bluuebunny/crossref_metadata_2025 huggingface dataset:
Axons of retinal ganglion cells are insulted in the optic nerve early in DBA/2J glaucoma
Authors: Howell Gareth R. , Libby Richard T. , Jakobs Tatjana C. , Smith Richard S. , Phalan F. Campbell , Barter Joseph W. , Barbay Jessica M. , Marchant Jeffrey K. , Mahesh Nagaraju , Porciatti Vittorio , Whitmore Alan V. , Masland Richard H. , John Simon W. M.
Published: December 2007

Here, we use a mouse model (DBA/2J) to readdress the location of insult(s) to retinal ganglion cells (RGCs) in glaucoma. We localize an early sign of axon damage to an astrocyte-rich region of the optic nerve just posterior to the retina, analogous to the lamina cribrosa. In this region, a network of astrocytes associates intimately with RGC axons. Using BAX-deficient DBA/2J mice, which retain all of their RGCs, we provide experimental evidence for an insult within or very close to the lamina in the optic nerve. We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina. In contrast, axon segments in the lamina and behind the eye degenerate. Finally, the Wlds allele, which is known to protect against insults to axons, strongly protects against DBA/2J glaucoma and preserves RGC activity as measured by pattern electroretinography. These experiments provide strong evidence for a local insult to axons in the optic nerve.
Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons
Authors: Dong Min , Tepp William H. , Liu Huisheng , Johnson Eric A. , Chapman Edwin R.
Published: December 2007

Botulinum neurotoxins (BoNTs) target presynaptic nerve terminals by recognizing specific neuronal surface receptors. Two homologous synaptic vesicle membrane proteins, synaptotagmins (Syts) I and II, bind toxins BoNT/B and G. However, a direct demonstration that Syts I/II mediate toxin binding and entry into neurons is lacking. We report that BoNT/B and G fail to bind and enter hippocampal neurons cultured from Syt I knockout mice. Wild-type Syts I and II (but not Syt I loss-of-function toxin-binding domain mutants) restored binding and entry of BoNT/B and G in Syt I–null neurons, thus demonstrating that Syts I/II are protein receptors for BoNT/B and G. Furthermore, mice lacking complex gangliosides exhibit reduced sensitivity to BoNT/G, and binding and entry of BoNT/A, B, and G into hippocampal neurons lacking gangliosides is diminished. These data suggest that gangliosides are the shared coreceptor for BoNT/A, B, and G, supporting a double-receptor model for these three BoNTs for which the protein receptors are known.
DNA-activated protein kinase functions in a newly observed S phase checkpoint that links histone mRNA abundance with DNA replication
Authors: Müller Berndt , Blackburn Jane , Feijoo Carmen , Zhao Xiujie , Smythe Carl
Published: December 2007

DNA and histone synthesis are coupled and ongoing replication is required to maintain histone gene expression. Here, we expose S phase–arrested cells to the kinase inhibitors caffeine and LY294002. This uncouples DNA replication from histone messenger RNA (mRNA) abundance, altering the efficiency of replication stress–induced histone mRNA down-regulation. Interference with caffeine-sensitive checkpoint kinases ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) does not affect histone mRNA down- regulation, which indicates that ATR/ATM alone cannot account for such coupling. LY294002 potentiates caffeine's ability to uncouple histone mRNA stabilization from replication only in cells containing functional DNA-activated protein kinase (DNA-PK), which indicates that DNA-PK is the target of LY294002. DNA-PK is activated during replication stress and DNA-PK signaling is enhanced when ATR/ATM signaling is abrogated. Histone mRNA decay does not require Chk1/Chk2. Replication stress induces phosphorylation of UPF1 but not hairpin-binding protein/stem-loop binding protein at S/TQ sites, which are preferred substrate recognition motifs of phosphatidylinositol 3-kinase–like kinases, which indicates that histone mRNA stability may be directly controlled by ATR/ATM- and DNA-PK–mediated phosphorylation of UPF1.
Huntingtin-interacting protein 14, a palmitoyl transferase required for exocytosis and targeting of CSP to synaptic vesicles
Authors: Ohyama Tomoko , Verstreken Patrik , Ly Cindy V. , Rosenmund Tanja , Rajan Akhila , Tien An-Chi , Haueter Claire , Schulze Karen L. , Bellen Hugo J.
Published: December 2007

Posttranslational modification through palmitoylation regulates protein localization and function. In this study, we identify a role for the Drosophila melanogaster palmitoyl transferase Huntingtin-interacting protein 14 (HIP14) in neurotransmitter release. hip14 mutants show exocytic defects at low frequency stimulation and a nearly complete loss of synaptic transmission at higher temperature. Interestingly, two exocytic components known to be palmitoylated, cysteine string protein (CSP) and SNAP25, are severely mislocalized at hip14 mutant synapses. Complementary DNA rescue and localization experiments indicate that HIP14 is required solely in the nervous system and is essential for presynaptic function. Biochemical studies indicate that HIP14 palmitoylates CSP and that CSP is not palmitoylated in hip14 mutants. Furthermore, the hip14 exocytic defects can be suppressed by targeting CSP to synaptic vesicles using a chimeric protein approach. Our data indicate that HIP14 controls neurotransmitter release by regulating the trafficking of CSP to synapses.
Regulation of liver regeneration and hepatocarcinogenesis by suppressor of cytokine signaling 3
Authors: Riehle Kimberly J. , Campbell Jean S. , McMahan Ryan S. , Johnson Melissa M. , Beyer Richard P. , Bammler Theo K. , Fausto Nelson
Published: December 2007

Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation at the early stages of liver regeneration (Campbell, J.S., L. Prichard, F. Schaper, J. Schmitz, A. Stephenson-Famy, M.E. Rosenfeld, G.M. Argast, P.C. Heinrich, and N. Fausto. 2001.J. Clin. Invest. 107:1285–1292). We developed Socs3 hepatocyte-specific knockout (Socs3 h-KO) mice to directly study the role of SOCS3 during liver regeneration after a two-thirds partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA replication and liver weight restoration after PH in comparison with littermate controls. Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation is prolonged, and activation of the mitogenic extracellular signal-regulated kinase 1/2 (ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 deficiency enhances hepatocyte proliferation in association with enhanced STAT3 and ERK activation after epidermal growth factor or interleukin 6 stimulation. Microarray analyses show that SOCS3 modulates a distinct set of genes, which fall into diverse physiological categories, after PH. Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop hepatocellular carcinoma at an accelerated rate. By acting on cytokines and multiple proliferative pathways, SOCS3 modulates both physiological and neoplastic proliferative processes in the liver and may act as a tumor suppressor.
Regulation of cellular zinc balance as a potential mechanism of EVER-mediated protection against pathogenesis by cutaneous oncogenic human papillomaviruses
Authors: Lazarczyk Maciej , Pons Christian , Mendoza José-Andrès , Cassonnet Patricia , Jacob Yves , Favre Michel
Published: December 2007

Epidermodysplasia verruciformis (EV) is a genodermatosis associated with skin cancers that results from a selective susceptibility to related human papillomaviruses (EV HPV). Invalidating mutations in either of two genes (EVER1 and EVER2) with unknown functions cause most EV cases. We report that EVER1 and EVER2 proteins form a complex and interact with the zinc transporter 1 (ZnT-1), as shown by yeast two-hybrid screening, GST pull-down, and immunoprecipitation experiments. In keratinocytes, EVER and ZnT-1 proteins do not influence intracellular zinc concentration, but do affect intracellular zinc distribution. EVER2 was found to inhibit free zinc influx to nucleoli. Keratinocytes with a mutated EVER2 grew faster than wild-type keratinocytes. In transiently and stably transfected HaCaT cells, EVER and ZnT-1 down-regulated transcription factors stimulated by zinc (MTF-1) or cytokines (c-Jun and Elk), as detected with luciferase assays. To get some insight into the control of EV HPV infection, we searched for interaction between EVER and ZnT-1 and oncoproteins of cutaneous (HPV5) and genital (HPV16) genotypes. HPV16 E5 protein binds to EVER and ZnT-1 and blocks their negative regulation. The lack of a functional E5 protein encoded by EV HPV genome may account for host restriction of these viruses.
Syndecan-4 protects against osteopontin-mediated acute hepatic injury by masking functional domains of osteopontin
Authors: Kon Shigeyuki , Ikesue Masahiro , Kimura Chiemi , Aoki Momoe , Nakayama Yosuke , Saito Yoshinari , Kurotaki Daisuke , Diao Hongyan , Matsui Yutaka , Segawa Tatsuya , Maeda Masahiro , Kojima Tetsuhito , Uede Toshimitsu
Published: December 2007

Osteopontin (OPN) is a T helper type 1 immunoregulatory cytokine that plays a critical role in various inflammatory disorders. OPN exerts proinflammatory reactions through interaction with integrin receptors. OPN function can be modulated by protease digestion. However, the molecular mechanisms that regulate OPN function in vivo have not been elucidated. There are two putative heparin-binding domains (HBDs) within the OPN molecule, which may bind both heparin and heparin-like glycosaminoglycans such as syndecan. We show that expression of OPN and syndecan-4 is significantly up-regulated after concanavalin-A (ConA) injection. Syndecan-4 binds to one of the HBDs of OPN, which overlaps with the thrombin cleavage site of OPN. When OPN is associated with syndecan-4, syndecan-4 masks both the thrombin cleavage and the integrin binding sites within OPN. Importantly, syndecan-4–deficient (Syn4KO) mice are more susceptible to hepatic injury, and the thrombin-cleaved form of OPN is significantly elevated in Syn4KO mice as compared with wild-type mice after ConA injection. Finally, we demonstrate that administration of purified syndecan-4 protects mice from ConA-induced hepatic injury. Thus, syndecan-4 is a critical intrinsic regulator of inflammatory reactions via its effects on OPN function and is a potential novel therapeutic tool for treating inflammatory diseases.
Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs
Authors: Wolf Andrea J. , Desvignes Ludovic , Linas Beth , Banaiee Niaz , Tamura Toshiki , Takatsu Kiyoshi , Ernst Joel D.
Published: December 2007

The onset of the adaptive immune response to Mycobacterium tuberculosis is delayed compared with that of other infections or immunization, and allows the bacterial population in the lungs to expand markedly during the preimmune phase of infection. We used adoptive transfer of M. tuberculosis Ag85B-specific CD4+ T cells to determine that the delayed adaptive response is caused by a delay in initial activation of CD4+ T cells, which occurs earliest in the local lung-draining mediastinal lymph node. We also found that initial activation of Ag85B-specific T cells depends on production of antigen by bacteria in the lymph node, despite the presence of 100-fold more bacteria in the lungs. Although dendritic cells have been found to transport M. tuberculosis from the lungs to the local lymph node, airway administration of LPS did not accelerate transport of bacteria to the lymph node and did not accelerate activation of Ag85B-specific T cells. These results indicate that delayed initial activation of CD4+ T cells in tuberculosis is caused by the presence of the bacteria in a compartment that cannot be mobilized from the lungs to the lymph node, where initial T cell activation occurs.
In vivo visualization of abdominal malignancies with acoustic radiation force elastography
Authors: Fahey B J , Nelson R C , Bradway D P , Hsu S J , Dumont D M , Trahey G E
Published: December 2007

