bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch
Authors: Lee Stephanie A. , Eyeson Rosemary , Cheever Matthew L. , Geng Jinming , Verkhusha Vladislav V. , Burd Christopher , Overduin Michael , Kutateladze Tatiana G.
Published: September 2005

Specific recognition of phosphatidylinositol 3-phosphate [PtdIns(3)P] by the FYVE domain targets cytosolic proteins to endosomal membranes during key signaling and trafficking events within eukaryotic cells. Here, we show that this membrane targeting is regulated by the acidic cellular environment. Lowering the cytosolic pH enhances PtdIns(3)P affinity of the FYVE domain, reinforcing the anchoring of early endosome antigen 1 (EEA1) to endosomal membranes. Reversibly, increasing the pH disrupts phosphoinositide binding and leads to cytoplasmic redistribution of EEA1. pH dependency is due to a pair of conserved His residues, the successive protonation of which is required for PtdIns(3)P head group recognition as revealed by NMR. Substitution of the His residues abolishes PtdIns(3)P binding by the FYVE domain
in vitro
and
in vivo
. Another PtdIns(3)P-binding module, the PX domain of Vam7 and p40
phox
is shown to be pH-independent. This provides the fundamental functional distinction between the two phosphoinositide-recognizing domains. The presented mode of FYVE regulation establishes the unique function of FYVE proteins as low pH sensors of PtdIns(3)P and reveals the critical role of the histidine switch in targeting of these proteins to endosomal membranes.
Crystal structure of human arginase I at 1.29-Å resolution and exploration of inhibition in the immune response
Authors: Di Costanzo Luigi , Sabio Guadalupe , Mora Alfonso , Rodriguez Paulo C. , Ochoa Augusto C. , Centeno Francisco , Christianson David W.
Published: September 2005

Human arginase I is a potential target for therapeutic intervention in diseases linked to compromisedl-arginine homeostasis. Here, we report high-affinity binding of the reaction coordinate analogue inhibitors 2(S)-amino-6-boronohexanoic acid (ABH,Kd= 5 nM) andS-(2-boronoethyl)-l-cysteine (BEC,Kd= 270 nM) to human arginase I, and we report x-ray crystal structures of the respective enzyme–inhibitor complexes at 1.29- and 1.94-Å resolution determined from crystals twinned by hemihedry. The ultrahigh-resolution structure of the human arginase I–ABH complex yields an unprecedented view of the binuclear manganese cluster and illuminates the structural basis for nanomolar affinity: bidentate inner-sphere boronate–manganese coordination interactions and fully saturated hydrogen bond networks with inhibitor α-amino and α-carboxylate groups. These interactions are therefore implicated in the stabilization of the transition state forl-arginine hydrolysis. Electron density maps also reveal that active-site residue H141 is protonated as the imidazolium cation. The location of H141 is such that it could function as a general acid to protonate the leaving amino group ofl-ornithine during catalysis, and this is a revised mechanistic proposal for arginase. This work serves as a foundation for studying the structural and chemical biology of arginase I in the immune response, and we demonstrate the inhibition of arginase activity by ABH in human and murine myeloid cells.
A model of antibiotic-resistant bacterial epidemics in hospitals
Authors: Webb Glenn F. , D'Agata Erika M. C. , Magal Pierre , Ruan Shigui
Published: September 2005

The emergence of drug-resistant strains of bacteria is an increasing threat to society, especially in hospital settings. Many antibiotics that were formerly effective in combating bacterial infections in hospital patients are no longer effective because of the evolution of resistant strains, which compromises medical care worldwide. In this article, we formulate a two-level population model to quantify key elements in nosocomial (hospital-acquired) infections. At the bacteria level, patients infected with these strains generate both nonresistant and resistant bacteria. At the patient level, susceptible patients are infected by infected patients at rates proportional to the total bacteria load of each strain present in the hospital. The objectives of this paper are to analyze the dynamic elements of nonresistant and resistant bacteria strains in epidemic populations in hospital environments and to provide understanding of measures to avoid the endemicity of resistant antibiotic strains.
A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM
Authors: Sun Yingli , Jiang Xiaofeng , Chen Shujuan , Fernandes Norvin , Price Brendan D.
Published: September 2005

The ataxia telangiectasia mutant (ATM) protein kinase regulates the cell's response to DNA damage through the phosphorylation of proteins involved in cell-cycle checkpoints and DNA repair. However, the signal-transduction pathway linking DNA strand breaks to activation of ATM's kinase activity is not clearly defined. Here, we demonstrate that DNA damage induces the rapid acetylation of ATM. This acetylation depends on the Tip60 histone acetyltransferase (HAT). Suppression of Tip60 blocks the activation of ATM's kinase activity and prevents the ATM-dependent phosphorylation of p53 and chk2. Further, inactivation of Tip60 sensitizes cells to ionizing radiation. ATM forms a stable complex with Tip60 through the conserved FATC domain of ATM. The interaction between ATM and Tip60 is not regulated in response to DNA damage. Instead, the HAT activity of the ATM–Tip60 complex is specifically activated by DNA damage. Furthermore, this activation of Tip60 by DNA damage and the recruitment of the ATM–Tip60 complex to sites of DNA damage is independent of ATM's kinase activity. The results demonstrate that the Tip60 HAT plays a key role in the activation of ATM's kinase activity in response to DNA damage.
Neural circuitry underlying the interaction between emotion and asthma symptom exacerbation
Authors: Rosenkranz Melissa A. , Busse William W. , Johnstone Tom , Swenson Cheri A. , Crisafi Gina M. , Jackson Maryjo M. , Bosch Jos A. , Sheridan John F. , Davidson Richard J.
Published: September 2005

