bluuebunny/crossref_metadata_2025 huggingface dataset:
The Orphan Nuclear Receptor, Steroidogenic Factor 1, Regulates Neuronal Nitric Oxide Synthase Gene Expression in Pituitary Gonadotropes
Authors: Wei Xueying , Sasaki Masayuki , Huang Hui , Dawson Valina L. , Dawson Ted M.
Published: November 2002

Steroidogenic factor 1 (SF-1), an essential nuclear receptor, plays key roles in steroidogenic cell function within the adrenal cortex and gonads. It also contributes to reproductive function at all three levels of the hypothalamic-pituitary-gonadal axis. SF-1 regulates genes in the steroidogenic pathway, such as LHβ, FSHβ, and steroid hydroxylase. Abundant evidence suggests that nitric oxide (NO) has an important role in the control of reproduction due to its ability to control GnRH secretion from the hypothalamus and the preovulatory LH surge in pituitary gonadotropes. Recently, we cloned and characterized the promoter of mouse neuronal NO synthase (nNOS). nNOS is localized at all three levels of the hypothalamic-pituitary-gonadal axis to generate NO. We find that its major promoter resides at exon 2 in the pituitary gonadotrope αT3–1 cell line and that there is a nuclear hormone receptor binding site in this region, to which SF-1 can bind and regulate nNOS transcription. Mutation of the nuclear hormone receptor binding site dramatically decreases basal promoter activity and abolishes SF-1 responsiveness. A dominant negative of SF-1, in which the transactivation (AF-2) domain of SF-1 was deleted, inhibits nNOS exon 2 promoter activity. Dosage-sensitive reversal- adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1), which colocalizes and interferes with SF-1 actions in multiple cell lineages, negatively modulates SF-1 regulation of nNOS transcription. These findings demonstrate that mouse nNOS gene expression is regulated by the SF-1 gene family in pituitary gonadotropes. nNOS, a member of the cytochrome p450 gene family, could be one of the downstream effector genes, which mediates SF-1’s reproductive function and developmental patterning.
A Novel Androgen Receptor Mutant, A748T, Exhibits Hormone Concentration-Dependent Defects in Nuclear Accumulation and Activity Despite Normal Hormone-Binding Affinity
Authors: James Alaina J. , Agoulnik Irina U. , Harris Jonathan M. , Buchanan Grant , Tilley Wayne D. , Marcelli Marco , Lamb Dolores J. , Weigel Nancy L.
Published: November 2002

Functional analysis of androgen receptor (AR) gene mutations isolated from prostate cancer has led to the identification of residues that play important roles in the structure and function of the receptor. Here we report the characteristics of a novel AR mutation A748T located in helix 5 of the ligand-binding domain, which was identified in metastatic prostate cancer. Despite a normal hormone-binding affinity, A748T causes hormone concentration-dependent defects in nuclear accumulation and transcriptional activation. Moreover, when equivalent amounts of DNA are transfected, the mutant is expressed at much lower levels than the wild-type AR (ARWT). Treatment with geldanamycin to disrupt receptor-heat shock protein complexes rapidly decreases the levels of ARWT but not A748T, suggesting that the lower expression and rapid degradation rate of A748T is due to weaker interactions with heat shock proteins. Further analysis revealed that hormone dissociates from A748T five times faster than from ARWT. Loss of the ability to form stable amino/carboxyl-terminal interactions causes accelerated dissociation rates in some AR mutants. However, A748T exhibits normal amino/carboxyl-terminal interactions at high hormone concentrations, suggesting that the mutation alters interactions with ligand. Consistent with this conclusion, our structural model predicts that A748T disrupts crucial contact points with ligand, thereby altering the conformation of the ligand-binding domain.
Interaction of Early Growth Response Protein 1 (Egr-1), Specificity Protein 1 (Sp1), and Cyclic Adenosine 3′5′-Monophosphate Response Element Binding Protein (CREB) at a Proximal Response Element Is Critical for Gastrin-Dependent Activation of the Chromogranin A Promoter
Authors: Raychowdhury Raktima , Schäfer Georgia , Fleming John , Rosewicz Stefan , Wiedenmann Bertram , Wang Timothy C. , Höcker Michael
Published: November 2002

Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at −92/−62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the −92/−62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA −92/−73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA −92/−62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.
Role of Specific Protein Kinase C Isozymes in Mediating Epidermal Growth Factor, Thyrotropin-Releasing Hormone, and Phorbol Ester Regulation of the Rat Prolactin Promoter in GH4/GH4C1 Pituitary Cells
Authors: Pickett Cheryl A. , Manning Nicole , Akita Yoshiko , Gutierrez-Hartmann Arthur
Published: November 2002

