bluuebunny/crossref_metadata_2025 huggingface dataset:
Soft lithography fabrication of all-organic bottom-contact and top-contact field effect transistors
Authors: Cosseddu P. , Bonfiglio A.
Published: January 2006

All-organic field effect transistors on flexible plastic substrates have been fabricated. A thin Mylar® foil acts both as substrate and gate dielectric. The contacts have been fabricated with poly(ethylene-dioxythiophene)/polystyrene sulfonate (PEDT/PSS) by means of soft lithography. The active layer (pentacene) is vacuum sublimed on the prepatterned film in case of bottom-contact devices or, in case of top-contact devices, the active layer sublimation is made in advance. On the opposite side of the foil, a thin PEDT/PSS film, acting as gate electrode, is spin coated. The comparison between top-contact and bottom-contact devices shows interesting characteristics as a marked difference in the ID versus VD curve that can be mainly attributed to a different quality of PEDT/PSS-semiconductor contact. The flexibility of the obtained structure and the easy scalability of the technological process open the way for economic production of high resolution organic devices.
Correction of the sickle cell mutation in embryonic stem cells
Authors: Chang Judy C. , Ye Lin , Kan Yuet Wai
Published: January 2006

Sickle cell anemia is one of the most common genetic diseases worldwide. Patients often suffer from anemia, painful crises, infections, strokes, and cardiopulmonary complications. Although current management has improved the quality of life and survival of patients, cure can be achieved only with bone marrow transplantation when histocompatible donors are available. The ES cell technology suggests that a therapeutic cloning approach may be feasible for treatment of this disease. Using a transgenic/knockout sickle cell anemia mouse model, which harbors 240 kb of human DNA sequences containing the β
S
-globin gene, we prepared ES cells from blastocysts that had the sickle cells anemia genotype and carried out homologous recombination with DNA constructs that contained the β
A
-globin gene. We obtained ES cells in which the β
S
was corrected to the β
A
sequence. Hematopoietic cells differentiated from these ES cells produced both hemoglobin A and hemoglobin S. This approach can be applied to human ES cells to correct the sickle mutation as well as β-thalassemia mutations.
Blood Transfusion Requirements for Patients With Sarcomas Undergoing Combined Radio‐ and Chemotherapy
Authors: Sherbourne Karen R. , Earl Helena M. , Whitehead Lynne , Jefferies Sarah J. , Burnet Neil G.
Published: January 2006

Patients with bony and soft tissue sarcomas may require intensive treatment with chemotherapy and radiotherapy,
which often leads to a fall in haemoglobin levels, requiring blood transfusion. There may be advantages in predicting
which patients will require transfusion, partly because anaemia and hypoxia may worsen the response of tumours to
chemotherapy and radiotherapy. Between 1997 and 2003, a total of 26 patients who received intensive treatment with
curative intent were identified. Transfusions were given to maintain the haemoglobin at 10g/dl or above during
chemotherapy, and at 12 g/dl or above during radiotherapy. Eighteen (69%) required a transfusion, the majority
as a result of both the chemotherapy and RT criteria. There were 78 transfusion episodes, and 181 units of blood given.
In the 18 patients who required transfusion, the average number of units was 10.1, but seven patients required more
blood than this. The most significant factor influencing blood transfusion was choice of intensive chemotherapy.
Intensive chemotherapy and presenting Hb less than 11.6 g/dl identified 13 out of 18 patients who needed transfusion.
Adding a drop in haemoglobin of greater than 1.7 g/dl after one cycle of chemotherapy identified 16 out of 18 patients
who required transfusion. The seven patients who had heavy transfusion requirements were identified by age 32 or less,
intensive chemotherapy and a presenting Hb of 12 g/dl or less. Erythropoietin might be a useful alternative to transfusion
in selected patient groups, especially those with heavy transfusion requirements.
Brain Metastases in Soft Tissue Sarcomas: Case Report and Literature Review
Authors: Gupta Tejpal , Laskar Siddhartha , Gujral Sumeet , Muckaden Mary Ann
Published: January 2006

Background and purpose: Brain metastasis is a relatively uncommon event in the natural history of soft tissue sarcomas.
The increasing use of chemotherapy may have caused a reduction in local relapses as well as distant failures leading to an
improvement in survival, thereby allowing metachronous seeding of the brain, a sanctuary site. The purpose of this report is
to increase awareness amongst clinicians regarding such a possibility.

Patients and methods: A review of the departmental sarcoma database following the presentation of this index case in the
clinic.