Asthma, like many inflammatory disorders, is affected by psychological stress, suggesting that reciprocal modulation may occur between peripheral factors regulating inflammation and central neural circuitry underlying emotion and stress reactivity. Despite suggestions that emotional factors may modulate processes of inflammation in asthma and, conversely, that peripheral inflammatory signals influence the brain, the neural circuitry involved remains elusive. Here we show, using functional magnetic resonance imaging, that activity in the anterior cingulate cortex and insula to asthma-relevant emotional, compared with valence-neutral stimuli, is associated with markers of inflammation and airway obstruction in asthmatic subjects exposed to antigen. This activation accounts for ≥40% of the variance in the peripheral markers and suggests a neural basis for emotion-induced modulation of airway disease in asthma. The anterior cingulate cortex and insula have been implicated in the affective evaluation of sensory stimulation, regulation of homeostatic responses, and visceral perception. In individuals with asthma and other stress-related conditions, these brain regions may be hyperresponsive to disease-specific emotional and afferent physiological signals, which may contribute to the dysregulation of peripheral processes, such as inflammation.
Significance analysis of time course microarray experiments
Authors: Storey John D. , Xiao Wenzhong , Leek Jeffrey T. , Tompkins Ronald G. , Davis Ronald W.
Published: September 2005

Characterizing the genome-wide dynamic regulation of gene expression is important and will be of much interest in the future. However, there is currently no established method for identifying differentially expressed genes in a time course study. Here we propose a significance method for analyzing time course microarray studies that can be applied to the typical types of comparisons and sampling schemes. This method is applied to two studies on humans. In one study, genes are identified that show differential expression over time in response toin vivoendotoxin administration. By using our method, 7,409 genes are called significant at a 1% false-discovery rate level, whereas several existing approaches fail to identify any genes. In another study, 417 genes are identified at a 10% false-discovery rate level that show expression changing with age in the kidney cortex. Here it is also shown that as many as 47% of the genes change with age in a manner more complex than simple exponential growth or decay. The methodology proposed here has been implemented in the freely distributed and open-sourceedgesoftware package.
Deficiency of Bruton's tyrosine kinase in B cell precursor leukemia cells
Authors: Feldhahn Niklas , Río Paula , Soh Bonaventure Ndikung Bejeng , Liedtke Stefanie , Sprangers Mieke , Klein Florian , Wernet Peter , Jumaa Hassan , Hofmann Wolf-Karsten , Hanenberg Helmut , Rowley Janet D. , Müschen Markus
Published: September 2005

Bruton's tyrosine kinase (BTK) deficiency results in a differentiation block at the pre-B cell stage. Likewise, acute lymphoblastic leukemia cells are typically arrested at early stages of B cell development. We therefore investigated BTK function in B cell precursor leukemia cells carrying a
BCR-ABL1, E2A-PBX1, MLL-AF4, TEL-AML1
, or
TEL-PDGFRB
gene rearrangement. Although somatic mutations of the
BTK
gene are rare in B cell precursor leukemia cells, we identified kinase-deficient splice variants of BTK throughout all leukemia subtypes. Unlike infant leukemia cells carrying an
MLL-AF4
gene rearrangement, where expression of full-length BTK was detectable in only four of eight primary cases, in leukemia cells harboring other fusion genes full-length BTK was typically coexpressed with kinase-deficient variants. As shown by overexpression experiments, kinase-deficient splice variants can act as a dominant-negative BTK in that they suppress BTK-dependent differentiation and pre-B cell receptor responsiveness of the leukemia cells. On the other hand, induced expression of full-length BTK rendered the leukemia cells particularly sensitive to apoptosis. Comparing BTK expression in surviving or preapoptotic leukemia cells after 10-Gy γ radiation, we observed selective survival of leukemia cells that exhibit expression of dominant-negative BTK forms. These findings indicate that lack of BTK expression or expression of dominant-negative splice variants in B cell precursor leukemia cells can (
i
) inhibit differentiation beyond the pre-B cell stage and (
ii
) protect from radiation-induced apoptosis.
Fine functional organization of auditory cortex revealed by Fourier optical imaging
Authors: Kalatsky Valery A. , Polley Daniel B. , Merzenich Michael M. , Schreiner Christoph E. , Stryker Michael P.
Published: September 2005

We provide an overall view of the functional tonotopic organization of the auditory cortex in the rat. We apply a recently developed technique for acquiring intrinsic signal optical maps, Fourier imaging, in the rat auditory cortex. These highly detailed maps, derived in a several-minute-long recording procedure, delineate multiple auditory cortical areas and demonstrate their shapes, sizes, and tonotopic order. Beyond the primary auditory cortex, there are at least three distinct areas with fine-scale tonotopic organization, as well as at least one additional high-frequency field. The arrangement of all of these cortical areas is consistent across subjects. The accuracy of these optical maps was confirmed by microelectrode mapping in the same subjects. This imaging method allows fast mapping of the auditory cortex at high spatial resolution comparable to that provided by conventional microelectrode technique. Although spiking activity is largely responsible for the evoked intrinsic signals, certain features of the optical signal cannot be explained by spiking activity only, and should probably be attributed to other mechanisms inducing metabolic activity, such as subthreshold membrane phenomena.
Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation
Authors: Iwata Atsushi , Christianson John C. , Bucci Mirella , Ellerby Lisa M. , Nukina Nobuyuki , Forno Lysia S. , Kopito Ron R.
Published: September 2005

CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous aggregates in mouse models of polyglutamine disease, but the cellular mechanism underlying this phenomenon is unknown. Here, we show that autophagy is essential for the elimination of aggregated forms of mutant huntingtin and ataxin-1 from the cytoplasmic but not nuclear compartments. Human orthologs of yeast autophagy genes, molecular determinants of autophagic vacuole formation, are recruited to cytoplasmic but not nuclear inclusion bodies
in vitro
and
in vivo
. These data indicate that autophagy is a critical component of the cellular clearance of toxic protein aggregates and may help to explain why protein aggregates are more toxic when directed to the nucleus.
Receptor-specific requirements for anthrax toxin delivery into cells
Authors: Rainey G. Jonah A. , Wigelsworth Darran J. , Ryan Patricia L. , Scobie Heather M. , Collier R. John , Young John A. T.
Published: September 2005

The three proteins that constitute anthrax toxin self-assemble into toxic complexes after one of these proteins, protective antigen (PA), binds to tumor endothelial marker 8 (TEM8) or capillary morphogenesis protein 2 (CMG2) cellular receptors. The toxin receptor complexes are internalized, and acidic endosomal pH triggers pore formation by PA and translocation of the catalytic subunits into the cytosol. In this study we show that the pH threshold for conversion of the PA prepore to the pore and for translocation differs by approximately a pH unit, depending on whether the TEM8 or CMG2 receptor is used. For TEM8-associated toxin, these events can occur at close to neutral pH values, and they show relatively low sensitivity to ammonium chloride treatment in cells. In contrast, with CMG2-associated toxin, these events require more acidic conditions and are highly sensitive to ammonium chloride. We show, furthermore, that PA dissociates from TEM8 and CMG2 upon pore formation. Our results are consistent with a model in which translocation depends on pore formation and pore formation, in turn, depends on release of PA from its receptor. We propose that because PA binds to CMG2 with much higher affinity than it does to TEM8, a lower pH is needed to attenuate CMG2 binding to allow pore formation. Our results suggest that toxin can form pores at different points in the endocytic pathway, depending on which receptor is used for entry.
RBR3, a member of the retinoblastoma-related family from maize, is regulated by the RBR1/E2F pathway
Authors: Sabelli Paolo A. , Dante Ricardo A. , Leiva-Neto João T. , Jung Rudolf , Gordon-Kamm William J. , Larkins Brian A.
Published: September 2005

Retinoblastoma-related (RBR) proteins regulate cell division in higher eukaryotes by controlling the adenovirus E2 promoter binding factor (E2F)/dimerization partner (DP) family of transcription factors that regulate expression of many genes involved in cell-cycle progression. We identified a previously undescribed member of the maize RBR family, RBR3, which has the characteristic structure and binding activities of pocket proteins, where interaction depends on a LxCxE motif in the partner proteins and a critical cysteine within the B pocket domain. Like other RBR proteins, RBR3 appears to be regulated by phosphorylation mediated by cyclin-dependent kinases. During endosperm development, RBR3 expression is restricted to the mitotic stage preceding the onset of endoreduplication. This finding suggests a role distinct from RBR1, which is constitutively expressed. Two sites in the RBR3 promoter bind to complexes containing maize E2F1 and DP proteins. Expression of wheat dwarf virus RepA protein, which blocks RBR1 activity and stimulates cell proliferation, dramatically up-regulates RBR3, but not RBR1, RNA in embryogenic maize calli. The results indicate that RBR3 expression is controlled by RBR1 through the activity of E2F/DP and that RBR3 is the maize equivalent of mammalian p107. Furthermore, maize and related grasses might have evolved a compensatory mechanism among distinct types of RBR proteins to ensure robust control of pocket protein activity.
Knockdown of
Pu.1
by small interfering RNA in CD34
+
embryoid body cells derived from mouse ES cells turns cell fate determination to pro-B cells
Authors: Zou Gang-Ming , Chen Jian-Jun , Yoder Mervin C. , Wu Wei , Rowley Janet D.
Published: September 2005