Epidermal growth factor (EGF) and TRH both produce enhanced prolactin (PRL) gene transcription and PRL secretion in GH4 rat pituitary tumor cell lines. These agents also activate protein kinase C (PKC) in these cells. Previous studies have implicated the PKCε isozyme in mediating TRH-induced PRL secretion. However, indirect studies using phorbol ester down-regulation to investigate the role of PKC in EGF- and TRH-induced PRL gene transcription have been inconclusive. In the present study, we examined the role of multiple PKC isozymes on EGF- and TRH-induced activation of the PRL promoter by utilizing general and selective PKC inhibitors and by expression of genes for wild-type and kinase-negative forms of the PKC isozymes. Multiple nonselective PKC inhibitors, including staurosporine, bisindolylmaleimide I, and Calphostin C, inhibited both EGF and TRH induced rat PRL promoter activity. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of diacylglycerol, whereas Go 6976, a selective inhibitor of Ca2+-dependent PKCs, produced a modest inhibition of EGF but no inhibition of TRH effects. Rottlerin, a specific inhibitor of the novel nPKCδ isozyme, significantly blocked both EGF and TRH effects. Overexpression of genes encoding PKCs α, βΙ, βΙΙ, δ, γ, and λ failed to enhance either EGF or TRH responses, whereas overexpression of nPKCη enhanced the EGF response. Neither stable nor transient overexpression of nPKCε produced enhancement of EGF- or TRH-induced PRL promoter activity, suggesting that different processes regulate PRL transcription and hormone secretion. Expression of a kinase inactive nPKCδ construct produced modest inhibition of EGF-mediated rPRL promoter activity. Taken together, these data provide evidence for a role of multiple PKC isozymes in mediating both EGF and TRH stimulated PRL gene transcription. Both EGF and TRH responses appear to require the novel isozyme, nPKCδ, whereas nPKCη may also be able to transmit the EGF response. Inhibitor data suggest that the EGF response may also involve Ca2+-dependent isozymes, whereas the TRH response appears to be more dependent on diacylglycerol.
Identification of a Functional Peroxisome Proliferator-Activated Receptor Response Element in the Rat Catalase Promoter
Authors: Girnun Geoffrey D. , Domann Frederick E. , Moore Steven A. , Robbins Mike E. C.
Published: November 2002

Peroxisomal proliferator-activated receptor (PPAR)γ has been shown to decrease the inflammatory response via transrepression of proinflammatory transcription factors. However, the identity of PPARγ responsive genes that decrease the inflammatory response has remained elusive. Because generation of the reactive oxygen species hydrogen peroxide (H2O2) plays a role in the inflammatory process and activation of proinflammatory transcription factors, we wanted to determine whether the antioxidant enzyme catalase might be a PPARγ target gene. We identified a putative PPAR response element (PPRE) containing the canonical direct repeat 1 motif, AGGTGA-A-AGTTGA, in the rat catalase promoter. In vitro translated PPARγ and retinoic X receptor-α proteins were able to bind to the catalase PPRE. Promoter deletion analysis revealed that the PPRE was functional, and a heterologous promoter construct containing a multimerized catalase PPRE demonstrated that the PPRE was necessary and sufficient for PPARγ-mediated activation. Treatment of microvascular endothelial cells with PPARγ ligands led to increases in catalase mRNA and activity. These results demonstrate that PPARγ can alter catalase expression; this occurs via a PPRE in the rat catalase promoter. Thus, in addition to transrepression of proinflammatory transcription factors, PPARγ may also be modulating catalase expression, and hence down-regulating the inflammatory response via scavenging of reactive oxygen species.
Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin
Authors: Chauvin Stephanie , Bencsik Margaret , Bambino Tom , Nissenson Robert A.
Published: November 2002

The recovery of PTH receptor (PTHR) function after acute homologous receptor desensitization and down-regulation in bone and kidney cells has been attributed to receptor recycling. To determine the role of receptor dephosphorylation in PTHR recycling, we performed morphological and functional assays on human embryonic kidney 293 cells stably expressing wild-type (wt) or mutant PTHRs. Confocal microscopy and ligand binding assays revealed that the wt PTHR is rapidly recycled back to the plasma membrane after removal of the agonist. Receptors that were engineered to either lack the sites of phosphorylation or to resemble constitutively phosphorylated receptors were able to recycle back to the plasma membrane with the same kinetics as the wt PTHR. The PTHR was found to be dephosphorylated by an enzyme apparently distinct from protein phosphatases 1 or 2A. The PTHR and β-arrestin-2-green fluorescent protein (GFP) were found to stably colocalize during PTHR internalization, whereas after agonist removal and during receptor recycling, the colocalization slowly disappeared. Experiments using phosphorylation-deficient PTHRs and a dominant-negative form of β-arrestin showed that β-arrestin does not regulate the efficiency of PTHR recycling. These studies indicate that, unlike many G protein-coupled receptors, PTHR recycling does not require receptor dephosphorylation or its dissociation from β-arrestin.
Mammary Epithelial Cells Are Not Able to Undergo Pregnancy-Dependent Differentiation in the Absence of the Helix-Loop-Helix Inhibitor Id2
Authors: Miyoshi Keiko , Meyer Barbara , Gruss Peter , Cui Yongzhi , Renou Jean-Pierre , Morgan Fanta V. , Smith Gilbert H. , Reichenstein Moshe , Shani Moshe , Hennighausen Lothar , Robinson Gertraud W.
Published: November 2002