The utility of acoustic radiation force impulse (ARFI) imaging for real-time visualization of abdominal malignancies was investigated. Nine patients presenting with suspicious masses in the liver (n= 7) or kidney (n= 2) underwent combined sonography/ARFI imaging. Images were acquired of a total of 12 tumors in the nine patients. In all cases, boundary definition in ARFI images was improved or equivalent to boundary definition in B-mode images. Displacement contrast in ARFI images was superior to echo contrast in B-mode images for each tumor. The mean contrast for suspected hepatocellular carcinomas (HCCs) in B-mode images was 2.9 dB (range: 1.5–4.2) versus 7.5 dB (range: 3.1–11.9) in ARFI images, with all HCCs appearing more compliant than regional cirrhotic liver parenchyma. The mean contrast for metastases in B-mode images was 3.1 dB (range: 1.2–5.2) versus 9.3 dB (range: 5.7–13.9) in ARFI images, with all masses appearing less compliant than regional non-cirrhotic liver parenchyma. ARFI image contrast (10.4 dB) was superior to B-mode contrast (0.9 dB) for a renal mass. To our knowledge, we present the firstin vivoimages of abdominal malignancies in humans acquired with the ARFI method or any other technique of imaging tissue elasticity.
Isc1p Plays a Key Role in Hydrogen Peroxide Resistance and Chronological Lifespan through Modulation of Iron Levels and Apoptosis
Authors: Almeida Teresa , Marques Marta , Mojzita Dominik , Amorim Maria A. , Silva Rui D. , Almeida Bruno , Rodrigues Pedro , Ludovico Paula , Hohmann Stefan , Moradas-Ferreira Pedro , Côrte-Real Manuela , Costa Vítor
Published: December 2007

The inositolphosphosphingolipid phospholipase C (Isc1p) of Saccharomyces cerevisiae belongs to the family of neutral sphingomyelinases that generates the bioactive sphingolipid ceramide. In this work the role of Isc1p in oxidative stress resistance and chronological lifespan was investigated. Loss of Isc1p resulted in a higher sensitivity to hydrogen peroxide that was associated with an increase in oxidative stress markers, namely intracellular oxidation, protein carbonylation, and lipid peroxidation. Microarray analysis showed that Isc1p deficiency up-regulated the iron regulon leading to increased levels of iron, which is known to catalyze the production of the highly reactive hydroxyl radicals via the Fenton reaction. In agreement, iron chelation suppressed hydrogen peroxide sensitivity of isc1Δ mutants. Cells lacking Isc1p also displayed a shortened chronological lifespan associated with oxidative stress markers and aging of parental cells was correlated with a decrease in Isc1p activity. The analysis of DNA fragmentation and caspase-like activity showed that Isc1p deficiency increased apoptotic cell death associated with oxidative stress and aging. Furthermore, deletion of Yca1p metacaspase suppressed the oxidative stress sensitivity and premature aging phenotypes of isc1Δ mutants. These results indicate that Isc1p plays an important role in the regulation of cellular redox homeostasis, through modulation of iron levels, and of apoptosis.
RNA Interference in J774 Macrophages Reveals a Role for Coronin 1 in Mycobacterial Trafficking but Not in Actin-dependent Processes
Authors: Jayachandran Rajesh , Gatfield John , Massner Jan , Albrecht Imke , Zanolari Bettina , Pieters Jean
Published: December 2007

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes. However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobacteria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a specific role in modulating phagosome–lysosome transport upon mycobacterial infection and that it is dispensable for most F-actin–mediated cytoskeletal rearrangements.
Identification of Novel Human Cdt1-binding Proteins by a Proteomics Approach: Proteolytic Regulation by APC/CCdh1
Authors: Sugimoto Nozomi , Kitabayashi Issay , Osano Satoko , Tatsumi Yasutoshi , Yugawa Takashi , Narisawa-Saito Mako , Matsukage Akio , Kiyono Tohru , Fujita Masatoshi
Published: December 2007

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it is central to the regulation of DNA replication during the cell cycle. In fact, unscheduled Cdt1 hyperfunction results in rereplication and/or chromosomal damage. Thus, it is important to understand its function and regulations precisely. We sought to comprehensively identify human Cdt1-binding proteins by a combination of Cdt1 affinity chromatography and liquid chromatography and tandem mass spectrometry analysis. Through this approach, we could newly identify 11 proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and IIα, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/CCdh1, SNF2H, topoisomerase I and IIα, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/CCdh1indicated that this ubiquitin ligase controls the levels of Cdt1 during the cell cycle via three destruction boxes in the Cdt1 N-terminus. Notably, elimination of these destruction boxes resulted in induction of strong rereplication and chromosomal damage. Thus, in addition to SCFSkp2and cullin4-based ubiquitin ligases, APC/CCdh1is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.
The MIQ‐RS: A Suitable Option for Examining Movement Imagery Ability
Authors: Gregg Melanie , Hall Craig , Butler Andrew
Published: December 2007