The factors that regulate murine ES cell-derived hematopoietic progenitor cell (HPC) commitment to the B lymphocyte lineage remain unclear.
Pu.1
plays an essential role in the development of all lymphoid lineages; however, it also regulates commitment to other blood cell lineages. In this study, we found evidence for early B cell lineage commitment as determined by coexpression of CD19 and CD45R (B220) when
Pu.1
expression was knocked down in HPC by specific small interfering RNA (siRNA); moreover, the expression of early B cell factor (Ebf) and paired box protein 5 (Pax-5) transcription factors was induced when cells were treated by
Pu.1
siRNA, but not by control siRNA. We also found that siRNA-mediated knockdown of
Pu.1
expression was more efficient in generating progenitor B cells (pro-B cells) compared with the more common
in vitro
method of B lymphoid development by means of coculture of CD34
+
embryoid body (EB) cells with OP9 stromal cells. To investigate whether this phenomenon also exists in HPC from other sources, we then knocked down
Pu.1
gene expression in CD34
+
murine bone marrow cells and found a similar effect of increased production of CD19
+
CD43
+
CD45R
+
progenitor B cells upon the siRNA-mediated decrease in
Pu.1
expression. We conclude that, in early B cell development from ES cell-derived HPC, constitutive
Pu.1
expression inhibits the earliest B cell development through repressing early B cell factor and paired box protein 5 expression, although lower levels of
Pu.1
expression in HPC play a key role in promoting B cell fate determination.
An expression signature for p53 status in human breast cancer predicts mutation status, transcriptional effects, and patient survival
Authors: Miller Lance D. , Smeds Johanna , George Joshy , Vega Vinsensius B. , Vergara Liza , Ploner Alexander , Pawitan Yudi , Hall Per , Klaar Sigrid , Liu Edison T. , Bergh Jonas
Published: September 2005

Perturbations of the p53 pathway are associated with more aggressive and therapeutically refractory tumors. However, molecular assessment of p53 status, by using sequence analysis and immunohistochemistry, are incomplete assessors of p53 functional effects. We posited that the transcriptional fingerprint is a more definitive downstream indicator of p53 function. Herein, we analyzed transcript profiles of 251 p53-sequenced primary breast tumors and identified a clinically embedded 32-gene expression signature that distinguishes p53-mutant and wild-type tumors of different histologies and outperforms sequence-based assessments of p53 in predicting prognosis and therapeutic response. Moreover, the p53 signature identified a subset of aggressive tumors absent of sequence mutations in p53 yet exhibiting expression characteristics consistent with p53 deficiency because of attenuated p53 transcript levels. Our results show the primary importance of p53 functional status in predicting clinical breast cancer behavior.
Orthogonal arrangement of rhythm-generating microcircuits in the hippocampus
Authors: Gloveli Tengis , Dugladze Tamar , Rotstein Horacio G. , Traub Roger D. , Monyer Hannah , Heinemann Uwe , Whittington Miles A. , Kopell Nancy J.
Published: September 2005

As a structure involved in learning and memory, the hippocampus functions as a network. The functional differentiation along the longitudinal axis of the hippocampus is poorly demarcated in comparison with the transverse axis. Using patch clamp recordings in conjunction with post hoc anatomy, we have examined the pattern of connectivity and the functional differentiation along the long axis of the hippocampus. Here, we provide anatomical and physiological evidence that the prominent rhythmic network activities of the hippocampus, the behavior-specific gamma and theta oscillations, are seen predominantly along the transverse and longitudinal axes respectively. This orthogonal relationship is the result of the axonal field trajectories and the consequential interaction of the principal cells and major interneuron subtypes involved in generating each rhythm. Thus, the axonal arborization patterns of hippocampal inhibitory cells may represent a structural framework for the spatiotemporal distribution of activity observed within the hippocampus.
A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells
Authors: Stegmeier Frank , Hu Guang , Rickles Richard J. , Hannon Gregory J. , Elledge Stephen J.
Published: September 2005

The advent of RNA interference has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. The expression of short hairpin RNA (shRNA) from polymerase III promoters can be encoded in transgenes and used to produce small interfering RNAs that down-regulate specific genes. In this study, we show that polymerase II-transcribed shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a microRNA context. Importantly, our shRNA expression system [called PRIME (potent RNA interference using microRNA
e
xpression) vectors] allows for the multicistronic cotranscription of a reporter gene, thereby facilitating the tracking of shRNA production in individual cells. Based on this system, we developed a series of lentiviral vectors that display tetracycline-responsive knockdown of gene expression at single copy. The high penetrance of these vectors will facilitate genomewide loss-of-function screens and is an important step toward using bar-coding strategies to follow loss of specific sequences in complex populations.
Structure of stochastic dynamics near fixed points
Authors: Kwon Chulan , Ao Ping , Thouless David J.
Published: September 2005

We analyze the structure of stochastic dynamics near either a stable or unstable fixed point, where the force can be approximated by linearization. We find that a cost function that determines a Boltzmann-like stationary distribution can always be defined near it. Such a stationary distribution does not need to satisfy the usual detailed balance condition but might have instead a divergence-free probability current. In the linear case, the force can be split into two parts, one of which gives detailed balance with the diffusive motion, whereas the other induces cyclic motion on surfaces of constant cost function. By using the Jordan transformation for the force matrix, we find an explicit construction of the cost function. We discuss singularities of the transformation and their consequences for the stationary distribution. This Boltzmann-like distribution may be not unique, and nonlinear effects and boundary conditions may change the distribution and induce additional currents even in the neighborhood of a fixed point.
The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin
Authors: Kudryashov Dmitry S. , Sawaya Michael R. , Adisetiyo Helty , Norcross Todd , Hegyi György , Reisler Emil , Yeates Todd O.
Published: September 2005