Mammary alveolar development during pregnancy is triggered by hormone signals. The prolactin receptor/Jak2/signal transducer and activator of transcription (Stat) 5 signal transduction pathway is the principal mediator of these cues and alveolar development is abrogated in its absence. The loss of the basic helix-loop-helix protein inhibitor of differentiation (Id)2 results in a similar defect. To investigate the role of Id2 in mammary epithelium, we performed structural and molecular analyses. Id2-null mammary epithelial cells were unable to form alveoli; the epithelial architecture was disorganized and dissimilar from early stages of alveologenesis in wild-type glands. The epithelial cells retained the ductal marker Na-K-Cl cotransporter (NKCC)1. Nuclear localization of Stat5a and down-regulation of NKCC1 was observed in some areas, indicating a limited response to pregnancy signals. The differentiation status of Id2-null tissue at term was further characterized with cDNA microarrays enriched in mammary specific sequences (mammochip). Some of the early differentiation markers for mammary epithelium were expressed in the Id2-null tissue, whereas genes that are expressed at later stages of pregnancy were not induced. From these results, we conclude that, in the absence of Id2, mammary epithelial development is arrested at an early stage of pregnancy.
Attenuation of Glucocorticoid Signaling through Targeted Degradation of p300 via the 26S Proteasome Pathway
Authors: Li Qiao , Su Anna , Chen Jihong , Lefebvre Yvonne A. , Haché Robert J. G.
Published: November 2002

The effects of acetylation on gene expression are complex, with changes in chromatin accessibility intermingled with direct effects on transcriptional regulators. For the nuclear receptors, both positive and negative effects of acetylation on specific gene transcription have been observed. We report that p300 and steroid receptor coactivator 1 interact transiently with the glucocorticoid receptor and that the acetyltransferase activity of p300 makes an important contribution to glucocorticoid receptor-mediated transcription. Treatment of cells with the deacetylase inhibitor, sodium butyrate, inhibited steroid-induced transcription and altered the transient association of glucocorticoid receptor with p300 and steroid receptor coactivator 1. Additionally, sustained sodium butyrate treatment induced the degradation of p300 through the 26S proteasome pathway. Treatment with the proteasome inhibitor MG132 restored both the level of p300 protein and the transcriptional response to steroid over 20 h of treatment. These results reveal new levels for the regulatory control of gene expression by acetylation and suggest feedback control on p300 activity.
Disruption of Steroid and Prolactin Receptor Patterning in the Mammary Gland Correlates with a Block in Lobuloalveolar Development
Authors: Grimm Sandra L. , Seagroves Tiffany N. , Kabotyanski Elena B. , Hovey Russell C. , Vonderhaar Barbara K. , Lydon John P. , Miyoshi Keiko , Hennighausen Lothar , Ormandy Christopher J. , Lee Adrian V. , Stull Malinda A. , Wood Teresa L. , Rosen Jeffrey M.
Published: November 2002

Targeted deletion of the bZIP transcription factor, CCAAT/enhancer binding protein-β (C/EBPβ), was shown previously to result in aberrant ductal morphogenesis and decreased lobuloalveolar development, accompanied by an altered pattern of progesterone receptor (PR) expression. Here, similar changes in the level and pattern of prolactin receptor (PrlR) expression were observed while screening for differentially expressed genes in C/EBPβnull mice. PR patterning was also altered in PrlRnull mice, as well as in mammary tissue transplants from both PrlRnull and signal transducer and activator of transcription (Stat) 5a/b-deficient mice, with concomitant defects in hormone-induced proliferation. Down-regulation of PR and activation of Stat5 phosphorylation were seen after estrogen and progesterone treatment in both C/EBPβnull and wild-type mice, indicating that these signaling pathways were functional, despite the failure of steroid hormones to induce proliferation. IGF binding protein-5, IGF-II, and insulin receptor substrate-1 all displayed altered patterns and levels of expression in C/EBPβnull mice, suggestive of a change in the IGF signaling axis. In addition, small proline-rich protein (SPRR2A), a marker of epidermal differentiation, and keratin 6 were misexpressed in the mammary epithelium of C/EBPβnull mice. Together, these data suggest that C/EBPβ is a master regulator of mammary epithelial cell fate and that the correct spatial pattern of PR and PrlR expression is a critical determinant of hormone-regulated cell proliferation.
A Genomic Approach to Identify Novel Progesterone Receptor Regulated Pathways in the Uterus during Implantation
Authors: Cheon Yong-Pil , Li Quanxi , Xu Xueping , DeMayo Francesco J. , Bagchi Indrani C. , Bagchi Milan K.
Published: November 2002

The cellular actions of steroid hormone progesterone (P) are mediated via its nuclear receptors, which regulate the expression of specific target genes. The identity of gene networks that are regulated by the P receptors (PRs) in the uterus at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. In this study, we have used oligonucleotide microarrays to identify mRNAs whose expression in the pregnant mouse uterus is modulated by RU486, a well-characterized PR antagonist, which is also an effective inhibitor of implantation. We found that, in response to RU486, expression of mRNAs corresponding to 78 known genes was down-regulated at least 2-fold in the preimplantation mouse uterus. The PR regulation of several of these genes was ascertained by administering P to ovariectomized wild-type and PR knockout (PRKO) mice. Detailed spatio-temporal analysis of these genes in the pregnant uterus indicated that their expression in the epithelium and stroma could be correlated with the expression of PR in those cell types. Furthermore, time-course studies suggested that many of these genes are likely primary targets of PR regulation. We also identified 70 known genes that were up-regulated at least 2-fold in the pregnant uterus in response to RU486. Interestingly, initial examination of a number of RU486-inducible genes reveals that their uterine expression is also regulated by estrogen. The identification of several novel PR-regulated gene pathways in the reproductive tract is an important step toward understanding how P regulates the physiological events leading to implantation.
Oxidative tyrosylation of high density lipoproteins impairs cholesterol efflux from mouse J774 macrophages: role of scavenger receptors, classes A and B
Authors: Suc Isabelle , Brunet Sylvain , Mitchell Grant , Rivard Georges-Etienne , Levy Emile
Published: November 2002