Within rehabilitation settings, mental imagery helps to promote long‐term recovery and facilitates compliance to rehabilitation exercises. Individuals who are able to effectively engage in imagery practice are likely to gain the most benefit from imagery training. Thus, a suitable imagery ability measurement tool for individuals with movement limitations is needed. The purpose of the present study was to evaluate the Movement Imagery Questionnaire—Revised second version (MIQ‐RS), and compare the results of this new version with Hall and Martin′s (1997) MIQ‐R. Three‐hundred and twenty participants from a variety of sports and performance levels agreed to take part. Results showed the internal consistency and test–retest reliability of the MIQ‐RS were satisfactory, the two‐factor structure of the MIQ‐RS was supported by confirmatory factor analysis, and Pearson correlations indicated a strong relationship between the MIQ‐R and MIQ‐RS. It appears the MIQ‐RS is a suitable option for examining movement imagery ability primarily aimed at the upper extremity.
Glutamate Receptor Subtypes Evidenced by Differences in Desensitization and Dependence on theGLR3.3andGLR3.4Genes
Authors: Stephens Nicholas R. , Qi Zhi , Spalding Edgar P.
Published: December 2007

Ionotropic glutamate (Glu) receptors in the central nervous system of animals are tetrameric ion channels that conduct cations across neuronal membranes upon binding Glu or another agonist. Plants possess homologous molecules encoded by GLR genes. Previous studies of Arabidopsis thaliana root cells showed that the amino acids alanine (Ala), asparagine (Asn), cysteine (Cys), Glu, glycine (Gly), and serine trigger transient Ca2+ influx and membrane depolarization by a mechanism that depends on the GLR3.3 gene. This study of hypocotyl cells demonstrates that these six effective amino acids are not equivalent agonists. Instead, they grouped into hierarchical classes based on their ability to desensitize the response mechanism. Sequential treatment with two different amino acids separated by a washout phase demonstrated that Glu desensitized the depolarization mechanism to Gly, but Gly did not desensitize the mechanism to Glu. All 36 possible pairs of agonists were tested to characterize the desensitization hierarchy. The results could be explained by a model in which one class of channels contained a subunit that was activated and therefore desensitized only by Glu, while a second class could be activated and desensitized by Ala, Cys, Glu, or Gly. A third class could be activated and desensitized by any of the six effective amino acids. Analysis of knockout mutants indicated that GLR3.3 was a required component of all three classes of channels, while the related GLR3.4 molecule specifically affected only two of the classes. The resulting model is an important step toward understanding the biological roles of these enigmatic ion channels.
Structural, Biochemical, and Phylogenetic Analyses Suggest That Indole-3-Acetic Acid Methyltransferase Is an Evolutionarily Ancient Member of the SABATH Family
Authors: Zhao Nan , Ferrer Jean-Luc , Ross Jeannine , Guan Ju , Yang Yue , Pichersky Eran , Noel Joseph P. , Chen Feng
Published: December 2007

The plant SABATH protein family encompasses a group of related small-molecule methyltransferases (MTs) that catalyze the S-adenosyl-l-methionine-dependent methylation of natural chemicals encompassing widely divergent structures. Indole-3-acetic acid (IAA) methyltransferase (IAMT) is a member of the SABATH family that modulates IAA homeostasis in plant tissues through methylation of IAA's free carboxyl group. The crystal structure of Arabidopsis (Arabidopsis thaliana) IAMT (AtIAMT1) was determined and refined to 2.75 Å resolution. The overall tertiary and quaternary structures closely resemble the two-domain bilobed monomer and the dimeric arrangement, respectively, previously observed for the related salicylic acid carboxyl methyltransferase from Clarkia breweri (CbSAMT). To further our understanding of the biological function and evolution of SABATHs, especially of IAMT, we analyzed the SABATH gene family in the rice (Oryza sativa) genome. Forty-one OsSABATH genes were identified. Expression analysis showed that more than one-half of the OsSABATH genes were transcribed in one or multiple organs. The OsSABATH gene most similar to AtIAMT1 is OsSABATH4. Escherichia coli-expressed OsSABATH4 protein displayed the highest level of catalytic activity toward IAA and was therefore named OsIAMT1. OsIAMT1 exhibited kinetic properties similar to AtIAMT1 and poplar IAMT (PtIAMT1). Structural modeling of OsIAMT1 and PtIAMT1 using the experimentally determined structure of AtIAMT1 reported here as a template revealed conserved structural features of IAMTs within the active-site cavity that are divergent from functionally distinct members of the SABATH family, such as CbSAMT. Phylogenetic analysis revealed that IAMTs from Arabidopsis, rice, and poplar (Populus spp.) form a monophyletic group. Thus, structural, biochemical, and phylogenetic evidence supports the hypothesis that IAMT is an evolutionarily ancient member of the SABATH family likely to play a critical role in IAA homeostasis across a wide range of plants.
Overexpression of AtMYB44 Enhances Stomatal Closure to Confer Abiotic Stress Tolerance in Transgenic Arabidopsis
Authors: Jung Choonkyun , Seo Jun Sung , Han Sang Won , Koo Yeon Jong , Kim Chung Ho , Song Sang Ik , Nahm Baek Hie , Choi Yang Do , Cheong Jong-Joo
Published: December 2007