The 2.5-Å resolution crystal structure is reported for an actin dimer, composed of two protomers cross-linked along the longitudinal (or vertical) direction of the F-actin filament. The crystal structure provides an atomic resolution view of a molecular interface between actin protomers, which we argue represents a near-native interaction in the F-actin filament. The interaction involves subdomains 3 and 4 from distinct protomers. The atomic positions in the interface visualized differ by 5–10 Å from those suggested by previous models of F-actin. Such differences fall within the range of uncertainties allowed by the fiber diffraction and electron microscopy methods on which previous models have been based. In the crystal, the translational arrangement of protomers lacks the slow twist found in native filaments. A plausible model of F-actin can be constructed by reintroducing the known filament twist, without disturbing significantly the interface observed in the actin dimer crystal.
Marine phosphate oxygen isotopes and organic matter remineralization in the oceans
Authors: Colman Albert S. , Blake Ruth E. , Karl David M. , Fogel Marilyn L. , Turekian Karl K.
Published: September 2005

We show that the isotopic composition of oxygen (δ
18
O) in dissolved inorganic phosphate (P
i
) reveals the balance between P
i
transport and biological turnover rates in marine ecosystems. Our δ
18
Op of P
i
(δ
18
Op) measurements herein indicate the importance of cell lysis in the regeneration of P
i
in the euphotic zone. Depth profiles of the δ
18
Op in the Atlantic and Pacific Oceans are near a temperature-dependent isotopic equilibrium with water. Small deviations from equilibrium below the thermocline suggest that P remineralization in the deep ocean is a byproduct of microbial carbon and energy requirements. However, isotope effects associated with phosphohydrolase enzymes involved in P remineralization are quite large and could potentially lead to significant disequilibration of P
i
oxygen. The observed near equilibration of deep water P
i
likely calls for continued slow rates of microbial uptake and release of P
i
and/or extracellular pyrophosphatase-mediated oxygen exchange between water and P
i
along the deep water flow path.
Crystal structure of the GTPase domain of rat dynamin 1
Authors: Reubold Thomas F. , Eschenburg Susanne , Becker Andreas , Leonard Marilyn , Schmid Sandra L. , Vallee Richard B. , Kull F. Jon , Manstein Dietmar J.
Published: September 2005

Here, we present the 1.9-Å crystal structure of the nucleotide-free GTPase domain of dynamin 1 from
Rattus norvegicus
. The structure corresponds to an extended form of the canonical GTPase fold observed in Ras proteins. Both nucleotide-binding switch motifs are well resolved, adopting conformations that closely resemble a GTP-bound state not previously observed for nucleotide-free GTPases. Two highly conserved arginines, Arg-66 and Arg-67, greatly restrict the mobility of switch I and are ideally positioned to relay information about the nucleotide state to other parts of the protein. Our results support a model in which switch I residue Arg-59 gates GTP binding in an assembly-dependent manner and the GTPase effector domain functions as an assembly-dependent GTPase activating protein in the fashion of RGS-type GAPs.
Role of PDZK1 in membrane expression of renal brush border ion exchangers
Authors: Thomson R. Brent , Wang Tong , Thomson Benjamin R. , Tarrats Luis , Girardi Adriana , Mentone SueAnn , Soleimani Manoocher , Kocher Olivier , Aronson Peter S.
Published: September 2005

Na-H exchanger NHE3 and Cl-anion exchanger CFEX (SLC26A6, PAT1) play principal roles in the reabsorption of Na and Cl in the proximal tubule of the mammalian kidney. The mechanisms by which NHE3 and CFEX are localized to and maintained in the brush border of the proximal tubule are largely unknown. To investigate the possible interaction of NHE3 and CFEX with the PDZ-domain-containing scaffolding protein PDZK1, we performed a series of
in vitro
interaction assays with GST-fusion proteins and native brush border membrane proteins. These studies demonstrated that, not only were NHE3 and CFEX capable of directly interacting with PDZK1, but that this interaction was mediated through their C-terminal PDZ-interaction sites. To determine whether PDZK1 interaction is essential for brush border localization of NHE3 and CFEX
in vivo
, we examined the expression of NHE3 and CFEX in kidneys of wild-type and PDZK1-null mutant mice by both Western analysis and immunocytochemistry. These studies indicated that, although brush border expression of NHE3 was unaffected by the loss of PDZK1, the expression of CFEX was markedly reduced. Finally, we assayed CFEX functional activity as Cl–oxalate exchange in brush border membrane vesicles and oxalate-stimulated volume absorption in microperfused proximal tubules. Consistent with the observed decrease in CFEX protein expression, both measures of CFEX functional activity were dramatically reduced in PDZK1-null animals. In conclusion, the scaffolding protein PDZK1 is essential for the normal expression and function of Cl-anion exchanger CFEX in the proximal tubule of the mammalian kidney.
Transcriptional regulation of phagocytosis-induced membrane biogenesis by sterol regulatory element binding proteins
Authors: Castoreno Adam B. , Wang Yan , Stockinger Walter , Jarzylo Larissa A. , Du Hong , Pagnon Joanne C. , Shieh Eugenie C. , Nohturfft Axel
Published: September 2005