Studies were designed to test whether tyrosylation of high-density lipoprotein (HDLT) modifies its metabolic features. HDLTwas less effective than native HDL in promoting cholesterol efflux from J774-AI macrophages. Cell association with fluorescent HDLT-apolipoprotein and the uptake of HDLT-[3H]cholesteryl hexadecyl ether were enhanced by 50% in comparison with native HDL. In addition, neutral cholesterol ester hydrolase (nCEH) activity in J774-AI, which controls the hydrolysis of cholesteryl ester stores to provide free cholesterol for cellular release,declined in the presence of HDLT. In vitro displacement experiments revealed the ability of HDLT to compete with oxidized and acetylated LDL, known as ligands of scavenger receptor (SR) class B type I/II. Similarly, treatment with a blocking antibody to SR-BI/II reduced the cell association of HDLT and native HDL by 50%. The addition of polyinosinic acid, an inhibitor of SR class A, reduced the cell association of HDLT without affecting that of native HDL. These findings provide evidence that HDLT can compete with modified LDL, bind SR-BI/BII and internalize cholesterol ester. Furthermore, the impaired capacity of HDLT in promoting cholesterol efflux from J774-AI was accompanied by diminished nCEH and enhanced recognition by SR-AI/II, which appears to involve the transport of cholesterol into cells.
Analysis of the DNA replication competence of thexrs-5 mutant cells defective in Ku86
Authors: Matheos Diamanto , Novac Olivia , Price Gerald B. , Zannis-Hadjopoulos Maria
Published: November 2002

The radiosensitive mutant xrs-5, a derivative of the Chinese hamster ovary (CHO) K1 cell line, is defective in DNA double-strand break repair and V(D)J recombination. The defective phenotypes of xrs-5 cells are complemented by the 86 kDa subunit of Ku antigen. OBA is a protein,previously purified from HeLa cells, that binds in a sequence-specific manner to mammalian origins of DNA replication. The DNA-binding subunit of OBA has been identified as Ku86. We tested the xrs-5 cell line for its ability to replicate a mammalian origin-containing plasmid, p186, in vivo and in vitro. In vivo, the p186 episomal DNA replication in transfected xrs-5 cells was reduced by 45% when compared with the CHO K1 cells transfected with p186. In vitro, although total and cytoplasmic cell extracts from xrs-5 cells replicated the p186 with the same efficiency as the parental CHO K1 cell extracts, xrs-5 nuclear extracts did not possess any detectable replication activity. Addition of affinity-purified OBA/Ku restored replication in the xrs-5 nuclear extract reaction. Western blot analyses showed that the levels of other replication proteins (Orc2,PCNA, DNA polymerase ϵ and δ, Primase and Topoisomerase IIα)were comparable in both the xrs-5 mutant and CHO K1 wild-type cell lines. In addition, the in vivo association of Ku with the DHFR origin-containing sequence (oriβ) was examined in both the CHO K1 and xrs-5 cell lines by a chromatin immunoprecipitation (ChIP) assay. Anti-Ku antibodies did not immunoprecipitate a detectable amount of Ku from the xrs-5 cells in the origin-containing sequence, in contrast to the CHO K1 cells, wherein Ku was found to be associated with the oriβ origin. The data implicate Ku antigen in in vivo and in vitro DNA replication and suggest the existence of another protein with Ku-like functions in the xrs-5 cells.
Nectin and afadin: novel organizers of intercellular junctions
Authors: Takai Yoshimi , Nakanishi Hiroyuki
Published: November 2002

The cadherin superfamily plays key roles in intercellular adhesion. An emerging intercellular adhesion system, consisting of nectin and afadin, also has roles in organization of a variety of intercellular junctions either in cooperation with, or independently of, cadherin. Nectin is a Ca2+-independent immunoglobulin-like intercellular adhesion molecule, and afadin is a nectin- and actin-filament-binding protein that connects nectin to the actin cytoskeleton. This novel intercellular adhesion system has roles in the organization of E-cadherin-based adherens junctions and claudin-based tight junctions in epithelial cells. The adhesion system is furthermore involved in the formation of synapses in neurons and the organization of heterotypic junctions between Sertoli cells and spermatids in the testis.
Nuclear RanGTP is not required for targeting small nucleolar RNAs to the nucleolus
Authors: Narayanan Aarthi , Eifert Julia , Marfatia Kavita A. , Macara Ian G. , Corbett Anita H. , Terns Rebecca M. , Terns Michael P.
Published: November 2002

