective sweep in the MHC class I gene may have resulted from a widespread viral infection. Based on the present results and the fact that chimpanzees have a natural resistance to the development of AIDS, we hypothesize that the selective sweep was caused by the chimpanzee-derived simian immunodeficiency virus (SIVcpz), the closest relative of HIV-1, or a closely related retrovirus. Hence, the contemporary chimpanzee populations represent the offspring of AIDS-resistant animals, the survivors of a HIV-like pandemic that took place in the distant past.
PPARα is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue
Authors: Lee Y. , Yu X. , Gonzales F. , Mangelsdorf D. J. , Wang May-Yun , Richardson C. , Witters L. A. , Unger R. H.
Published: September 2002

Adenovirus-induced hyperleptinemia causes rapid disappearance of body fat in normal rats, presumably by up-regulating fatty acid oxidation within white adipocytes. To determine the role of peroxisomal proliferation-activated receptor (PPAR)α expression, which was increased during the rapid loss of fat, we infused adenovirus–leptin into PPARα
−/−
and PPARα
+/+
mice. Despite similar degrees of hyperleptinemia and reduction in food intake, epididymal fat pad weight declined 55% in wild-type but only 6% in PPARα
−/−
mice; liver triacylglycerol fell 39% in the wild-type group but was unchanged in PPAR
−/−
mice. Carnitine palmitoyl transferase-1 mRNA rose 52% in the wild-type mice but did not increase in PPARα
−/−
mice. PPARγ coactivator-1α rose 3-fold in the fat and 46% in the liver of wild-type mice but was unchanged in PPARα
−/−
mice. Although AMP-activated protein kinase could not be implicated in the lipopenic actions of hyperleptinemia, acetyl CoA carboxylase protein was reduced in the liver of wild-type but not in PPARα
−/−
mice. Thus, in PPARα
−/−
mice, up-regulation of carnitine palmitoyl transferase-1 mRNA in fat, down-regulation of acetyl CoA carboxylase in liver, and up-regulation of PPARγ coactivator-1α mRNA in both tissues are abolished, as is the reduction in their triacylglycerol content.
Circadian genes in a blind subterranean mammal II: Conservation and uniqueness of the threePeriodhomologs in the blind subterranean mole rat,Spalax ehrenbergisuperspecies
Authors: Avivi Aaron , Oster Henrik , Joel Alma , Beiles Avigdor , Albrecht Urs , Nevo Eviatar
Published: September 2002

We demonstrated that a subterranean, visually blind mammal has a functional set of threePergenes that are important components of the circadian clockwork in mammals. The mole rat superspeciesSpalax ehrenbergiis a blind subterranean animal that lives its entire life underground in darkness. It has degenerated eyes, but the retina and highly hypertrophic harderian gland are involved in photoperiodic perception. All threePergenes oscillate with a periodicity of 24 h in the suprachiasmatic nuclei, eye, and harderian gland and are expressed in peripheral organs. This oscillation is maintained under constant conditions. The light inducibility ofsPer1andsPer2, which are similar in structure to those of other mammals, indicates the role of these genes in clock resetting. However,sPer3is unique in mammals and has two truncated isoforms, and its expressional analysis leaves its function unresolved.Per's expression analysis in the harderian gland suggests an important participation of this organ in the stabilization and resetting mechanism of the central pacemaker in the suprachiasmatic nuclei and in unique adaptation to life underground.
Dysregulated T helper cell differentiation in the absence of interferon regulatory factor 4
Authors: Lohoff Michael , Mittrücker Hans-Willi , Prechtl Stefan , Bischof Susi , Sommer Frank , Kock Sonja , Ferrick David A. , Duncan Gordon S. , Gessner Andre , Mak Tak W.
Published: September 2002

Certain IFN regulatory factor (IRF) transcription factors indirectly influence T helper (Th) cell differentiation by regulating the production of IL-12. Here, we show that IRF4 directly regulates Th cell differentiationin vitroandin vivoduring murine leishmaniasis. In the absence of IRF4, IL-12-induced Th1 cell differentiation was compromised, while IL-4 failed to induce Th2 cell differentiation. Instead, IL-4 tended to induce Th1 cells, defined by production of IFN-γ and TNF. Although early IL-4 signaling was normal in IRF4−/−Th cells, the protein GATA-3, a transcription factor critical for Th2 development, was not up-regulated following IL-4 treatment. Retroviral overexpression of GATA-3 rescued Th2 differentiation. Therefore, IRF4 deficiency manifests itself as severely dysregulated Th cell differentiation.
Identification of a transcriptionally active peroxisome proliferator-activated receptor α-interacting cofactor complex in rat liver and characterization of PRIC285 as a coactivator
Authors: Surapureddi Sailesh , Yu Songtao , Bu Hengfu , Hashimoto Takashi , Yeldandi Anjana V. , Kashireddy Papreddy , Cherkaoui-Malki Mustapha , Qi Chao , Zhu Yi-Jun , Rao M. Sambasiva , Reddy Janardan K.
Published: September 2002