In the process of membrane biogenesis several dozen proteins must operate in precise concert to generate ≈100 lipids at appropriate concentrations. To study the regulation of bilayer assembly in a cell cycle-independent manner, we have exploited the fact that phagocytes replenish membranes expended during particle engulfment in a rapid phase of lipid synthesis. In response to phagocytosis of latex beads, human embryonic kidney 293 cells synthesized cholesterol and phospholipids at amounts equivalent to the surface area of the internalized particles. Lipid synthesis was accompanied by increased transcription of several lipogenic proteins, including the low-density lipoprotein receptor, enzymes required for cholesterol synthesis (3-hydroxy-3-methylglutaryl CoA synthase, 3-hydroxy-3-methylglutaryl CoA reductase), and fatty acid synthase. Phagocytosis triggered the proteolytic activation of two lipogenic transcription factors, sterol regulatory element binding protein-1a (SREBP-1a) and SREBP-2. Proteolysis of SREBPs coincided with the appearance of their transcriptionally active N termini in the nucleus and 3-fold activation of an SREBP-specific reporter gene. In previous studies with cultured cells, proteolytic activation of SREBP-1a and SREBP-2 has been observed in response to selective starvation of cells for cholesterol and unsaturated fatty acids. However, under the current conditions, SREBP-1a and SREBP-2 are induced without lipid deprivation. SREBP activation is inhibited by high levels of the SREBP-interacting proteins Insig1 or the cytosolic domain of SREBP cleavage-activating protein. Upon overexpression of these proteins, phagocytosis-induced transcription and lipid synthesis were blocked. These results identify SREBPs as essential regulators of membrane biogenesis and provide a useful system for further studies on membrane homeostasis.
ETHY. A Theory of Fruit Climacteric Ethylene Emission
Authors: Génard Michel , Gouble Barbara
Published: September 2005

A theory of fruit climacteric ethylene emission was developed and used as the basis of a simulation model called ETHY. According to the theory, the biosynthetic pathway of ethylene is supplied by ATP and is regulated by 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. The conjugation of ACC with malonate to form MACC was taken into account as a way to decrease the availability of ACC. Because of the seasonal increase of fruit volume, the dilution of biochemical compounds used in ETHY was taken into account. Finally, the ethylene diffusion across the skin was considered. The theory took into account the effect of temperature and O2 and CO2 internal concentrations on ethylene. The model was applied to peach (Prunus persica) fruit over 3 years, several leaf:fruit ratios, and irrigation conditions. An adequate ethylene increase was predicted without considering any increase in respiration during the ripening period, which suggests that the respiratory climacteric may not be required for ripening. Another important result of this study is the high sensitivity of ETHY to the parameters involved in the calculation of ACC oxidase and ACC synthase activities, ATP production, and skin surface and permeability. ETHY was also highly sensitive to changes in fruit growth and temperature.
ThewpMutation ofGlycine maxCarries a Gene-Fragment-Rich Transposon of the CACTA Superfamilyw⃞
Authors: Zabala Gracia , Vodkin Lila O.
Published: September 2005

We used soybean (Glycine max) cDNA microarrays to identify candidate genes for a stable mutation at the Wp locus in soybean, which changed a purple-flowered phenotype to pink, and found that flavanone 3-hydroxylase cDNAs were overexpressed in purple flower buds relative to the pink. Restriction fragment length polymorphism analysis and RNA gel blots of purple and pink flower isolines, as well as the presence of a 5.7-kb transposon insertion in the wp mutant allele, have unequivocally shown that flavanone 3-hydroxylase gene 1 is the Wp locus. Moreover, the 5.7-kb insertion in wp represents a novel transposable element (termed Tgm-Express1) with inverted repeats closely related to those of other Tgms (transposable-like elements, G. max) but distinct in several characteristics, including the lack of subterminal inverted repeats. More significantly, Tgm-Express1 contains four truncated cellular genes from the soybean genome, resembling the Pack-MULEs (Mutator-like transposable elements) found in maize (Zea mays), rice (Oryza sativa), and Arabidopsis thaliana and the Helitrons of maize. The presence of the Tgm-Express1 element causing the wp mutation, as well as a second Tgm-Express2 element elsewhere in the soybean genome, extends the ability to acquire and transport host DNA segments to the CACTA family of elements, which includes both Tgm and the prototypical maize Spm/En.
RiceUndeveloped Tapetum1Is a Major Regulator of Early Tapetum Development
Authors: Jung Ki-Hong , Han Min-Jung , Lee Yang-Seok , Kim Yong-Woo , Hwang Inhwan , Kim Min-Jeong , Kim Yeon-Ki , Nahm Baek Hie , An Gynheung
Published: September 2005

The tapetum, the innermost of four sporophytic layers in the anther wall, comes in direct contact with the developing male gametophyte and is thought to play a crucial role in the development and maturation of microspores. Here, we report the identification of rice (Oryza sativa) Undeveloped Tapetum1 (Udt1), which is required for the differentiation of secondary parietal cells to mature tapetal cells. T-DNA or retrotransposon Tos17 insertions in the Udt1 gene caused male sterility. The anther walls and meiocytes of the mutants were normal during the early premeiosis stage, but their tapeta failed to differentiate and became vacuolated during the meiotic stage. In addition, meiocytes did not develop to microspores, and middle layer degeneration was inhibited. Consequently, the anther locules contained no pollen. The UDT1:green fluorescent protein fusion protein was localized to the nucleus. This, together with its homology with other basic helix-loop-helix proteins, suggests that UDT1 is a transcription factor. DNA microarray analysis identified 958 downregulated and 267 upregulated genes in the udt1-1 anthers, suggesting that Udt1 plays a major role in maintaining tapetum development, starting in early meiosis.
RNA Immunoprecipitation and Microarray Analysis Show a Chloroplast Pentatricopeptide Repeat Protein to Be Associated with the 5′ Region of mRNAs Whose Translation It Activates
Authors: Schmitz-Linneweber Christian , Williams-Carrier Rosalind , Barkan Alice
Published: September 2005