Results and discussion: An adolescent male who had previously been treated with surgery and radiotherapy for a spindle cell
sarcoma of the left thigh developed a space‐occupying lesion in the brain within 6 months of treatment of the primary
tumor. He subsequently underwent resection of the presumed solitary brain metastasis followed by whole brain
radiotherapy. On radiation he was detected to have pulmonary metastases too, for which he was offered palliative
chemotherapy. The patient died of brain metastasis within 4 months. A review of the departmental sarcoma database,
restricted to soft tissue sarcomas purely, maintained prospectively from 2000 till date, could not identify any other such
case.

Conclusion: Brain metastases from soft tissue sarcomas are rare. Patients with neurological symptoms, however, should
be appropriately investigated. Surgical resection of brain metastasis could be considered for solitary brain metastasis
in non‐eloquent areas. Palliative radiotherapy is appropriate for patients with multiple brain metastases or co‐existing
extra‐cranial disease.
An Association of Multiple Well Differentiated Liposarcomas, Lipomatous Tissue and Hereditary Retinoblastoma
Authors: O′Neill J. K. , Stone C. A. , Sarsfield P. , Smith M. , Smithson S. F. , Silver D. , Devaraj V. S.
Published: January 2006

Well differentiated liposarcoma (atypical lipomatous tumour) is a low grade tumour, with no metastatic potential unless
dedifferentiation supervenes. When superficial, it recurs locally only occasionally after marginal excision. We present a
patient in whom bilateral childhood retinoblastoma was followed by later development of massive confluent areas of low
grade liposarcoma and lipomatous tissue affecting the upper extremities and trunk. We discuss the role of mutations in the
retinoblastoma gene (RB1) in linking these conditions and demonstrate the surgical management of an extremely unusual
and challenging case.
Neurotized Free Muscle Flaps can Produce MRI Changes Mimicking Tumour Recurrence
Authors: O’Neill J. K. , Barrett J. A. , Cobley T. , Devaraj V. , Silver D. A. T.
Published: January 2006

Soft tissue sarcomas are investigated by magnetic resonance imaging (MRI) both for initial staging and follow‐up. We
describe the presence of increased signal on T2‐weighted images caused by a neurotized muscle flap following reconstructive
surgery. This raised concern about possible sarcoma recurrence that was not clinically evident. On post‐operative imaging
of sarcomas the presence of recurrent tumour is indicated by a mass and high signal intensity on T2‐weighted images.
However, high signal changes in skeletal muscle on T2‐weighted images are not specific. In this case, the free functioning
muscle transfer with neurotization of the flap mimicked recurrence on MR scan. High signal intensity on T2‐weighted
images in muscle is an indication of either a physiological change or a pathological condition and must be taken in
context of the clinical picture.
Management of Skin Toxicity Related to the Use of ImatinibMesylate (STI571, Glivec™) for Advanced Stage GastrointestinalStromal Tumours
Authors: Scott Lucy C. , White Jeff D. , Reid Robin , Cowie Fiona
Published: January 2006

Skin toxicity is a common side‐effect of treatment with imatinib mesylate (STI571, Glivec™) in advanced gastrointestinal
stromal tumours (GIST) and chronic myeloid leukaemia. The optimal duration of treatment with imatinib mesylate in
GIST has not yet been established, as durable remissions have been observed in patients. It is, therefore, important to
develop strategies to deal with common side‐effects of what may be a long‐term treatment. Here we report the case of a
patient with advanced GIST who developed a cutaneous drug reaction secondary to imatinib mesylate and the various
management options that may be employed depending upon the severity of the toxicity. The case and literature are
discussed.
Angiosarcoma With Malignant Peripheral Nerve Sheath TumourDeveloping in a Patient With Klippel–Trénaunay–Weber Syndrome
Authors: Ploegmakers Marieke J. M. , Pruszczynski Maciej , De Rooy Jacky , Kusters Benno , Veth René P. H.
Published: January 2006

Purpose: We discuss the coexistence of Klippel–Trénaunay–Weber syndrome with various malignancies, the possible
histogenetic pathways and therapeutic implications.

Patient: We report on a 46‐year‐old man presenting with increasing pain and swelling of his right lower leg after fracturing
his fibula. Since birth he was known as having the uncommon syndrome of Klippel–Trénaunay–Weber of his right lower leg.

Methods: Our patient underwent an above‐knee amputation for biopsy‐proven malignant vascular tumour, first thought
to be a composite hemangio‐endothelioma and/or angiosarcoma with lung metastases.