Peroxisome proliferator-activated receptor α (PPARα) plays a central role in the cell-specific pleiotropic responses induced by structurally diverse synthetic chemicals designated as peroxisome proliferators. Transcriptional regulation by liganded nuclear receptors involves the participation of cofactors that form multiprotein complexes to achieve cell- and gene-specific transcription. Here we report the identification of such a transcriptionally active PPARα-interacting cofactor (PRIC) complex from rat liver nuclear extracts that interacts with full-length PPARα in the presence of ciprofibrate, a synthetic ligand, and leukotriene B4, a natural ligand. The liganded PPARα-PRIC complex enhanced transcription from a peroxisomal enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme gene promoter template that contains peroxisome proliferator response elements. Rat liver PRIC complex comprises some 25 polypeptides, and their identities were established by mass spectrometry and limited sequence analysis. Eighteen of these peptides contain one or more LXXLL motifs necessary for interacting with nuclear receptors. PRIC complex includes known coactivators or coactivator-binding proteins (CBP, SRC-1, PBP, PRIP, PIMT, TRAP100, SUR-2, and PGC-1), other proteins that have not previously been described in association with transcription complexes (CHD5, TOG, and MORF), and a few novel polypeptides designated PRIC300, -285, -215, -177, and -145. We describe the cDNA for PRIC285, which contains five LXXLL motifs. It interacts with PPARα and acts as a coactivator by moderately stimulating PPARα-mediated transcription in transfected cells. We conclude that liganded PPARα recruits a distinctive multiprotein complex from rat liver nuclear extracts. The composition of this complex may provide insight into the basis of tissue and species sensitivity to peroxisome proliferators.
The tobacco salicylic acid-binding protein 3 (SABP3) is the chloroplast carbonic anhydrase, which exhibits antioxidant activity and plays a role in the hypersensitive defense response
Authors: Slaymaker David H. , Navarre Duroy A. , Clark Daniel , del Pozo Olga , Martin Gregory B. , Klessig Daniel F.
Published: September 2002

In plants, salicylic acid (SA) plays an important role in signaling both local and systemic defense responses. Previous efforts to identify SA effector proteins in tobacco have led to the isolation of two soluble cytoplasmic SA-binding proteins (SABPs): catalase, SABP, and an ≈25-kDa protein, SABP2. Here we describe the identification of an SA-binding protein, SABP3, in the stroma of tobacco chloroplasts. SABP3 bound SA with an apparent dissociation constant (
K
d
) of 3.7 μM and exhibited much greater affinity for biologically active than inactive analogs. Purification and partial sequencing of SABP3 indicated that it is the chloroplast carbonic anhydrase (CA). Confirming this finding, recombinant tobacco chloroplast CA exhibited both CA enzymatic and SA-binding activities. Expression of this protein in yeast also demonstrated that CA/SABP3 has antioxidant activity. A second gene encoding CA was also cloned, and its encoded protein was shown to behave similarly to that purified as SABP3. Finally, silencing of CA gene expression in leaves suppressed the
Pto
:
avrPto
-mediated hypersensitive response in disease resistance. These results demonstrate that SA may act through multiple effector proteins in plants and shed further light on the function of CA in chloroplasts.
The expression of growth hormone-releasing hormone (GHRH) and splice variants of its receptor in human gastroenteropancreatic carcinomas
Authors: Busto Rebeca , Schally Andrew V. , Varga Jozsef L. , Garcia-Fernandez M. Olga , Groot Kate , Armatis Patricia , Szepeshazi Karoly
Published: September 2002