AtMYB44 belongs to the R2R3 MYB subgroup 22 transcription factor family in Arabidopsis (Arabidopsis thaliana). Treatment with abscisic acid (ABA) induced AtMYB44 transcript accumulation within 30 min. The gene was also activated under various abiotic stresses, such as dehydration, low temperature, and salinity. In transgenic Arabidopsis carrying an AtMYB44 promoter-driven β-glucuronidase (GUS) construct, strong GUS activity was observed in the vasculature and leaf epidermal guard cells. Transgenic Arabidopsis overexpressing AtMYB44 is more sensitive to ABA and has a more rapid ABA-induced stomatal closure response than wild-type and atmyb44 knockout plants. Transgenic plants exhibited a reduced rate of water loss, as measured by the fresh-weight loss of detached shoots, and remarkably enhanced tolerance to drought and salt stress compared to wild-type plants. Microarray analysis and northern blots revealed that salt-induced activation of the genes that encode a group of serine/threonine protein phosphatases 2C (PP2Cs), such as ABI1, ABI2, AtPP2CA, HAB1, and HAB2, was diminished in transgenic plants overexpressing AtMYB44. By contrast, the atmyb44 knockout mutant line exhibited enhanced salt-induced expression of PP2C-encoding genes and reduced drought/salt stress tolerance compared to wild-type plants. Therefore, enhanced abiotic stress tolerance of transgenic Arabidopsis overexpressing AtMYB44 was conferred by reduced expression of genes encoding PP2Cs, which have been described as negative regulators of ABA signaling.
Arabidopsis Branched-Chain Aminotransferase 3 Functions in Both Amino Acid and Glucosinolate Biosynthesis
Authors: Knill Tanja , Schuster Joachim , Reichelt Michael , Gershenzon Jonathan , Binder Stefan
Published: December 2007

In Arabidopsis thaliana, transamination steps in the leucine biosynthetic and catabolic pathways and the methionine (Met) chain elongation cycle of aliphatic glucosinolate formation are catalyzed by branched-chain aminotransferases (BCATs) that are encoded by a small gene family of six members. One member of this family, the plastid-located BCAT3, was shown to participate in both amino acid and glucosinolate metabolism. In vitro activity tests with the recombinant protein identified highest activities with the 2-oxo acids of leucine, isoleucine, and valine, but also revealed substantial conversion of intermediates of the Met chain elongation pathway. Metabolite profiling of bcat3-1 single and bcat3-1/bcat4-2 double knockout mutants showed significant alterations in the profiles of both amino acids and glucosinolates. The changes in glucosinolate proportions suggest that BCAT3 most likely catalyzes the terminal steps in the chain elongation process leading to short-chain glucosinolates: the conversion of 5-methylthiopentyl-2-oxo and 6-methylthiohexyl-2-oxo acids to their respective Met derivatives, homomethionine and dihomo-methionine, respectively. The enzyme can also at least partially compensate for the loss of BCAT4, which catalyzes the initial step of Met chain elongation by converting Met to 4-methylthio-2-oxobutanoate. Our results show the interdependence of amino acid and glucosinolate metabolism and demonstrate that a single enzyme plays a role in both processes.
Specificity of RCN1-Mediated Protein Phosphatase 2A Regulation in Meristem Organization and Stress Response in Roots
Authors: Blakeslee Joshua J. , Zhou Hong-Wei , Heath Jeffrey T. , Skottke Kyle R. , Barrios Jorge A. Rodriguez , Liu Su-Yang , DeLong Alison
Published: December 2007

Protein dephosphorylation by the serine/threonine protein phosphatase 2A (PP2A) modulates a broad array of cellular functions. PP2A normally acts as a heterotrimeric holoenzyme complex comprising a catalytic subunit bound by regulatory A and B subunits. Characterization of the regulatory A subunit isoforms (ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 [RCN1], PP2AA2, and PP2AA3) of Arabidopsis thaliana PP2A has shown that RCN1 plays a primary role in controlling root and hypocotyl PP2A activity in seedlings. Here we show that hypocotyl and root growth exhibit different requirements for RCN1-mediated regulation of PP2A activity. Roots of rcn1 mutant seedlings exhibit characteristic abnormalities in cell division patterns at the root apical meristem, as well as reduced growth under ionic, osmotic, and oxidative stress conditions. We constructed chimeric A subunit genes and found that restoration of normal root tip development in rcn1 plants requires both regulatory and coding sequences of RCN1, whereas the hypocotyl elongation defect of rcn1 plants can be complemented by either RCN1 or PP2AA3 transgenes. Furthermore, the RCN1 and PP2AA3 proteins exhibit ubiquitous subcellular localization patterns in seedlings and both associate with membrane compartments. Together, these results show that RCN1-containing PP2A has unique functions that cannot be attributed to isoform-specific expression and localization patterns. Postembryonic RCN1 function is required to maintain normal auxin distribution and stem cell function at the root apex. Our data show that RCN1-regulated phosphatase activity plays a unique role in regulating postembryonic root development and stress response.
High Temperature-Induced Abscisic Acid Biosynthesis and Its Role in the Inhibition of Gibberellin Action in Arabidopsis Seeds
Authors: Toh Shigeo , Imamura Akane , Watanabe Asuka , Nakabayashi Kazumi , Okamoto Masanori , Jikumaru Yusuke , Hanada Atsushi , Aso Yukie , Ishiyama Kanako , Tamura Noriko , Iuchi Satoshi , Kobayashi Masatomo , Yamaguchi Shinjiro , Kamiya Yuji , Nambara Eiji , Kawakami Naoto
Published: December 2007