Results: In the amputated extremity, a vascular malformation was found with tumour showing various components with
foci of angiosarcoma adjacent to diffuse neurofibroma and areas with high‐grade malignant peripheral nerve sheath
tumour. Amputation and palliative chemotherapy were indicated, but he died of pulmonary and cerebral metastases
2 months postoperatively.

Discussion: This case describes an angiosarcoma with malignant peripheral nerve sheath tumour developing in a patient
with Klippel–Trénaunay–Weber syndrome. A case never described before in literature and probably, as in our case,
difficult to diagnose at first biopsy.
Epithelioid Angiosarcoma of the Small Intestine After OccupationalExposure to Radiation and Polyvinyl Chloride: A case Report and Review of Literature
Authors: Khalil Maged F. , Thomas Asha , Aassad Adel , Rubin Moshe , Taub Robert N.
Published: January 2006

Angiosarcomas represent 1–2% of soft tissue sarcomas and most frequently occur in the subcutis. They may affect internal
organs, such as the heart, liver, and spleen, and only rarely do they emerge in the gastrointestinal tract. The association
between angiosarcomas and certain toxic chemical substances or previous external‐beam radiation therapy is well
documented.
A Unique Radiation Scheme for the Treatment of High‐Grade Non‐Metastatic Soft Tissue Sarcoma: The Detroit Medical Center Experience
Authors: Fontanesi James , Mott Michael P. , Kraut Michael J. , Lucas David R. , Miller Peter R.
Published: January 2006

Purpose:This is the initial report on the utilization of combined photon irradiation followed by a neutron boost irradiation
for the initial management of patients with high‐grade non‐metastatic soft tissue sarcoma (STS). We present data on local
control, complications, disease‐free survival and overall survival in patients at high risk for local relapse.

Methods and materials: Between 1/1/1995 and 10/31/02, twenty‐three patients with high‐grade non‐metastatic soft tissue
sarcoma were referred to the Department of Radiation Oncology at the Detroit Medical Center. These patients were
referred for consultation due to surgical margin status (tumor within 3mm of surgical margin (n=11)), or gross residual
disease (n=12). There were 14 males and nine females whose ages ranged from 12 to 75 at the time of diagnosis (med=44
years). The most common histology was malignant fibrous histiocytoma (n=6), followed by liposarcoma (n=5), synovial
sarcoma (n=4), and angiosarcoma (n=2). Twenty‐one of 23 patients also received multi‐agent multi‐cyclic cyto‐reductive
therapy. Treatment consisted of initial daily photon irradiation delivered either using twice daily fractions of 120 cGy
(n=10) or once daily 200 cGy/fx (n=13).Total photon dose was 36–39.6 Gy. Neutron irradiation was initiated
immediately following the photon irradiation and consisted of fraction sizes of 1.0–1.25NGy to a total dose of 6–10 NGy.
The neutrons were given once daily. Follow‐up is calculated from the day of last radiation treatment.

Results: No patient has been lost to follow‐up, which has ranged from 18 to 82 months (med=36 months). To date there
have been two local relapses and three patients with distant disease development without local relapse. Each of the patients
with distant disease has died. The local failures occurred at 9 and 12 months. The 36‐month local control is 91%. Thirtysix
month disease‐free survival was 78%. Overall survival at 36 months was 87%. Three patients had unusual complications
consisting of delayed wound healing, and in one of these patients a fracture of the tibia has been noted.

Conclusion: The use of this unique radiation sequence post‐surgically in patients at high risk for local relapse has resulted
in an exciting 36‐month local control rate of 91%. The 3‐year disease‐free survival of 78% and overall survival rate of 87%
are exciting but need to mature. The low complication rate is similar to that reported in other large institutional series that
have not utilized neutrons. We continue to evaluate the role of combined photon and once‐off neutron irradiation in the
treatment of patients with high‐grade STS that are risk for local recurrence.
Identification of Novel In Vivo Raf-1 Phosphorylation Sites Mediating Positive Feedback Raf-1 Regulation by Extracellular Signal-regulated Kinase
Authors: Balan Vitaly , Leicht Deborah T. , Zhu Jun , Balan Karina , Kaplun Alexander , Singh-Gupta Vinita , Qin Jun , Ruan Hong , Comb Michael J. , Tzivion Guri
Published: January 2006

The Ras–Raf–mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using two-dimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERK-phosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation.
Chromokinesin Xklp1 Contributes to the Regulation of Microtubule Density and Organization during Spindle Assembly
Authors: Castoldi Mirco , Vernos Isabelle
Published: January 2006