The small GTPase Ran is the central regulator of macromolecular transport between the cytoplasm and the nucleus. Recent work has suggested that RanGTP also plays an important role in regulating some intra-nuclear processes. In this study, we have investigated whether RanGTP is required for the intra-nuclear transport of RNAs. Specifically, we directly analyzed the nucleolar localization of Box C/D and Box H/ACA small nucleolar RNAs (snoRNAs)in mammalian (tsBN2) cells, Saccharomyces cerevisiae and Xenopus oocytes under conditions that deplete nuclear RanGTP and prevent RNA export to the cytoplasm. Our data suggest that depletion of nuclear RanGTP does not significantly alter the nucleolar localization of U3 snoRNA in tsBN2 cells. Complementary studies in the budding yeast S. cerevisiae using conditional Ran mutants as well as mutants in Ran regulatory proteins also indicate that disruption of the Ran gradient or of Ran itself does not detectably affect the nucleolar localization of snoRNAs. Finally, microinjection into Xenopus oocytes was used to clearly demonstrate that a specific pool of snoRNAs could still be efficiently targeted to the nucleolus even when the RanGTP gradient was disrupted by microinjection of mutant Ran proteins. Taken together, our data from three phylogenetically distinct experimental systems suggest that nuclear RanGTP,which is essential for trafficking of RNAs between the nuclear and cytoplasmic compartments, is not required for nuclear retention or nucleolar localization of snoRNAs.
An insulin-related peptide expressed in 3T3L1 adipocytes is localized in GLUT4 vesicles and secreted in response to exogenous insulin, which augments the insulin-stimulated glucose uptake
Authors: Nakamichi Yoko , Ohara-Imaizumi Mica , Ishida Hitoshi , Nagamatsu Shinya
Published: November 2002

If an adipocyte is programmed to secrete insulin, then the insulin released may amplify the insulin action by an autocrine manner. To examine this hypothesis in vitro, we investigated the effects of expressing the preproinsulin gene in 3T3L1 adipocytes on (pro)insulin release and glucose uptake. The human preproinsulin gene was transferred into 3T3L1 adipocytes by infecting the cells with recombinant adenovirus Adex1CA human preproinsulin. Immunocytochemical studies showed that (pro)insulin is associated with vesicular structures that colocalize with GLUT4 vesicles but not with GLUT1 vesicles. We then examined insulin-induced proinsulin release from 3T3L1 adipocytes expressing the insulin gene. The exogenously administered insulin stimulated proinsulin release from these cells in a dose-dependent manner. HPLC determination revealed the existence of mature human insulin in these cells, which suggested the release of mature insulin into the medium. Further we monitored the (pro)insulin release from these cells with confocal laser microscopy using the expression of a fusion protein between insulin and green fluorescent protein (GFP). Time-lapse confocal laser-scanning microscopy revealed that the total number of vesicles containing insulin-GFP was decreased by the addition of 10-7 M insulin within 1 minute. Finally, we examined the insulin-stimulated glucose uptake by these cells. The data showed that insulin-stimulated glucose uptake increased to about 150% of that of control cells in response to exogenously administered insulin,indicating that the insulin released augmented the insulin-stimulated glucose uptake in an autocrine manner. Thus, the data support our hypothesis,indicating that we could construct the insulin-regulated insulin release system in adipocytes by introducing the preproinsulin gene.
Multiple connexins contribute to intercellular communication in theXenopusembryo
Authors: Landesman Yosef , Postma Friso R. , Goodenough Daniel A. , Paul David L.
Published: November 2002

To explore the role of gap junctional intercellular communication (GJIC)during Xenopus embryogenesis, we utilized the host-transfer and antisense techniques to specifically deplete Cx38, the only known maternally expressed connexin. Cx38-depleted embryos developed normally but displayed robust GJIC between blastomeres at 32-128 cell stages, suggesting the existence of other maternal connexins. Analysis of embryonic cDNA revealed maternal expression of two novel connexins, Cx31 and Cx43.4, and a third,Cx43, that had been previously identified as a product of zygotic transcription. Thus, the early Xenopus embryo contains at least four maternal connexins. Unlike Cx38, expression of Cx31, Cx43 and Cx43.4 continue zygotically. Of these, Cx43.4 is the most abundant, accumulating significantly in neural structures including the brain, the eyes and the spinal cord.
Nitric oxide signaling specificity — the heart of the problem
Authors: Bredt David S.
Published: November 2002

Nitric oxide (NO) is a gaseous free radical that functions as an endogenous mediator in numerous tissues. Because NO is both reactive and highly diffusible, its formation must be tightly regulated to control its synthesis and to specify its signaling. Indeed, molecular studies of the NO synthase(NOS) family of enzymes have elaborated a variety of mechanisms, including protein interactions, lipid modifications and protein phosphorylation cascades that spatially and temporally control NO biosynthesis. These mechanisms determine both the upstream cellular signals that stimulate NO formation and the downstream molecular targets for NO. Understanding these cellular pathways that control NOS will help us to elucidate the functional roles of NO and provide novel strategies to treat diseases associated with NO abnormalities.
Synaptotagmin III is a critical factor for the formation of the perinuclear endocytic recycling compartment and determination of secretory granules size
Authors: Grimberg Elena , Peng Ze , Hammel Ilan , Sagi-Eisenberg Ronit
Published: November 2002

