bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a NifS-Like Chloroplast Protein from Arabidopsis. Implications for Its Role in Sulfur and Selenium Metabolism
Authors: Pilon-Smits Elizabeth A.H. , Garifullina Gulnara F. , Abdel-Ghany Salah , Kato Shin-Ichiro , Mihara Hisaaki , Hale Kerry L. , Burkhead Jason L. , Esaki Nobuyoshi , Kurihara Tatsuo , Pilon Marinus
Published: December 2002

NifS-like proteins catalyze the formation of elemental sulfur (S) and alanine from cysteine (Cys) or of elemental selenium (Se) and alanine from seleno-Cys. Cys desulfurase activity is required to produce the S of iron (Fe)-S clusters, whereas seleno-Cys lyase activity is needed for the incorporation of Se in selenoproteins. In plants, the chloroplast is the location of (seleno) Cys formation and a location of Fe-S cluster formation. The goal of these studies was to identify and characterize chloroplast NifS-like proteins. Using seleno-Cys as a substrate, it was found that 25% to 30% of the NifS activity in green tissue in Arabidopsis is present in chloroplasts. A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS, was cloned, and its chloroplast localization was confirmed using immunoblot analysis and in vitro import. AtCpNIFS is expressed in all major tissue types. The protein was expressed in Escherichia coli and purified. The enzyme contains a pyridoxal 5′ phosphate cofactor and is a dimer. It is a type II NifS-like protein, more similar to bacterial seleno-Cys lyases than to Cys desulfurases. The enzyme is active on both seleno-Cys and Cys but has a much higher activity toward the Se substrate. The possible role of AtCpNifS in plastidic Fe-S cluster formation or in Se metabolism is discussed.
A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice
Authors: Weinmann Pamela , Scharffetter-Kochanek Karin , Forlow S. Bradley , Peters Thorsten , Walzog Barbara
Published: December 2002

The control of neutrophil turnover in the circulation is a key event in homeostasis and inflammation. Using CD18- deficient (CD18−/−) mice that show a 19-fold increase of blood neutrophil counts when compared with wild-type animals (CD18+/+), we found that apoptosis of peripheral neutrophils was significantly reduced from 27.4% in the wild-type to 4.8% inCD18−/− mice within 4 hours after isolation as measured by analysis of DNA content. This was confirmed by detecting CD16 expression, nuclear morphology, and internucleosomal DNA degradation. In contrast, no difference in apoptosis was observed in neutrophils derived from the bone marrow. Neutrophilia and delayed neutrophil apoptosis were also present inCD18−/−/interleukin 6 (IL-6−/−) double knockout mice. Moreover, plasma ofCD18−/− mice was not able to delay apoptosis of CD18+/+neutrophils and plasma ofCD18+/+ mice did not augment apoptosis of CD18−/−neutrophils. However,CD18−/− neutrophils revealed an up-regulation of the antiapoptotic gene bcl-Xl and a down-regulation of the proapoptotic gene bax-α compared withCD18+/+ neutrophils suggesting that this delayed apoptosis. Accordingly, down-regulation of Bax-α using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18+/+ mice by a 1:1 mixture of CD18+/+ andCD18−/− hematopoietic cells abolished the delay of apoptosis in peripheralCD18−/− neutrophils and prevented neutrophilia. Altogether, this suggests that a delay of neutrophil apoptosis inCD18−/− mice causes an alteration of neutrophil homeostasis, which may induce the massive increase of peripheral neutrophil counts. Thus, apoptosis seems to be critically involved in the control of neutrophil turnover in the circulation.
Dual-specific Src and Abl kinase inhibitors, PP1 and CGP76030, inhibit growth and survival of cells expressing imatinib mesylate–resistant Bcr-Abl kinases
Authors: Warmuth Markus , Simon Nicola , Mitina Olga , Mathes Ruth , Fabbro Doriano , Manley Paul W. , Buchdunger Elisabeth , Forster Karin , Moarefi Ismail , Hallek Michael
Published: December 2002