Xklp1 is a chromosome-associated kinesin required for Xenopus early embryonic cell division. Function blocking experiments in Xenopus egg extracts suggested that it is required for spindle assembly. We have reinvestigated Xklp1 function(s) by monitoring spindle assembly and microtubule behavior under a range of Xklp1 concentrations in egg extracts. We found that in the absence of Xklp1, bipolar spindles form with a reduced efficiency and display abnormalities associated with an increased microtubule mass. Likewise, centrosomal asters assembled in Xklp1-depleted extract show an increased microtubule mass. Conversely, addition of recombinant Xklp1 to the extract reduces the microtubule mass associated with spindles and asters. Our data suggest that Xklp1 affects microtubule polymerization during M-phase. We propose that these attributes, combined with Xklp1 plus-end directed motility, contribute to the assembly of a functional bipolar spindle.
The Heat-Shock Protein Apg-2 Binds to the Tight Junction Protein ZO-1 and Regulates Transcriptional Activity of ZONAB
Authors: Tsapara Anna , Matter Karl , Balda Maria S.
Published: January 2006

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1–ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G1/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1–ZONAB signaling in epithelial cells in response to cellular stress.
Identification of the Junctional Plaque Protein Plakophilin 3 in Cytoplasmic Particles Containing RNA-binding Proteins and the Recruitment of Plakophilins 1 and 3 to Stress Granules
Authors: Hofmann Ilse , Casella Marialuisa , Schnölzer Martina , Schlechter Tanja , Spring Herbert , Franke Werner W.
Published: January 2006

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell–cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25–35 S and 45–55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in “stress granules” known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.
Survivin Modulates Microtubule Dynamics and Nucleation throughout the Cell Cycle
Authors: Rosa Jack , Canovas Pedro , Islam Ashraful , Altieri Dario C. , Doxsey Stephen J.
Published: January 2006

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-α-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.
Proteomic Analysis of the Yeast Mitochondrial Outer Membrane Reveals Accumulation of a Subclass of Preproteins
Authors: Zahedi Rene P. , Sickmann Albert , Boehm Andreas M. , Winkler Christiane , Zufall Nicole , Schönfisch Birgit , Guiard Bernard , Pfanner Nikolaus , Meisinger Chris
Published: January 2006

Mitochondria consist of four compartments–outer membrane, intermembrane space, inner membrane, and matrix—with crucial but distinct functions for numerous cellular processes. A comprehensive characterization of the proteome of an individual mitochondrial compartment has not been reported so far. We used a eukaryotic model organism, the yeast Saccharomyces cerevisiae, to determine the proteome of highly purified mitochondrial outer membranes. We obtained a coverage of ∼85% based on the known outer membrane proteins. The proteome represents a rich source for the analysis of new functions of the outer membrane, including the yeast homologue (Hfd1/Ymr110c) of the human protein causing Sjögren–Larsson syndrome. Surprisingly, a subclass of proteins known to reside in internal mitochondrial compartments were found in the outer membrane proteome. These seemingly mislocalized proteins included most top scorers of a recent genome-wide analysis for mRNAs that were targeted to mitochondria and coded for proteins of prokaryotic origin. Together with the enrichment of the precursor form of a matrix protein in the outer membrane, we conclude that the mitochondrial outer membrane not only contains resident proteins but also accumulates a conserved subclass of preproteins destined for internal mitochondrial compartments.
Differential Distribution of Exosome Subunits at the Nuclear Lamina and in Cytoplasmic Foci
Authors: Graham Amy C. , Kiss Daniel L. , Andrulis Erik D.
Published: January 2006

The exosome complex plays important roles in RNA processing and turnover. Despite significant mechanistic insight into exosome function, we still lack a basic understanding of the subcellular locales where exosome complex biogenesis and function occurs. Here, we employ a panel of Drosophila S2 stable cell lines expressing epitope-tagged exosome subunits to examine the subcellular distribution of exosome complex components. We show that tagged Drosophila exosome subunits incorporate into complexes that recover endogenous nuclear and cytoplasmic exosome subunits. Immunolocalization analyses demonstrate that subsets of both epitope-tagged and endogenous exosome subunits are enriched in discrete subcellular compartments. In particular, dRrp4, dRrp42, dRrp46, and dCsl4 are enriched in cytoplasmic foci. Although dRrp4 and dRrp42 sometimes colocalize with dCsl4, these subunits are predominantly found in distinct cytoplasmic compartments. Strikingly, dRrp44/dDis3 and dRrp41/dSki6 colocalize with the nuclear lamina and often exhibit a restricted and asymmetric distribution at the nuclear periphery. Taken together, these observations indicate that individual exosome subunits have distinct localizations in vivo. These different distribution patterns presumably reflect distinct exosome subunit subcomplexes with correspondingly specialized functions.
Cip1 and Cip2 Are Novel RNA-Recognition-Motif Proteins That Counteract Csx1 Function during Oxidative Stress
Authors: Martín Victoria , Rodríguez-Gabriel Miguel A. , McDonald W. Hayes , Watt Stephen , Yates John R. , Bähler Jürg , Russell Paul
Published: January 2006