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.
Silica-supported Ag-Au Bimetallic Nanosystems by XPS
Authors: Barreca Davide , Gasparotto Alberto , Maragno Cinzia , Tondello Eugenio
Published: December 2007

Bimetallic Ag-Au/SiO2 nanosystems were synthesized by sequential deposition of Au and Ag on silica substrates via rf-sputtering. Sample preparation was accomplished at 60 °C from Ar plasmas, using a constant sputtering time for gold (10 min), followed by silver deposition at different times (5-20 min) in order to vary the Ag/Au ratio as well as the total metal content. A proper choice of the synthesis conditions allowed us to obtain a controlled dispersion of silica-supported bimetallic nanoparticles with tailored size and shape, as evidenced by X-ray Photoelectron Spectroscopy (XPS), X-ray Excited Auger Electron Spectroscopy (XE-AES), Transmission Electron Microscopy (TEM), Selected Area Electron Diffraction (SAED), and optical absorption spectroscopy. This work is devoted to the XPS and XE-AES characterization of a representative Ag-Au/SiO2 specimen with an average nanoparticle size of 9 ± 5 nm. In particular, detailed scans for the C 1s, O 1s, Si 2s, Au 4f, Ag 3d, and Ag MNN regions and related data are presented and discussed.
Nanostructured CeO2 Powders by XPS
Authors: Natile Marta Maria , Glisenti Antonella
Published: December 2007

Two different synthetic routes were used to synthesize three nanostructured cerium (IV) oxide powders: Two samples were obtained by precipitation from a basic solution of cerium nitrate and treated at 523 and 923 K, respectively, the third one was prepared by a microwave-assisted heating hydrolysis method and treated at 523 K. Compared with the first method, microwave synthesis has the advantage of very short reaction times and produces small particles with a narrower particle size distribution. All of these advantages can be attributed to the fast and homogeneous microwave heating which gives rise to a rapid and more simultaneous nucleation and ready dissolution of the gel. The present work focuses on the XPS analysis: The influence of the heat treatment and the preparation procedure on the surface properties are deeply investigated. Besides the wide scan spectrum, detailed spectra for the Ce 3d, Ce 4d, O 1s, and C 1s regions and related data are presented and discussed.
La0.6Sr0.4Co0.8Fe0.2O3-δ and Fe2O3/La0.6Sr0.4Co0.8Fe0.2O3-δ Powders: XPS Characterization
Authors: Galenda Alessandro , Natile Marta Maria , Glisenti Antonella
Published: December 2007

Perovskite based oxides have been attracting great interest because of the possible applications in several technological fields such as energy production and gas sensing. The present work focuses on the XPS analysis of three perovskite-based powders: the first is a nanosized La0.6Sr0.4Co0.8Fe0.2O3-δ (LaSrCoFeO) system; the other two are Fe2O3/LaSrCoFeO nanocomposite powders which differ in the amount of deposited Fe2O3. The nanosized LaSrCoFeO perovskite was obtained by the Pechini method and treated at 1173 K for 26 h. Nanocomposite Fe2O3/LaSrCoFeO powder samples (Fe2O3/LaSrCoFeO = 1:9 and 1:1 wt) were obtained by wet impregnation.
Interferon at 50: New Molecules, New Potential, New (and Old) Questions
Authors: Langer Jerome A.
Published: December 2007

Type I interferons (IFNs) are a family of cytokines defined by their antiviral activity but with a broad spectrum of biological activities, including antiproliferative, antitumor, and immunomodulatory effects. Mirroring these activities are diverse therapeutic applications to viral infections, antitumor therapy, and multiple sclerosis. The type I IFNs all signal through a common heterodimeric receptor. The existence of such a large family of cytokines (17 human IFNs) activating a common receptor is unusual. Moreover, the IFNs vary in their relative potency in different assays and are not functionally equivalent. How this functional variation is mediated through a common receptor has not been understood. Reports have now highlighted the interaction of IFNs with the low-affinity receptor subunit IFNAR-1 as a surprising key to their differential activity, particularly regarding antiproliferative and antitumor activities. Two groups have used contrasting approaches to produce variant IFN-α proteins with novel activity profiles. These advances portend enhanced therapeutic possibilities based on the better understanding of IFN-receptor interactions, while raising interesting mechanistic questions.
The Spark of Life: Electricity and Regeneration
Authors: Slack Jonathan M. W.
Published: December 2007

A recent paper shows that a proton pump, the vacuolar-type adenosine triphosphatase (V-ATPase), is necessary for
Xenopus
tadpole tail regeneration. The V-ATPase is synthesized in distal cells of the regeneration bud as an early response to amputation, and its role in regeneration is specifically to pump H
+
out of cells. Although the downstream effects of proton movement are not known, they may be mediated through changes in membrane potential. This work could reopen a consideration of the role of electric fields in regeneration, an issue that has caused much controversy in the past.
Opposing Signals Working Together
Authors: Adler Elizabeth M.
Published: December 2007