Splice variants (SVs) of receptors for growth hormone-releasing hormone (GHRH) have been found in primary human prostate cancers and diverse human cancer cell lines. GHRH antagonists inhibit growth of various experimental human cancers, including pancreatic and colorectal, xenografted into nude mice or cultured
in vitro
, and their antiproliferative action could be mediated in part through SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and for SVs of its receptors in tumors of human pancreatic, colorectal, and gastric cancer cell lines grown in nude mice. mRNA for both GHRH and SV
1
isoform of GHRH receptors was expressed in tumors of pancreatic (SW1990, PANC-1, MIA PaCa-2, Capan-1, Capan-2, and CFPAC1), colonic (COLO 320DM and HT-29), and gastric (NCI-N87, HS746T, and AGS) cancer cell lines; mRNA for SV
2
was also present in Capan-1, Capan-2, CFPAC1, HT-29, and NCI-N87 tumors. In proliferation studies
in vitro
, the growth of pancreatic, colonic, and gastric cancer cells was stimulated by GHRH(1–29)NH
2
and inhibited by GHRH antagonist JV-1–38. The stimulation of some gastroenteropancreatic cancer cells by GHRH was followed by an increase in cAMP production, and GHRH antagonist JV-1–38 competitively inhibited this effect. Our study indicates the presence of an autocrine/paracrine stimulatory loop based on GHRH and SV
1
of GHRH receptors in human pancreatic, colorectal, and gastric cancers. The finding of SV
1
receptor in human cancers provides an approach to an antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.
Epstein–Barr virus EBNA2 blocks Nur77- mediated apoptosis
Authors: Lee Jae Myun , Lee Kyoung-Ho , Weidner Magdalena , Osborne Barbara A. , Hayward S. Diane
Published: September 2002

Epstein–Barr virus infectionin vitroimmortalizes primary B cells. EBNA2 is an Epstein–Barr virus-encoded transcriptional transactivator that mimics the effects of activated Notch signaling and is essential for this proliferative response. An assay using Sindbis virus (SV) as a cell death inducer revealed that, like Notch, EBNA2 also has antiapoptotic activity. We show that Nur77 is a mediator of SV-induced cell death and that EBNA2 antiapoptotic activity results from interaction with Nur77. EBNA2 colocalized with Nur77 in transfected cells and coprecipitated with Nur77 in IB4 B cells. EBNA2 binds to Nur77 through sequences in the EBNA2 amino acid 123–147 conserved domain and an EBNA2 mutant unable to bind Nur77 also lost the ability to protect cells from SV-induced apoptosis. EBNA2 exerted its antideath function by retaining Nur77 in the nucleus and preventing Nur77 from targeting mitochondria in response to apoptotic stimuli. Thus, targeting of Nur77 can be added to the list of strategies used by viruses to counter apoptosis.
The Alzheimer's Aβ-peptide is deposited at sites of complement activation in pathologic deposits associated with aging and age-related macular degeneration
Authors: Johnson Lincoln V. , Leitner William P. , Rivest Alexander J. , Staples Michelle K. , Radeke Monte J. , Anderson Don H.
Published: September 2002

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in older individuals worldwide. The disease is characterized by abnormal extracellular deposits, known as drusen, that accumulate along the basal surface of the retinal pigmented epithelium. Although drusen deposition is common in older individuals, large numbers of drusen and/or extensive areas of confluent drusen represent a significant risk factor for AMD. Widespread drusen deposition is associated with retinal pigmented epithelial cell dysfunction and degeneration of the photoreceptor cells of the neural retina. Recent studies have shown that drusen contain a variety of immunomodulatory molecules, suggesting that the process of drusen formation involves local inflammatory events, including activation of the complement cascade. Similar observations in Alzheimer's disease (AD) have lead to the hypothesis that chronic localized inflammation is an important element of AD pathogenesis, with significant neurodegenerative consequences. Accordingly, the amyloid beta (Aβ) peptide, a major constituent of neuritic plaques in AD, has been implicated as a primary activator of complement in AD. Here we show that Aβ is associated with a substructural vesicular component within drusen. Aβ colocalizes with activated complement components in these “amyloid vesicles,” thereby identifying them as potential primary sites of complement activation. Thus, Aβ deposition could be an important component of the local inflammatory events that contribute to atrophy of the retinal pigmented epithelium, drusen biogenesis, and the pathogenesis of AMD.
Functional plasticity of an antigen-specific memory CD4 T cell population
Authors: Ahmadzadeh Mojgan , Farber Donna L.
Published: September 2002

The protective nature of memory immune responses is attributed largely to terminally differentiated memory T cells that retain memory of the antigen via the antigen receptor and memory of the effector functions that initially cleared the pathogen. It is not known whether a given population of antigen-specific memory T cells is endowed with functional flexibility to provide protective responses against antigens reencountered in different immunological contexts. Here, we examine functional properties of influenza hemagglutinin (HA)-specific memory CD4 T cells recovered from adoptive hosts that receivedin vitro-activated HA-specific T cell receptor-transgenic CD4 T cells 2 months to 1 year previously. We demonstrate that this HA-specific memory CD4 T cell population bearing a clonal T cell receptor can produce predominantly T helper 1 or T helper 2 effector cytokines depending on the nature of the recall stimulus. Our findings reveal remarkable functional plasticity within an antigen-specific memory T cell population and have direct implications for modulating memory T cell function in vaccine design and treatments for autoimmune diseases.
Cooperative activities of hematopoietic regulators recruit RNA polymerase II to a tissue-specific chromatin domain
Authors: Johnson Kirby D. , Grass Jeffrey A. , Boyer Meghan E. , Kiekhaefer Carol M. , Blobel Gerd A. , Weiss Mitchell J. , Bresnick Emery H.
Published: September 2002