Early endosomes and a perinuclear, Rab-11-positive compartment have been implicated in the recycling of internalized receptors. In this study, we show that synaptotagmin III (Syt III), a member of the Syt family of proteins, is required for the formation and delivery of cargo to the perinuclear endocytic recycling compartment (ERC). We demonstrate that rat basophilic leukemia(RBL-2H3) mast cells endogenously express Syt III, and >70% of this protein colocalizes with early endosomal markers, such as EEA1, annexin II and syntaxin 7, and the remaining protein colocalizes with secretory granule (SG)markers such as β-hexosaminidase, histamine and serotonin. To study the functional role of Syt III, we stably transfected RBL cells with Syt III antisense cDNA and monitored the route of transferrin (Tfn) internalization in cells that displayed substantially reduced (<90%) levels of Syt III(RBL-Syt III-). In these cells, Tfn binding and internalization into early endosomes were unaltered. However, whereas in the mock-transfected cells Tfn was subsequently delivered to the ERC, in the RBL-Syt III- cells, Tfn remained associated with dispersed peripheral vesicles and Rab 11 remained cytosolic. Nevertheless, the rates of Tfn internalization and recycling were not affected. RBL-Syt III- cells also displayed enlarged SGs, reminiscent of the SGs present in Chediak-Higashi(beige) mice. However, morphometric analyses suggested that granule formation was unaltered and that the calculated unit granule volume is the same in both cell lines. Therefore, our results implicate Syt III as a critical factor for the generation and delivery of internalized cargo to the perinuclear endocytic recycling compartment and suggest a possible link between ERC and recycling from immature SGs during the process of SG maturation.
EhPAK, a member of the p21-activated kinase family, is involved in the control ofEntamoeba histolyticamigration and phagocytosis
Authors: Labruyère Elisabeth , Zimmer Christophe , Galy Vincent , Olivo-Marin Jean-Christophe , Guillén Nancy
Published: November 2002

Entamoeba histolytica migration is essential for the development of amoebiasis, a human disease characterised by invasion and destruction of tissues. Amoebic motility requires both polarisation of the cell and formation of a predominant pseudopod. As p21-activated kinases PAKs are known to regulate eukaryotic cell motility and morphology, we investigated the role of PAK in E. histolytica. We showed that the C-terminal domain of EhPAK comprised a constitutive kinase activity in vitro and that overproduction of this fragment, in E. histolytica, caused a significant reduction in amoeboid migration, as measured by dynamic image analysis, indicating an involvement of EhPAK in this process. A dramatic loss of polarity, as indicated by the increased number of membrane extensions all around E. histolytica, was also observed, suggesting that the N-terminal domain of EhPAK was necessary for maintenance of cell polarity. To support this view, we showed that despite the absence of the consensus motif to bind to Rac and Cdc42, the N-terminal domain of EhPAK bound to Rac1, suggesting that the N-terminal region was a regulatory domain. In addition, we also found an increased rate of human red blood cell phagocytosis, suggesting for the first time an active role for a PAK protein in this process. Taking together, the results suggest strongly that EhPAK is a key regulatory element in polarity, motility and phagocytosis of E. histolytica.
Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex ofPlasmodiumsporozoites
Authors: Bergman Lawrence W. , Kaiser Karine , Fujioka Hisashi , Coppens Isabelle , Daly Thomas M. , Fox Sarah , Matuschewski Kai , Nussenzweig Victor , Kappe Stefan H. I.
Published: November 2002

Apicomplexan host cell invasion and gliding motility depend on the parasite's actomyosin system located beneath the plasma membrane of invasive stages. Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein. A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex. Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA. Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP. In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA. The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane. The head domain of the immobilized MyoA moves actin filaments that,directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family. The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.
Myosin II contributes to the posterior contraction and the anterior extension during the retraction phase in migratingDictyosteliumcells
Authors: Uchida Kazuhiko S. K. , Kitanishi-Yumura Toshiko , Yumura Shigehiko
Published: November 2002

Cells must exert force against the substrate to migrate. We examined the vectors (both the direction and the magnitude) of the traction force generated by Dictyostelium cells using an improved non-wrinkling silicone substrate. During migration, the cells showed two `alternate' phases of locomotory behavior, an extension phase and a retraction phase. In accordance with these phases, two alternate patterns were identified in the traction force. During the extension phase, the cell exerted a `pulling force' toward the cell body in the anterior and the posterior regions and a `pushing force'in the side of the cell (pattern 1). During the retraction phase, the cell exerted a `pushing force' in the anterior region, although the force disappeared in the side and the posterior regions of the cell (pattern 2). Myosin II heavy chain null cells showed a single pattern in their traction force comparable to `pattern 1', although they still had the alternate biphasic locomotory behavior similar to the wild-type cells. Therefore, the generation of `pushing force' in the anterior and the cancellation of the traction force in the side and the posterior during the retraction phase were deficient in myosin knock-out mutant cells, suggesting that these activities depend on myosin II via the posterior contraction. Considering all these results, we hypothesized that there is a highly coordinated, biphasic mechanism of cell migration in Dictyostelium.
Overexpression of theSuURgene induces reversible modifications at pericentric, telomeric and intercalary heterochromatin ofDrosophila melanogasterpolytene chromosomes
Authors: Zhimulev Igor F. , Belyaeva Elena S. , Semeshin Valerii F. , Shloma Victor V. , Makunin Igor V. , Volkova Elena I.
Published: November 2002