The leukemogenic tyrosine kinase Bcr-Abl contains a highly conserved inhibitor-binding pocket (IBP), which serves as a binding site for imatinib mesylate. Mutations at the IBP may lead to resistance of the Abl kinase against imatinib mesylate. To examine the mechanisms of imatinib mesylate binding and resistance in more detail, we created several point mutations at amino acid positions 315 and 380 of Abl, blocking the access to the IBP and rendering Bcr-Abl imatinib mesylate–resistant. Moreover, introduction of a mutation destabilizing the inactive conformation of Abl (Asp276Ser/Glu279Ser) also led to imatinib mesylate resistance, suggesting that the inhibitor required inactivation of the kinase prior to binding. These Bcr-Abl mutants were then used to evaluate the binding mode and specificity of 2 compounds, PP1 and CGP76030, originally characterized as Src kinase inhibitors. Both compounds inhibited Bcr-Abl in a concentration-dependent manner by overlapping binding modes. However, in contrast to imatinib mesylate, PP1 and CGP76030 blocked cell growth and survival in cells expressing various inhibitor-resistant Abl mutants. Studies on the potential signaling mechanisms demonstrated that in cells expressing inhibitor-resistant Bcr-Abl mutants, PP1 and CGP76030 inhibited the activity of Src family tyrosine kinases and Akt but not signal transducer and activator of transcription–5 (STAT5) and JUN kinase (Jnk). The results suggest that the use of Src kinase inhibitors is a potential strategy to prevent or overcome clonal evolution of imatinib mesylate resistance in Bcr-Abl+ leukemia.
Fetal hemoglobin modulation during human erythropoiesis: stem cell factor has “late” effects related to the expression pattern of CD117
Authors: Wojda Urszula , Leigh Kristina R. , Njoroge Joyce M. , Jackson Kaedrea A. , Natarajan Bhanu , Stitely Michael , Miller Jeffery L.
Published: December 2002

A cytokine-screening assay of cultured peripheral blood cells obtained using immune rosetting and separation of progenitors was developed to identify determinants of fetal hemoglobin (HbF) modulation during adult erythropoiesis. Among the 12 erythroid growth-promoting cytokines tested, stem cell factor (SCF) at a concentration of 50 ng/mL resulted in the most significant increase in cell proliferation and HbF content. The average HbF/hemoglobin A (HbA) ratio was 30.9% ± 18.7% in cultures containing SCF compared with 4.1% ± 2.2% in those grown with erythropoietin (EPO) alone (P = 8.5E-8). To further investigate the hemoglobin-modulating effects of SCF, we examined the surface expression pattern of the SCF receptor, CD117, among maturing erythroblasts. CD117 expression increased during the first week of culture and peaked on culture days 7 to 9. After culture day 9, the level of CD117 declined to lower levels. The rise in CD117 expression to high levels mirrored that of the transferrin receptor (CD71), and the subsequent reduction in CD117 was inversely related to increases in expression of glycophorin A. SCF-related increases in the HbF/HbA ratio correlated with the expression pattern of CD117. SCF added during days 7 to 14 resulted in a more pancellular distribution of HbF on day 14 compared with the heterocellular distribution present in cultures supplemented with SCF on days 0 to 7. A significant SCF-mediated increase in HbF was also measured using progenitors derived from cord blood. These results suggest that the HbF response to SCF is greatest at the late progenitor stage as a function of surface CD117 expression.
Role of the intracellular domains of CXCR4 in SDF-1–mediated signaling
Authors: Roland Joachim , Murphy Brendan J. , Ahr Barbara , Robert-Hebmann Véronique , Delauzun Vincent , Nye Keith E. , Devaux Christian , Biard-Piechaczyk Martine
Published: December 2002

The CXCR4 chemokine receptor is a Gi protein–coupled receptor that triggers multiple intracellular signals in response to stromal cell-derived factor 1 (SDF-1), including calcium mobilization and p44/42 extracellular signal-regulated kinases (ERK1/2). Transduced signals lead to cell chemotaxis and are terminated through receptor internalization depending on phosphorylation of the C terminus part of CXCR4. Receptor endocytosis is also required for some receptors to stimulate ERK1/2 and to migrate through a chemokine gradient. In this study, we explored the role played by the 3 intracellular loops (ICL1-3) and the C terminus domain of CXCR4 in SDF-1–mediated signaling by using human embryonic kidney (HEK)–293 cells stably expressing wild-type or mutated forms of CXCR4. ICL3 of CXCR4 is specifically involved in Gi-dependent signals such as calcium mobilization and ERK activation, but does not trigger CXCR4 internalization after SDF-1 binding, indicating that ERK phosphorylation is independent of CXCR4 endocytosis. Surprisingly, ICL2, with or without the aspartic acid, arginine, and tyrosine (DRY) motif, is dispensable for Gi signaling. However, ICL2 and ICL3, as well as the C terminus part of CXCR4, are needed to transduce SDF-1–mediated chemotaxis, suggesting that this event involves multiple activation pathways and/or cooperation of several cytoplasmic domains of CXCR4.
Late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants: importance of viral load and T-cell immunity
Authors: Boeckh Michael , Leisenring Wendy , Riddell Stanley R. , Bowden Raleigh A. , Huang Meei-Li , Myerson David , Stevens-Ayers Terry , Flowers Mary E. D. , Cunningham Terri , Corey Lawrence
Published: December 2002