Fertilization of the vertebrate eggs elicits a transient increase in the intracellular Ca
2+
concentration, thereby releasing the egg from cell cycle arrest in the second meiotic metaphase (see Jessus and Haccard). Two research groups--Mochida and Hunt and Nishiyama
et al
.--now show that, in addition to the known role of Ca
2+
-calmodulin-dependent protein kinase II (CaMKII), the calcium-dependent phosphatase calcineurin is crucial to this process. In
Xenopus
egg extracts, calcineurin was transiently activated in response to Ca
2+
independently of CaMKII. Its inhibition affected various Ca
2+
-dependent biochemical and morphological phenomena associated with activation, including cyclin destruction, dephosphorylation of phosphoproteins dephosphorylated at the metaphase-interphase transition, decondensation of sperm chromatin, and the formation of a nuclear envelope. Nishiyama
et al
. found that a constitutively active form of calcineurin attenuated the formation of sperm asters and inhibited the migration toward each other of male and female pronuclei in fertilized eggs. Mochida and Hunt found that Ca
2+
elicited a second wave of noncalcineurin-mediated phosphatase activity after the initial transient increase in calcineurin activity, which shut down with the next entry into mitosis in "cycling" extract from activated eggs and reappeared when mitosis was complete. Thus, both studies indicate that the Ca
2+
signal in fertilization independently activates calcineurin, as well as CaMKII, and that these two opposing signals work together to break metaphase arrest, allow the cell cycle to proceed, and initiate embryonic development.

S. Mochida, T. Hunt, Calcineurin is required to release
Xenopus
egg extracts from meiotic M phase.
Nature
449
, 336-340 (2007).
[PubMed]

T. Nishiyama, N. Yoshizaki, T. Kishimoto, K. Ohsumi, Transient activation of calcineurin is essential to initiate embryonic development in
Xenopus laevis
.
Nature
449
, 341-345 (2007).
[PubMed]

C. Jessus, O. Haccard, Calcium's double punch.
Nature
449
, 297-298 (2007).
[PubMed]
Control of a Metamorphic Switch
Authors: Ray L. Bryan
Published: December 2007

When a single hormone--such as the steroid hormone ecdysone in insects--can cause a metamorphic reorganization of the organism from a larva to a pupa or from the pupal stage to the adult form, accidental activation of its signaling could be a catastrophe. Varghese and Cohen present findings showing that a microRNA-mediated feedback loop appears to help form a reliable switch mechanism for activating ecdysone signaling. The authors noted that the
Drosophila
gene encoding the ecdysone receptor contains predicted binding sites for the microRNA
miR-14
. In transfected cells, expression of
miR-14
inhibited expression of a reporter construct containing the ecdysone receptor promoter sequence. Mutant flies lacking
miR-14
showed increased abundance of the ecdysone receptor, and some of the defects in
miR-14
mutants were alleviated in flies in which one functional copy of the ecdysone receptor gene was removed. Thus,
miR-14
appears to limit expression of the ecdysone receptor. Experiments with explanted larval fat bodies indicated that ecdysone increased the abundance of mRNA encoding the ecdysone receptor, an effect that was lost in cells depleted of the ecdysone receptor by RNAi (the primary transcript from the ecdysone receptor gene was not affected by the RNAi construct, so its abundance could be measured to estimate transcriptional activity of the gene). Furthermore, treatment of explants with ecdysone decreases the abundance of
miR-14
, again with a requirement for the ecdysone receptor. The authors interpret these findings in light of the dramatic all-or-none biological effects of ecdysone. The hormone itself participates in a positive feedback loop by promoting transcription of the ecdysone receptor gene. Expression of the ecdysone receptor gene is normally opposed by
miR-14
, but expression of
miR-14
is also subject to repression by ecdysone signals. Thus, noise in the system--in the form of a transient increase in ecdysone receptor expression--may be buffered by the need to overcome
miR-14
-mediated repression of ecdysone signaling. A more sustained activation of ecdysone signaling that lasts long enough for the decay of existing
miR-14
is expected to exceed this threshold and release full activation of the self-sustaining ecdysone transcriptional autoregulatory loop.

J. Varghese, S. M. Cohen, MicroRNA miR-14 acts to modulate a positive autoregulatory loop controlling steroid hormone signaling in
Drosophila
.
Genes Dev.
21
, 2277-2282 (2007).
[Abstract]
[Full Text]
Tag Team
Authors: Foley John F.
Published: December 2007

Ubiquitin and ubiquitin-like proteins (Ubls), including small ubiquitin-like modifier (SUMO), are covalently attached to target proteins through a coordinated series of reactions. These posttranslational modifications are important in regulating many signaling pathways and cellular processes, including DNA repair and maintenance of chromosome structure. Whereas ubiquitination is often associated with targeting proteins for degradation, SUMOylation enables new protein-protein interactions to occur and was thought to perhaps increase protein stability by blocking ubiquitination. To the contrary, however, two papers now identify a family of eukaryotic ubiquitin ligases that target SUMOylated proteins and proteins containing SUMO-like domains (SLDs). Prudden
et al
. used yeast two-hybrid studies to identify Rfp1 [really interesting new gene (RING) finger protein 1] and Rfp2 and found that both proteins formed heterodimers with another RING finger protein, the ubiquitin E3 ligase, Slx8. Sequence analysis showed that Rfp1 and Rfp2 also contained N-terminal SUMO-interacting motifs (SIMs). Yeast deficient in both Rfp1 and Rfp2 exhibited poor cell growth and had fragmented chromosomes, a phenotype similar to that seen in Slx8-deficient yeast. Cells deficient in Slx8-Rfp showed genomic instability, accumulated SUMOylated proteins, and had defects in DNA repair. Expression of the mouse Rfp ortholog, RNF4, rescued this phenotype. Further, the yeast DNA repair factor Rad60, which contains an SLD, was a target for Slx8-Rfp. Sun
et al
. showed that whereas the Rfps must recruit Slx8 in order to have ubiquitin ligase activity, RNF4 is itself a ubiquitin ligase that targets SUMOylated proteins through its SIM, consistent with the previous study. In vitro ubiquitin ligase assays showed that target proteins were ubiquitinated in a SUMO binding-dependent fashion. The discovery of ubiquitin ligases that target SUMOylated proteins provides a link between these two important pathways of posttranslational modification.