The SuUR (suppressor of underreplication) gene controls late replication and underreplication of DNA in Drosophila melanogaster polytene chromosomes: its mutation suppresses DNA underreplication whereas additional doses of the normal allele strongly enhances underreplication. The SuUR protein is localized in late replicating and underreplicating regions. The N-terminal part of the SuUR protein shares modest similarity with the ATPase/helicase domain of SWI2/SNF2 chromatin remodeling factors, suggesting a role in modification of chromatin structure.

Here we describe novel structural modifications of polytene chromosomes(swellings) and show that SuUR controls chromatin organization in polytene chromosomes. The swellings develop as the result of SuURectopic expression in the transgene system Sgs3-GAL4; UAS-SuUR+. They are reminiscent of chromosome puffs and appear in ∼190 regions of intercalary, pericentric and telomeric heterochromatin; some of them attain tremendous size. The swellings are temperature sensitive: they are maximal at 29°C and are barely visible at 18°C. Shifting from 29°C to 18°C results in the complete recovery of the normal structure of chromosomes. The swellings are transcriptionally inactive, since they do not incorporate [3H]uridine. The SuUR protein is not visualized in regions of maximally developed swellings. Regular ecdysone-inducible puffs are not induced in cells where these swellings are apparent.
Free oxygen radicals regulate plasma membrane Ca2+- and K+-permeable channels in plant root cells
Authors: Demidchik Vadim , Shabala Sergey N. , Coutts Katherine B. , Tester Mark A. , Davies Julia M.
Published: November 2002

Free oxygen radicals are an irrefutable component of life, underlying important biochemical and physiological phenomena in animals. Here it is shown that free oxygen radicals activate plasma membrane Ca2+- and K+-permeable conductances in Arabidopsis root cell protoplasts, mediating Ca2+ influx and K+ efflux,respectively. Free oxygen radicals generate increases in cytosolic Ca2+ mediated by a novel population of nonselective cation channels that differ in selectivity and pharmacology from those involved in toxic Na+ influx. Analysis of the free oxygen radical-activated K+ conductance showed its similarity to the Arabidopsisroot K+ outward rectifier. Significantly larger channel activation was found in cells responsible for perceiving environmental signals and undergoing elongation. Quenching root free oxygen radicals inhibited root elongation, confirming the role of radical-activated Ca2+ influx in cell growth. Net free oxygen radical-stimulated Ca2+ influx and K+ efflux were observed in root cells of monocots, dicots, C3 and C4 plants, suggesting conserved mechanisms and functions. In conclusion, two functions for free oxygen radical cation channel activation are proposed:initialization/amplification of stress signals and control of cell elongation in root growth.
Cell swelling increases intracellular calcium in Necturuserythrocytes
Authors: Light Douglas B. , Attwood Andrew J. , Siegel Corryn , Baumann Nicole L.
Published: November 2002

This study examined the role of Ca2+ in regulatory volume decrease by Necturus erythrocytes. Hypotonic shock (50% tonicity)stimulated an increase in cytosolic free Ca2+, detected using epi-fluorescence microscopy and the fluorescent Ca2+ indicator fluo-4-AM (10 μM). A similar increase in fluorescence did not occur under isosmotic conditions, unless cells were exposed to the Ca2+ionophore A23187 (0.5 μM). In addition, a low Ca2+ medium(amphibian Ringer solution with 5 mM EGTA), hexokinase (2.5 U/ml, an ATP scavenger), suramin (100 μM, a P2 receptor antagonist) and gadolinium (10μM, a stretch-activated channel blocker) each inhibited the swelling-induced increase in Ca2+. Consistent with these studies, a low Ca2+ Ringer solution increased osmotic fragility, whereas volume recovery following hypotonic shock (measured with a Coulter counter)was potentiated with A23187 (0.5 μM). By contrast, a low Ca2+extracellular medium or buffering intracellular Ca2+ with BAPTA-AM(100 μM) reduced the rate of volume recovery following hypotonic challenge. Finally, a low Ca2+ extracellular Ringer solution inhibited whole-cell currents that are activated during cell swelling (measured with the whole-cell patch clamp technique). Our results are most consistent with hypotonic shock causing an increase in cytosolic free Ca2+, thereby stimulating subsequent volume decrease.
Anomalous centriole configurations are detected inDrosophilawing disc cells upon Cdk1 inactivation
Authors: Vidwans Smruti J. , Wong Mei Lie , O'Farrell Patrick H.
Published: November 2002