Ganciclovir effectively prevents cytomegalovirus (CMV) disease in the first 100 days after allogeneic hematopoietic stem cell transplantation (HSCT), but late-onset CMV disease is increasingly observed. We designed a prospective cohort study to define the incidence and risk factors for late CMV infection in patients who undergo HSCT. CMV-seropositive patients were studied prospectively for CMV infection (quantitative pp65 antigenemia, quantitative CMV-DNA, blood culture), T-cell immunity (CMV-specific CD4+ T-helper and CD8+ cytotoxic T-lymphocyte responses, CD4 and CD8 T-cell count, absolute lymphocyte count), and other transplantation-related factors. Univariate and multivariable analyses were used to assess the risk for late CMV infection and disease and to assess overall survival. Late CMV disease developed in 26 of 146 (17.8%) patients a median of 169 days after transplantation (range, 96-784 days); the mortality rate was 46%. Thirty-eight percent of patients surviving late disease had a second episode a median of 79 days after the first episode. At 3 months after transplantation, preceding detection of CMV pp65 antigenemia, CD4 T-cell counts lower than 50 cells/mm3, postengraftment absolute lymphopenia levels lower than 100 lymphocytes/mm3, undetectable CMV-specific T-cell responses, and graft-versus-host disease (GVHD) were associated with late CMV disease or death. After 3 months, continued detection of pp65 antigenemia or CMV DNA in plasma or peripheral blood leukocytes and lymphopenia (fewer than 300 lymphocytes/mm3) were strong predictors of late CMV disease and death. In conclusion, CMV viral load, lymphopenia, and CMV-specific T-cell immunodeficiency are predictors of late CMV disease and death after allogeneic stem cell transplantation. Prevention strategies should be targeted at patients in whom CMV reactivated during the first 3 months and those with poor CMV-specific immunity or low CD4 counts.
Inhibition of maturation and function of dendritic cells by intravenous immunoglobulin
Authors: Bayry Jagadeesh , Lacroix-Desmazes Sébastien , Carbonneil Cedric , Misra Namita , Donkova Vladimira , Pashov Anastas , Chevailler Alain , Mouthon Luc , Weill Bernard , Bruneval Patrick , Kazatchkine Michel D. , Kaveri Srini V.
Published: December 2002

Normal immunoglobulin G for therapeutic use (intravenous immunoglobulin [IVIg]) is used in an increasing number of immune-mediated conditions, including acute and chronic/relapsing autoimmune diseases, transplantation, and systemic inflammatory disorders. Several mutually nonexclusive mechanisms of action account for the immunoregulatory effects of IVIg. Although IVIg inhibits T-cell proliferation and T-cell cytokine production, it is unclear whether these effects are directly dependent on the effects of IVIg on T cells or they are dependent through the inhibition of antigen-presenting cell activity. Here, we examined the effects of IVIg on differentiation, maturation, and function of dendritic cells (DCs). We show that IVIg inhibits the differentiation and maturation of DCs in vitro and abrogates the capacity of mature DC to secrete interleukin-12 (IL-12) on activation while enhancing IL-10 production. IVIg-induced down-regulation of costimulatory molecules associated with modulation of cytokine secretion resulted in the inhibition of autoreactive and alloreactive T-cell activation and proliferation. Modulation of DC maturation and function by IVIg is of potential relevance to its immunomodulatory effects in controlling specific immune responses in autoimmune diseases, transplantation, and other immune-mediated conditions.
In vitro adherence of lymphocytes to dermal endothelium under shear stress: implications in pathobiology and steroid therapy of acute cutaneous GVHD
Authors: Sackstein Robert , Messina Jane L. , Elfenbein Gerald J.
Published: December 2002