Eukaryotic cells reprogram their global patterns of gene expression in response to stress. Recent studies in Schizosaccharomyces pombe showed that the RNA-binding protein Csx1 plays a central role in controlling gene expression during oxidative stress. It does so by stabilizing atf1+ mRNA, which encodes a subunit of a bZIP transcription factor required for gene expression during oxidative stress. Here, we describe two related proteins, Cip1 and Cip2, that were identified by multidimensional protein identification technology (MudPIT) as proteins that coprecipitate with Csx1. Cip1 and Cip2 are cytoplasmic proteins that have RNA recognition motifs (RRMs). Neither protein is essential for viability, but a cip1Δ cip2Δ strain grows poorly and has altered cellular morphology. Genetic epistasis studies and whole genome expression profiling show that Cip1 and Cip2 exert posttranscriptional control of gene expression in a manner that is counteracted by Csx1. Notably, the sensitivity of csx1Δ cells to oxidative stress and their inability to induce expression of Atf1-dependent genes are partially rescued by cip1Δ and cip2Δ mutations. This study emphasizes the importance of a modulated mRNA stability in the eukaryotic stress response pathways and adds new information to the role of RNA-binding proteins in the oxidative stress response.
Conditional Dominant Mutations in theCaenorhabditis elegansGeneact-2Identify Cytoplasmic and Muscle Roles for a Redundant Actin Isoform
Authors: Willis John H. , Munro Edwin , Lyczak Rebecca , Bowerman Bruce
Published: January 2006

Animal genomes each encode multiple highly conserved actin isoforms that polymerize to form the microfilament cytoskeleton. Previous studies of vertebrates and invertebrates have shown that many actin isoforms are restricted to either nonmuscle (cytoplasmic) functions, or to myofibril force generation in muscle cells. We have identified two temperature-sensitive and semidominant embryonic-lethal Caenorhabditis elegans mutants, each with a single mis-sense mutation in act-2, one of five C. elegans genes that encode actin isoforms. These mutations alter conserved and adjacent amino acids predicted to form part of the ATP binding pocket of actin. At the restrictive temperature, both mutations resulted in aberrant distributions of cortical microfilaments associated with abnormal and striking membrane ingressions and protrusions. In contrast to the defects caused by these dominant mis-sense mutations, an act-2 deletion did not result in early embryonic cell division defects, suggesting that additional and redundant actin isoforms are involved. Accordingly, we found that two additional actin isoforms, act-1 and act-3, were required redundantly with act-2 for cytoplasmic function in early embryonic cells. The act-1 and -3 genes also have been implicated previously in muscle function. We found that an ACT-2::GFP reporter was expressed cytoplasmically in embryonic cells and also was incorporated into contractile filaments in adult muscle cells. Furthermore, one of the dominant act-2 mutations resulted in uncoordinated adult movement. We conclude that redundant C. elegans actin isoforms function in both muscle and nonmuscle contractile processes.
An Essential Role for SNX1 in Lysosomal Sorting of Protease-activated Receptor-1: Evidence for Retromer-, Hrs-, and Tsg101-independent Functions of Sorting Nexins
Authors: Gullapalli Anuradha , Wolfe Breann L. , Griffin Courtney T. , Magnuson Terry , Trejo JoAnn
Published: January 2006

Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.
Control of Bro1-Domain Protein Rim20 Localization by External pH, ESCRT Machinery, and theSaccharomyces cerevisiaeRim101 Pathway
Authors: Boysen Jacob H. , Mitchell Aaron P.
Published: January 2006