J. Prudden, S. Pebernard, G. Raffa, D. A. Slavin, J. J. P. Perry, J. A. Tainer, C. H. McGowan, M. N. Boddy, SUMO-targeted ubiquitin ligases in genome stability.
EMBO J.
26
, 4089-4101 (2007).
[PubMed]

H. Sun, J. D. Leverson, T. Hunter, Conserved function of RNF4 family proteins in eukaryotes: Targeting a ubiquitin ligase to SUMOylated proteins.
EMBO J.
26
, 4102-4112 (2007).
[PubMed]
Gating ASICs
Authors: Adler Elizabeth M.
Published: December 2007

Acid-sensing ion channels (ASICs) are members of a family of cation-selective ion channels that includes epithelial sodium channels and the degenerins, which shows a remarkable diversity of function and in the mechanisms of activation (see Canessa). Implicated in sensory perception, ASICs are sodium-selective and activated by extracellular protons independently of membrane potential. Jasti
et al
. combined crystallographic analysis with electrophysiology to explore the low-pH structure of ΔASIC1, a chicken ASIC deletion mutant (in a desensitized nonconducting state), and investigated the mechanism underlying its pH-dependent gating. Unexpectedly, they found that ΔASIC1 formed a homotrimer. Each subunit had two transmembrane helices and a large extracellular domain. Describing the overall domain structure of ΔASIC1 subunits as resembling an "upright forearm and clenched hand" (with the extracellular domain constituting the "hand"), the authors identified a group of acidic residues as proton-binding sites that constitute the pH sensor. They suggest that proton binding leads to movement of the disulfide-rich thumb region, which transmits a long-range conformational change to allow the channel to open.

J. Jasti, H. Furukawa, E. B. Gonzales, E. Gouaux, Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH.
Nature
449
, 316-323 (2007).
[PubMed]

C. M. Canessa, Unexpected opening.
Nature
449
, 293-294 (2007).
[PubMed]
Transporting Zinc
Authors: Chin Gilbert
Published: December 2007

The divalent zinc cation is found in multiple guises in prokaryotic and eukaryotic cells. Lu and Fu (see the Perspective by Nies) present the 3.8 angstrom structure of YiiP, a bacterial membrane protein that imports Zn
2+
by exchanging it for H
+
. The structure of YiiP is a Y-shaped homodimer, through which a presumptive pathway for Zn
2+
/H
+
exchange can be discerned. YiiP is a member of the cation diffusion facilitator family of transport proteins. Another family member, ZnT-8, is expressed solely in pancreatic β cells and has recently been associated with risk for type 2 diabetes.

M. Lu, D. Fu, Structure of the zinc transporter YiiP.
Science
317
, 1746-1748 (2007).
[Abstract]
[Full Text]

D. H. Nies, How cells control zinc homeostasis.
Science
317
, 1695-1696 (2007).
[Summary]
[Full Text]
Moving Through an Electrical Field
Authors: Gough Nancy R.
Published: December 2007

Breast cancer cells tend to first move into the lumen of the mammary ducts, where they proliferate. Then, after tissue deformation occurs, the cells breach the basement membrane and metastasize through the lymph and blood vessels. Pu
et al
. propose that the transepithelial potential, with the duct lumen having a relatively positive charge, creates an endogenous electric field that contributes to the movement of cancer cells. The authors used electric fields that were smaller than those that should exist across the breast duct epithelium based on the transepithelial potential to test whether breast cancer cell lines responded to the presence of such an electric field. The authors noted a correlation between the metastatic potential of human and rat mammary cancer cell lines and directional migration in the presence of an electric field (the cells moved toward the anode). The abundance of the epidermal growth factor receptor ErbB1 also correlated with directed migration in an electric field, and inhibition of ErbB1 with AG1478 profoundly reduced electric field-induced directed migration. Overexpression of ErbB1, ErbB2, or ErbB3 increased directed migration of two mammary tumor cell lines in response to an electric field. Pharmacological inhibitors were used to explore the signaling pathway responsible for migration, and tyrosine kinase activity, phosphatidylinositol 3-kinase, RhoA, Cdc42, and extracellular signal-regulated kinase (ERK) were all involved.

J. Pu, C. D. McCaig, L. Cao, Z. Zhao, J. E. Segall, M. Zhao, EGF receptor signaling is essential for electric-field-directed migration of breast cancer cells.
J. Cell Sci.
120
, 3395-3403 (2007).
[Abstract]
[Full Text]
Gene-Hormone-Environment Interactions in the Regulation of Aggressive Responses: Elegant Analysis of Complex Behavior
Authors: Pfaff Donald , Silver Rae
Published: December 2007

Photoperiodic influences on the relations between levels of a steroid hormone and aggressive behavior add a layer of complexity to our ideas about how genes influence behavior. The effects of estrogens on aggression are modulated by day length and involve both genomic and nongenomic routes of hormone action.