Plant nuclear genomes encode hundreds of predicted organellar RNA binding proteins, few of which have been connected with their physiological RNA substrates and functions. In fact, among the largest family of putative RNA binding proteins in plants, the pentatricopeptide repeat (PPR) family, no physiologically relevant RNA ligands have been firmly established. We used the chloroplast-splicing factor CAF1 to demonstrate the fidelity of a microarray-based method for identifying RNAs associated with specific proteins in chloroplast extract. We then used the same method to identify RNAs associated with the maize (Zea mays) PPR protein CRP1. Two mRNAs whose translation is CRP1-dependent were strongly and specifically enriched in CRP1 coimmunoprecipitations. These interactions establish CRP1 as a translational regulator by showing that the translation defects in crp1 mutants are a direct consequence of the absence of CRP1. Additional experiments localized these interactions to the 5′ untranslated regions and suggested a possible CRP1 interaction motif. These results enhance understanding of the PPR protein family by showing that a PPR protein influences gene expression through association with specific mRNAs in vivo, suggesting an unusual mode of RNA binding for PPR proteins, and highlighting the possibility that translational regulation may be a particularly common function of PPR proteins. Analogous methods should have broad application for the study of native RNA–protein interactions in both mitochondria and chloroplasts.
The Nuclear Actin-Related Protein ARP6 Is a Pleiotropic Developmental Regulator Required for the Maintenance ofFLOWERING LOCUS CExpression and Repression of Flowering inArabidopsis w⃞
Authors: Deal Roger B. , Kandasamy Muthugapatti K. , McKinney Elizabeth C. , Meagher Richard B.
Published: September 2005

Actin-related proteins (ARPs) are found in the nuclei of all eukaryotic cells, but their functions are generally understood only in the context of their presence in various yeast and animal chromatin-modifying complexes. Arabidopsis thaliana ARP6 is a clear homolog of other eukaryotic ARP6s, including Saccharomyces cerevisiae ARP6, which was identified as a component of the SWR1 chromatin remodeling complex. We examined the subcellular localization, expression patterns, and loss-of-function phenotypes for this protein and found that Arabidopsis ARP6 is localized to the nucleus during interphase but dispersed away from the chromosomes during cell division. ARP6 expression was observed in all vegetative tissues as well as in a subset of reproductive tissues. Null mutations in ARP6 caused numerous defects, including altered development of the leaf, inflorescence, and flower as well as reduced female fertility and early flowering in both long- and short-day photoperiods. The early flowering of arp6 mutants was associated with reduced expression of the central floral repressor gene FLOWERING LOCUS C (FLC) as well as MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. In addition, arp6 mutations suppress the FLC-mediated late flowering of a FRIGIDA-expressing line, indicating that ARP6 is required for the activation of FLC expression to levels that inhibit flowering. These results indicate that ARP6 acts in the nucleus to regulate plant development, and we propose that it does so through modulation of chromatin structure and the control of gene expression.
Shadowed reality or the ‘Prometheus‐complex’: analytical psychotherapy after political imprisonment and persecution
Authors: Hölter Reinhild
Published: September 2005

Abstract:  Traumatic experiences of violence like those that were brought about by the communist regime of the former German Democratic Republic (GDR) are still present as if they happened yesterday, causing severe disadvantages in everyday life even after all those years. Consequently, in cases of extreme traumatization induced by political imprisonment, persecution and applied psychological methods of disintegration, regressive processes take place which corrode the ego. The breakdown of cathexis, i.e., the failure of empathic connection at the time of the trauma, is the strongest characteristic of severe traumatization. As a result of this destruction for the traumatized there is an inability to keep upright an inner empathic, emotional connection.

As we try to grasp, from a Jungian point of view, the psychodynamics of severe traumatization, we can speak of a powerful pathogenous complex, which I call the ‘Prometheus‐complex’. Gustav Bovensiepen speaks of this complex as a sub‐network, a limited fragment of the matrix of all internalized experiences, consisting of internal working models, feelings and patterns of anticipation which interact mutually. The human being, captured in such isolation and paralysis of the mind, can find himself ‘in the therapist’ on a mental level.
Influence of rearing temperature during the larval and nursery periods on growth and sex differentiation in two Mediterranean strains of Dicentrarchus labrax
Authors: Mylonas C. C. , Anezaki L. , Divanach P. , Zanuy S. , Piferrer F. , Ron B. , Peduel A. , Ben Atia I. , Gorshkov S. , Tandler A.
Published: September 2005