Bro1-domain proteins such as yeast Bro1 and mammalian AIP1/Alix are well-established participants in endosome metabolism. The Bro1-domain interacts with endosomal surface protein Snf7/Vps32 in yeast, a subunit of the ESCRT complex. Yeast Bro1-domain protein Rim20 has no role in endosome function, but is required for alkaline pH-stimulated cleavage of transcription factor Rim101. Rim20-GFP is cytoplasmic under acidic conditions but concentrated in punctate foci under alkaline conditions. Bro1-GFP also accumulates in foci, but they are more numerous under acidic than alkaline conditions. Colocalization experiments indicate that some Rim20-GFP foci correspond to Bro1-RFP foci, whereas others do not. Rim8, Rim9, Rim21, Dfg16, Snf7, Vps20, Vps23, and Vps25, which are required for Rim101 cleavage, are required for appearance of Rim20-GFP foci. ESCRT complexes accumulate on endosome-derived compartments in cells that lack the AAA-ATPase Vps4. We find that Rim20-GFP foci accumulate in a vps4 mutant background independently of external pH, Rim101 pathway-specific genes, and most ESCRT subunit genes except for SNF7. Rim20-GFP foci seem to represent endosomes, because they colocalize with Snf7-RFP and because they correspond to a perivacuolar compartment in the vps4 strain. We propose that alkaline growth conditions alter the endosomal surface to favor Rim20-Snf7 interaction and Rim101 cleavage. Our findings raise the possibility that Bro1-domain proteins may be differentially regulated in the same cell, thereby coupling endosome metabolism to signaling.
PGAP2 Is Essential for Correct Processing and Stable Expression of GPI-anchored Proteins
Authors: Tashima Yuko , Taguchi Ryo , Murata Chie , Ashida Hisashi , Kinoshita Taroh , Maeda Yusuke
Published: January 2006

Biosynthesis of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the ER has been extensively studied, whereas the molecular events during the transport of GPI-APs from the ER to the cell surface are poorly understood. Here, we established new mutant cell lines whose surface expressions of GPI-APs were greatly decreased despite normal biosynthesis of GPI-APs in the ER. We identified a gene responsible for this defect, designated PGAP2 (for Post-GPI-Attachment to Proteins 2), which encoded a Golgi/ER-resident membrane protein. The low surface expression of GPI-APs was due to their secretion into the culture medium. GPI-APs were modified/cleaved by two reaction steps in the mutant cells. First, the GPI anchor was converted to lyso-GPI before exiting the trans-Golgi network. Second, lyso-GPI-APs were cleaved by a phospholipase D after transport to the plasma membrane. Therefore, PGAP2 deficiency caused transport to the cell surface of lyso-GPI-APs that were sensitive to a phospholipase D. These results demonstrate that PGAP2 is involved in the processing of GPI-APs required for their stable expression at the cell surface.
BioShell—a package of tools for structural biology computations
Authors: Gront Dominik , Kolinski Andrzej
Published: January 2006

Summary: BioShell is a suite of programs performing common tasks accompanying protein structure modeling. BioShell design is based on UNIX shell flexibility and should be used as its extension. Using BioShell various molecular modeling procedures can be integrated in a single pipeline.

Availability: BioShell package can be downloaded from its website and these pages provide many examples and a detailed documentation for the newest version.

Contact:  dgront@chem.uw.edu.pl
Why Do Entrepreneurs Enter Politics? Evidence from China
Authors: Li Hongbin , Meng Lingsheng , Zhang Junsen
Published: January 2006

This article examines the determinants of the entrepreneur's political participation by employing a unique matched firm‐institution data set from China. We find that the likelihood of an entrepreneur's participation can be explained by the underdevelopment of markets and market‐supporting institutions. According to our estimates, the probability of entering politics decreases by 8–20% from the mean when the institutional indices improve by one standard deviation. Our findings support the view that the institutional environment shapes the private entrepreneur's motivation to participate in politics; they also provide an example of how private entrepreneurs respond to state/market failure in developing and transition countries. (JEL G1, H00, O10, P2, P3)
The Deterrent Effect of Capital Punishment: Evidence from a “Judicial Experiment”
Authors: Dezhbakhsh Hashem , Shepherd Joanna M.
Published: January 2006

We use panel data for 50 states during the 1960–2000 period to examine the deterrent effect of capital punishment, using the moratorium as a “judicial experiment.” We compare murder rates immediately before and after changes in states' death penalty laws, drawing on cross‐state variations in the timing and duration of the moratorium. The regression analysis supplementing the before‐and‐after comparisons disentangles the effect of lifting the moratorium on murder from the effect of actual executions on murder. Results suggest that capital punishment has a deterrent effect, and that executions have a distinct effect which compounds the deterrent effect of merely (re)instating the death penalty. The finding is robust across 96 regression models. (JEL C1, K1)
The Regulation ofDWARF4Expression Is Likely a Critical Mechanism in Maintaining the Homeostasis of Bioactive Brassinosteroids in Arabidopsis
Authors: Kim Ho Bang , Kwon Mi , Ryu Hojin , Fujioka Shozo , Takatsuto Suguru , Yoshida Shigeo , An Chung Sun , Lee Ilha , Hwang Ildoo , Choe Sunghwa
Published: January 2006