The hematopoietic transcription factor GATA-1 regulates erythropoiesis and β-globin expression. Although consensus GATA-1 binding sites exist throughout the murine β-globin locus, we found that GATA-1 discriminates among these sites
in vivo
. Conditional expression of GATA-1 in GATA-1-null cells recapitulated the occupancy pattern. GATA-1 induced RNA polymerase II (pol II) recruitment to subregions of the locus control region and to the β-globin promoters. The hematopoietic factor NF-E2 cooperated with GATA-1 to recruit pol II to the promoters. We propose that only when GATA-1 attracts pol II to the locus control region can pol II access the promoter in a NF-E2-dependent manner.
Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides
Authors: Li Jing , Xu Qiang , Cortes D. Marien , Perozo Eduardo , Laskey Aaron , Karlin Arthur
Published: September 2002

Single cysteine-substitution mutants of KcsA, a K
+
channel from
Streptomyces lividans
, were expressed in
Escherichia coli
, and inner membranes were isolated. The rate constants for the reactions of these cysteines with three maleimides of increasing hydrophobicity, 4-(
N
-maleimido)phenyltrimethylammonium,
N-
phenylmaleimide, and
N-
(1-pyrenyl)maleimide, were determined by back titration of the remaining cysteines with methoxypolyethylene glycol-2-pyridine disulfide (
M
r
3,000) and quantitation of the fraction of gel-shifted KcsA as a function of reaction time. The patterns of the rate constants for the reactions of all three reagents with eight consecutive cysteines in the partially lipid-immersed amphipathic N-terminal tail helix were the same, with cysteines on the hydrophilic side of the helix reacting faster than Cys on the hydrophobic side. The results are consistent with the tail helix lying with its long axis in the lipid-water interface and with the orientation of the helix fluctuating around this axis. The patterns of the rate constants for the three reagents were similar to the pattern of the probabilities that the substituted cysteines were exposed to water, based on the sum of the free energies of transfer from water to octanol of all of the residues exposed to lipid in each orientation of the helix.
Regulation of membrane fusion by the membrane-proximal coil of the t-SNARE during zippering of SNAREpins
Authors: Melia Thomas J. , Weber Thomas , McNew James A. , Fisher Lillian E. , Johnston Robert J. , Parlati Frank , Mahal Lara K. , Söllner Thomas H. , Rothman James E.
Published: September 2002

We utilize structurally targeted peptides to identify a “tC fusion switch” inherent to the coil domains of the neuronal t-SNARE that pairs with the cognate v-SNARE. The tC fusion switch is located in the membrane-proximal portion of the t-SNARE and controls the rate at which the helical bundle that forms the SNAREpin can zip up to drive bilayer fusion. When the fusion switch is “off” (the intrinsic state of the t-SNARE), zippering of the helices from their membrane-distal ends is impeded and fusion is slow. When the tC fusion switch is “on,” fusion is much faster. The tC fusion switch can be thrown by a peptide that corresponds to the membrane-proximal half of the cognate v-SNARE, and binds reversibly to the cognate region of the t-SNARE. This structures the coil in the membrane-proximal domain of the t-SNARE and accelerates fusion, implying that the intrinsically unstable coil in that region is a natural impediment to the completion of zippering, and thus, fusion. Proteins that stabilize or destabilize one or the other state of the tC fusion switch would exert fine temporal control over the rate of fusion after SNAREs have already partly zippered up.
Reconstitution of nuclear protein export in isolated nuclear envelopes
Authors: Siebrasse Jan Peter , Coutavas Elias , Peters Reiner
Published: September 2002

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPγS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPγS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.
Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells
Authors: Kallio Marko J. , Beardmore Victoria A. , Weinstein Jasminder , Gorbsky Gary J.
Published: September 2002

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.
Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle
Authors: Rogers Stephen L. , Rogers Gregory C. , Sharp David J. , Vale Ronald D.
Published: September 2002

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.
N-cadherin–dependent cell–cell contact regulates Rho GTPases and β-catenin localization in mouse C2C12 myoblasts
Authors: Charrasse Sophie , Meriane Mayya , Comunale Franck , Blangy Anne , Gauthier-Rouvière Cécile
Published: September 2002

N-cadherin, a member of the Ca2+-dependent cell–cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin–dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin–mediated signals involved in myogenesis, we investigated whether N-cadherin–dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin–dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin–mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for β-catenin accumulation at cell–cell contact sites. We propose that cell–cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.