European sea bass Dicentrarchus labrax of the north‐western (NW) and south‐eastern (SE) Mediterranean Sea strains were exposed to different temperatures (13, 17 or 21° C) during the larval rearing (11–51 days post hatching, dph) or nursery periods (55–95 dph), in order to examine the effects of temperature on sex differentiation and subsequent growth during the first year of life. Higher growth was observed during exposure to higher temperatures, but fish of the NW strain exposed to 13 or 17° C during larval rearing exhibited compensatory growth once exposure to the lower temperatures finished, and as a result their final size at 300 dph was similar or greater to the group exposed to 21° C. Fish exposed to 17° C during the nursery period also had similar size to fish exposed to 21° C after 300 days of rearing, but the fish exposed to 13° C remained significantly smaller (ANOVA, n = 55–100, P < 0·05). There were significant differences in the sex ratio among the fish exposed to different temperatures during the two periods of rearing, with high temperature (21° C) resulting in a significantly higher percentage of males in the population, both in the NW (ANOVA, n = 2, P < 0·04) and SE populations (ANOVA, n = 2, P < 0·01). The masculinization effect of high temperature was significantly stronger during the larval rearing stage, both in the NW (ANOVA, n = 2, P < 0·005) and SE populations (ANOVA, n = 2, P < 0·01). None of the temperature manipulations could produce 100% females, suggesting that there is a part of the genetic component in sex differentiation which is not labile to environmental influence.
The influence of differential swimming speeds on composition of multi‐species fish shoals
Authors: Krause J. , Ward A. J. W. , Jackson A. L. , Ruxton G. D. , James R. , Currie S.
Published: September 2005

The importance of species‐specific swim speeds for the formation of multi‐species fish shoals in a Canadian freshwater system was investigated. Swim speed was positively correlated with total length and differed between species but was unrelated to shoal size.
Clutch characteristics of two populations of nine‐spined stickleback from south‐central Alaska
Authors: Heins D. C. , Baker J. A. , De Silva G. , Birden E. L.
Published: September 2005

Clutch size of nine‐spined sticklebackPungitius pungitiusfrom Airolo and Dog Bone Lakes, Alaska, showed small but significant differences between lakes and across 2 years. The variation in clutch size was independent of variation in egg mass, which did not differ appreciably between lakes or across years.
Plasma osmolarity, glucose concentration and erythrocyte responses of two Antarctic nototheniid fishes to acute and chronic thermal change
Authors: Lowe C. J. , Davison W.
Published: September 2005

Haematocrit, haemoglobin concentration, plasma osmolarity and plasma glucose concentration of the Antarctic nototheniid fishes Pagothenia borchgrevinki and Trematomus bernacchii were monitored during 24 h periods of exposure to 3 and 6° C. The same haematological variables were also measured in P. borchgrevinki following a 5–6 week period of 4° C acclimation. The first plasma glucose measurements in acutely thermally‐stressed Antarctic nototheniids revealed a delayed hyperglycaemia which related well to the relatively slow stress‐related elevation of plasma cortisol in these species. Plasma osmolarity of both species was unchanged by acute 3° C exposure, but exhibited a delayed and transient increase during acute exposure to 6° C. Haematocrit was unaltered in T. bernacchii during the acute temperature increases but was elevated in the relatively active P. borchgrevinki. Following 5–6 weeks of warm‐acclimation (4° C) the plasma glucose concentration, haematocrit and haemoglobin concentration of P. borchgrevinki were not significantly different from fish at −1° C, but plasma osmolarity decreased toward the level found in temperate‐water teleosts.
Widespread hybridization among species of Indian major carps in hatcheries, but not in the wild
Authors: Simonsen V. , Hansen M. M. , Mensberg K.‐L. D. , Sarder R. I. , Alam S.
Published: September 2005

Twenty‐one allozyme loci in samples of wild‐caught and hatchery‐reared Indian major carps from Bangladesh were analysed. Bayesian model‐based clustering analysis revealed the presence of four taxa, corresponding to the three known species along with a fourth unknown taxon present in two hatchery samples. Individual admixture coefficients showed that 24% of all hatchery‐reared fishes were hybrids, whereas a single hybrid was observed in the wild‐caught samples. Only catla Catla catla× rohu Labeo rohita and mrigal Cirrhinus cirrhosus× rohu hybrids were observed, the vast majority of which were F1 hybrids, though five individuals represented putative backcrosses. Mitochondrial DNA analysis revealed that catla × rohu hybridization primarily involved catla males and rohu females, whereas mrigal × rohu hybrids primarily resulted from rohu males and mrigal females. Despite the high percentage of F1‐hybrids in hatchery samples, reproductive barriers among species have so far precluded widespread introgression. Continued hybridization may eventually lead to a breakdown of species barriers, thereby compromising the genetic integrity of the species in the wild, and leading to production losses in aquaculture.
Lack of Ku80 Alteration in Multiple Myeloma
Authors: Kato Miyuki , Iida Shinsuke , Komatsu Hirokazu , Ueda Ryuzo
Published: September 2005

Chromosomal rearrangement involving the immunoglobulin gene locus, as a result of marked chromosomal instability, is the hallmark of human multiple myeloma (MM) cells. Since Ku80 plays a key role in the non‐homologous end‐joining (NHEJ) system, we investigated whether Ku80 alteration contributes to this genetic instability by examining its status in 16 MM cell lines. Our study demonstrated a lack of Ku80 alterations at the protein, mRNA and gene level in 15 out of the 16 cell lines. Only the U266 cell line carried a missense mutation of Ser335Leu in one allele of the cDNA. Six marrow samples derived from myeloma patients also did not show any aberrant Ku80 protein, in terms of size. Accordingly, Ku80 alteration is unlikely to be involved in MM, in disagreement with a previous study reporting frequent presence of a 69‐kD Ku80 variant (Ku86v) with reduced DNA binding activity in MM cells.