Mutants that are defective in brassinosteroid (BR) biosynthesis or signaling display severely retarded growth patterns due to absence of growth-promoting effects by BRs. Arabidopsis (Arabidopsis thaliana) DWARF4 (DWF4) catalyzes a flux-determining step in the BR biosynthetic pathways. Thus, it is hypothesized that the tissues of DWF4 expression may represent the sites of BR biosynthesis in Arabidopsis. Here we show that DWF4 transcripts accumulate in the actively growing tissues, such as root, shoot apices with floral clusters, joint tissues of root and shoot, and dark-grown seedlings. Conforming to the RNA gel-blot analysis, DWF4:β-glucuronidase (GUS) histochemical analyses more precisely define the tissues that express the DWF4 gene. Examination of the endogenous levels of BRs in six and seven different tissues of wild type and brassinosteroid insensitive1-5 mutant, respectively, revealed that BRs are significantly enriched in roots, shoot tips, and joint tissues of roots and shoots. In addition, DWF4:GUS expression was negatively regulated by BRs. DWF4:GUS activity was increased by treatment with brassinazole, a BR biosynthetic inhibitor, and decreased by exogenous application of bioactive BRs. When DWF4:GUS was expressed in a different genetic background, its level was down-regulated in brassinazole resistant1-D, confirming that BRASSINAZOLE RESISTANT1 acts as a negative regulator of DWF4. Interestingly, in the brassinosteroid insensitive2/dwf12-1D background, DWF4:GUS expression was intensified and delocalized to elongating zones of root, suggesting that BRASSINOSTEROID INSENSITIVE2 is an important factor that limits DWF4 expression. Thus, it is likely that the DWF4 promoter serves as a focal point in maintaining homeostasis of endogenous bioactive BR pools in specific tissues of Arabidopsis.
Glutamine Synthetase-Glutamate Synthase Pathway and Glutamate Dehydrogenase Play Distinct Roles in the Sink-Source Nitrogen Cycle in Tobacco
Authors: Masclaux-Daubresse Céline , Reisdorf-Cren Michèle , Pageau Karine , Lelandais Maud , Grandjean Olivier , Kronenberger Joceline , Valadier Marie-Hélène , Feraud Magali , Jouglet Tiphaine , Suzuki Akira
Published: January 2006

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.
Nitrogen Fixation Mutants ofMedicago truncatulaFail to Support Plant and Bacterial Symbiotic Gene Expression
Authors: Starker Colby G. , Parra-Colmenares Adriana L. , Smith Lucinda , Mitra Raka M. , Long Sharon R.
Published: January 2006

The Rhizobium-legume symbiosis culminates in the exchange of nutrients in the root nodule. Bacteria within the nodule reduce molecular nitrogen for plant use and plants provide bacteria with carbon-containing compounds. Following the initial signaling events that lead to plant infection, little is known about the plant requirements for establishment and maintenance of the symbiosis. We screened 44,000 M2 plants from fast neutron-irradiated Medicago truncatula seeds and isolated eight independent mutant lines that are defective in nitrogen fixation. The eight mutants are monogenic and represent seven complementation groups. To monitor bacterial status in mutant nodules, we assayed Sinorhizobium meliloti symbiosis gene promoters (nodF, exoY, bacA, and nifH) in the defective in nitrogen fixation mutants. Additionally, we used an Affymetrix oligonucleotide microarray to monitor gene expression changes in wild-type and three mutant plants during the nodulation process. These analyses suggest the mutants can be separated into three classes: one class that supports little to no nitrogen fixation and minimal bacterial expression of nifH; another class that supports no nitrogen fixation and minimal bacterial expression of nodF, bacA, and nifH; and a final class that supports low levels of both nitrogen fixation and bacterial nifH expression.
Mitosis-Specific Promoter of the Alfalfa Cyclin-Dependent Kinase Gene (Medsa;CDKB2;1) Is Activated by Wounding and Ethylene in a Non-Cell Division-Dependent Manner
Authors: Zhiponova Miroslava K. , Pettkó-Szandtner Aladár , Stelkovics Éva , Neer Zsuzsanna , Bottka Sándor , Krenács Tibor , Dudits Dénes , Fehér Attila , Szilák László
Published: January 2006

Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.
The Role of OsBRI1 and Its Homologous Genes, OsBRL1 and OsBRL3, in Rice
Authors: Nakamura Ayako , Fujioka Shozo , Sunohara Hidehiko , Kamiya Noriko , Hong Zhi , Inukai Yoshiaki , Miura Kotaro , Takatsuto Suguru , Yoshida Shigeo , Ueguchi-Tanaka Miyako , Hasegawa Yasuko , Kitano Hidemi , Matsuoka Makoto
Published: January 2006

Since first identifying two alleles of a rice (Oryza sativa) brassinosteroid (BR)-insensitive mutant, d61, that were also defective in an orthologous gene in Arabidopsis (Arabidopsis thaliana) BRASSINOSTEROID INSENSITIVE1 (BRI1), we have isolated eight additional alleles, including null mutations, of the rice BRI1 gene OsBRI1. The most severe mutant, d61-4, exhibited severe dwarfism and twisted leaves, although pattern formation and differentiation were normal. This severe shoot phenotype was caused mainly by a defect in cell elongation and the disturbance of cell division after the determination of cell fate. In contrast to its severe shoot phenotype, the d61-4 mutant had a mild root phenotype. Concomitantly, the accumulation of castasterone, the active BR in rice, was up to 30-fold greater in the shoots, while only 1.5-fold greater in the roots. The homologous genes for OsBRI1, OsBRL1 and OsBRL3, were highly expressed in roots but weakly expressed in shoots, and their expression was higher in d61-4 than in the wild type. Based on these observations, we conclude that OsBRI1 is not essential for pattern formation or organ initiation, but is involved in organ development through controlling cell division and elongation. In addition, OsBRL1 and OsBRL3 are at least partly involved in BR perception in the roots.
Evidence for Functional Conservation, Sufficiency, and Proteolytic Processing of the CLAVATA3 CLE Domain
Authors: Ni Jun , Clark Steven E.
Published: January 2006

Arabidopsis (Arabidopsis thaliana) CLAVATA3 (CLV3) is hypothesized to act as a ligand for the CLV1 receptor kinase in the regulation of stem cell specification at shoot and flower meristems. CLV3 is a secreted protein, with an amino-terminal signal sequence and a conserved C-terminal domain of 15 amino acids, termed the CLE (CLV3/ESR-related) domain, based on its similarity to a largely unstudied protein family broadly present in land plants. We have tested the function of 13 Arabidopsis CLEs in vivo and found a significant variability in the ability of CLEs to replace CLV3, ranging from complete to no complementation. The best rescuing CLE depends on CLV1 for function, while other CLEs act independently of CLV1. Domain-swap experiments indicate that differences in function can be traced to the CLE domain within these proteins. Indeed, when the CLE domain of CLV3 is placed downstream of an unrelated signal sequence, it is capable of fully replacing CLV3 function. Finally, we have detected proteolytic activity in extracts from cauliflower (Brassica oleracea) that process both CLV3 and CLE1 at their C termini. For CLV3, processing appears to occur at the absolutely conserved arginine-70 found at the beginning of the CLE domain. We propose that CLV3 and other CLEs are C-terminally processed to generate an active CLE peptide.
Evidence for Involvement of Photosynthetic Processes in the Stomatal Response to CO2
Authors: Messinger Susanna M. , Buckley Thomas N. , Mott Keith A.
Published: January 2006

Stomatal conductance (g  s) typically declines in response to increasing intercellular CO2 concentration (c  i). However, the mechanisms underlying this response are not fully understood. Recent work suggests that stomatal responses to c  i and red light (RL) are linked to photosynthetic electron transport. We investigated the role of photosynthetic electron transport in the stomatal response to c  i in intact leaves of cocklebur (Xanthium strumarium) plants by examining the responses of g  s and net CO2 assimilation rate to c  i in light and darkness, in the presence and absence of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and at 2% and 21% ambient oxygen. Our results indicate that (1) g  s and assimilation rate decline concurrently and with similar spatial patterns in response to DCMU; (2) the response of g  s to c  i changes slope in concert with the transition from Rubisco- to electron transport-limited photosynthesis at various irradiances and oxygen concentrations; (3) the response of g  s to c  i is similar in darkness and in DCMU-treated leaves, whereas the response in light in non-DCMU-treated leaves is much larger and has a different shape; (4) the response of g  s to c  i is insensitive to oxygen in DCMU-treated leaves or in darkness; and (5) stomata respond normally to RL when c  i is held constant, indicating the RL response does not require a reduction in c  i by mesophyll photosynthesis. Together, these results suggest that part of the stomatal response to c  i involves the balance between photosynthetic electron transport and carbon reduction either in the mesophyll or in guard cell chloroplasts.