The centriole, organizer of the centrosome, duplicates by assembling a unique daughter identical to itself in overall organization and length. The centriole is a cylindrical structure composed of nine sets of microtubules and is thus predicted to have nine-fold symmetry. During duplication, a daughter lacking discrete microtubular organization first appears off the wall of the mother centriole. It increases in length perpendicularly away from the mother and terminates growth when it matches the length of the mother. How a unique daughter of the correct length and overall organization is assembled is unknown. Here, we describe three types of unusual centriole configurations observed in wing imaginal discs of Drosophila following inactivation of Cdk1. First, we observed centriole triplets consisting of one mother and two daughters, which suggested that centrioles have more than one potential site for the assembly of daughters. Second, we observed centriole triplets comprising a grandmother, mother and daughter, which suggested that subsequent centriole duplication cycles do not require separation of mother and daughter centrioles. Finally, we observed centriole pairs in which the daughter is longer than its mother. These findings suggest that regulatory events rather than rigid structural constraints dictate features of the stereotyped duplication program of centrioles.
Autonomous and phosphorylation-responsive microtubule-regulating activities of the N-terminus of Op18/stathmin
Authors: Segerman Bo , Holmfeldt Per , Morabito Justin , Cassimeris Lynne , Gullberg Martin
Published: November 2002

Op18 is the prototypical member of a family of phosphorylation-responsive regulators of microtubule (MT) dynamics. Previous dissection of Op18 has suggested that it has a functional dichotomy in which an intact N-terminus is required for catastrophe promotion (i.e. transition from growing to shrinking MTs), whereas an intact C-terminus is required for efficient ternary Op18-tubulin complex formation and the resultant tubulin-sequestering activity. Here we have expressed and functionally analyzed the properties of the N-terminus of Op18. The data show that the N-terminal 57 residues are sufficient for low-affinity tubulin interactions, as shown by inhibition of basal GTP hydrolysis of soluble heterodimers. In addition, high concentrations of the Op18 N-terminal portion increased the catastrophe rate during MT assembly in vitro. Overexpression of the N-terminus in a human cell line results in MT destabilization in interphase and phosphorylation-modulated accumulation of metaphase-arrested cells with dense short MTs. These results demonstrate that the N-terminus of Op18 has autonomous activity. Evidently,this activity is enhanced by the increase in tubulin affinity that is provided by the extended α-helical portion of native Op18.
Mob2p interacts with the protein kinase Orb6p to promote coordination of cell polarity with cell cycle progression
Authors: Hou Ming-Chin , Wiley David J. , Verde Fulvia , McCollum Dannel
Published: November 2002

The molecular mechanisms that temporally and spatially coordinate cell morphogenesis with the cell cycle remain poorly understood. Here we describe the characterization of fission yeast Mob2p, a novel protein required for regulating cell polarity and cell cycle control. Deletion of mob2 is lethal and causes cells to become spherical, with depolarized actin and microtubule cytoskeletons. A decrease in Mob2p protein level results in a defect in the activation of bipolar growth. This phenotype is identical to that of mutants defective in the orb6 protein kinase gene, and we find that Mob2p physically interacts with Orb6p. In addition, overexpression of Mob2p, like that of Orb6p, results in a delay in the onset of mitosis. Mob2p localizes to the cell periphery and cytoplasm throughout the cell cycle and to the division site during late anaphase and telophase. Mob2p is unable to localize to the cell middle in mutants defective in actomyosin ring and septum formation. Our results suggest that Mob2p, along with Orb6p, is required for coordinating polarized cell growth during interphase with the onset of mitosis.
Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth
Authors: Abe Tomoyuki , Kato Masayoshi , Miki Hiroaki , Takenawa Tadaomi , Endo Takeshi
Published: November 2002

Rho family small GTPases regulate multiple cellular functions through reorganization of the actin cytoskeleton. Among them, Cdc42 and Tc10 induce filopodia or peripheral processes in cultured cells. We have identified a member of the family, designated as RhoT, which is closely related to Tc10. Tc10 was highly expressed in muscular tissues and brain and remarkably induced during differentiation of C2 skeletal muscle cells and neuronal differentiation of PC12 and N1E-115 cells. On the other hand, RhoT was predominantly expressed in heart and uterus and induced during neuronal differentiation of N1E-115 cells. Tc10 exogenously expressed in fibroblasts generated actin-filament-containing peripheral processes longer than the Cdc42-formed filopodia, whereas RhoT produced much longer and thicker processes containing actin filaments. Furthermore, both Tc10 and RhoT induced neurite outgrowth in PC12 and N1E-115 cells, but Cdc42 did not do this by itself. Tc10 and RhoT as well as Cdc42 bound to the N-terminal CRIB-motif-containing portion of N-WASP and activated N-WASP to induce Arp2/3-complex-mediated actin polymerization. The formation of peripheral processes and neurites by Tc10 and RhoT was prevented by the coexpression of dominant-negative mutants of N-WASP. Thus, N-WASP is essential for the process formation and neurite outgrowth induced by Tc10 and RhoT. Neuronal differentiation of PC12 and N1E-115 cells induced by dibutyryl cyclic AMP and by serum starvation, respectively, was prevented by dominant-negative Cdc42,Tc10 and RhoT. Taken together, all these Rho family proteins are required for neuronal differentiation, but they exert their functions differentially in process formation and neurite extension. Consequently, N-WASP activated by these small GTPases mediates neuronal differentiation in addition to its recently identified role in glucose uptake.