The extravasation of leukocytes at sites of inflammation critically depends on initial shear-resistant adhesive interactions between leukocytes in blood flow and target tissue endothelium. Dermal lymphocytic infiltrates are a hallmark feature of acute cutaneous graft-versus-host disease (acGVHD) following allogeneic hematopoietic stem cell (allo-HSC) transplantation. These infiltrates occur commonly during periods of profound lymphopenia, suggesting that the dermal endothelial adhesive mechanism(s) promoting lymphocyte emigration in acGVHD are highly efficient. To examine this issue, we performed Stamper-Woodruff assays on frozen sections of biopsy specimens of cutaneous lesions occurring within 100 days of HSC transplantation in 22 autologous hematopoietic stem cell transplant (auto-HSCT) and 25 allo-HSCT recipients. By using this shear-based assay, we observed lymphocyte adherence to papillary dermal vascular structures in all punch biopsy specimens of allo-HSCT recipients who had clinicohistologic evidence of acGVHD and who were not receiving steroids, whereas no lymphocyte adherence was observed within skin specimens from allo-HSCT recipients who did not develop acGVHD. Within the group of auto-HSCT recipients, 2 of 22 skin biopsies demonstrated lymphocyte binding to dermal vessels. Among allo-HSCT patients receiving steroid therapy for acGVHD, lymphocyte binding to dermal endothelium was abrogated prior to resolution of rash in those who responded, yet binding was persistent in skin from one patient whose rash did not respond to steroid therapy. Collectively, these data indicate that the papillary endothelium of skin in acGVHD displays heightened capacity to support lymphocyte adhesion under shear stress conditions and suggest that down-modulation of this endothelial adhesive capability may be one mechanism by which steroids abrogate acGVHD reactions.
A critical role of placental growth factor in the induction of inflammation and edema formation
Authors: Oura Hajimu , Bertoncini Jennifer , Velasco Paula , Brown Lawrence F. , Carmeliet Peter , Detmar Michael
Published: December 2002

Angiogenesis is a prominent feature of a number of inflammatory human diseases, including rheumatoid arthritis, psoriasis, and cutaneous delayed-type hypersensitivity (DTH) reactions. Up-regulation of placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, has been found in several conditions associated with pathologic angiogenesis; however, its distinct role in the control of angiogenesis has remained unclear. To directly investigate the biologic function of PlGF in cutaneous inflammation and angiogenesis, DTH reactions were investigated in the ear skin of wild-type mice, of PlGF-deficient mice, and of transgenic mice with targeted overexpression of human PlGF-2 in epidermal keratinocytes, driven by a keratin 14 promoter expression construct. Chronic transgenic delivery of PlGF-2 to murine epidermis resulted in a significantly increased inflammatory response, associated with more pronounced vascular enlargement, edema, and inflammatory cell infiltration than seen in wild-type mice. Conversely, PlGF deficiency resulted in a diminished and abbreviated inflammatory response, together with a reduction of inflammatory angiogenesis and edema formation. VEGF expression was up-regulated at a comparable level in the inflamed skin of all genotypes. These findings reveal that placental growth factor plays a critical role in the control of cutaneous inflammation, and they suggest inhibition of PlGF bioactivity as a potential new approach for anti-inflammatory therapy.
Levels of phospho-Smad2/3 are sensors of the interplay between effects of TGF-β and retinoic acid on monocytic and granulocytic differentiation of HL-60 cells
Authors: Cao Zhouhong , Flanders Kathleen C. , Bertolette Daniel , Lyakh Lyudmila A. , Wurthner Jens U. , Parks W. Tony , Letterio John J. , Ruscetti Francis W. , Roberts Anita B.
Published: December 2002

We have investigated the role of Smad family proteins, known to be important cytoplasmic mediators of signals from the transforming growth factor–β (TGF-β) receptor serine/threonine kinases, in TGF-β–dependent differentiation of hematopoietic cells, using as a model the human promyelocytic leukemia cell line, HL-60. TGF-β–dependent differentiation of these cells to monocytes, but not retinoic acid–dependent differentiation to granulocytes, was accompanied by rapid phosphorylation and nuclear translocation of Smad2 and Smad3. Vitamin D3 also induced phosphorylation of Smad2/3 and monocytic differentiation; however the effects were indirect, dependent on its ability to induce expression of TGF-β1. Simultaneous treatment of these cells with TGF-β1 and all-trans-retinoic acid (ATRA), which leads to almost equal numbers of granulocytes and monocytes, significantly reduced the level of phospho–Smad2/3 and its nuclear accumulation, compared with that in cells treated with TGF-β1 alone. TGF-β1 and ATRA activate P42/44 mitogen-activated protein (MAP) kinase with nearly identical kinetics, ruling out its involvement in these effects on Smad phosphorylation. Addition of the inhibitor-of-protein serine/threonine phosphatases, okadaic acid, blocks the ATRA-mediated reduction in TGF-β–induced phospho-Smad2 and shifts the differentiation toward monocytic end points. In HL-60R mutant cells, which harbor a defective retinoic acid receptor–α (RAR-α), ATRA is unable to reduce levels of TGF-β–induced phospho-Smad2/3, coincident with its inability to differentiate these cells along granulocytic pathways. Together, these data suggest a new level of cross-talk between ATRA and TGF-β, whereby a putative RAR-α–dependent phosphatase activity limits the levels of phospho-Smad2/3 induced by TGF-β, ultimately reducing the levels of nuclear Smad complexes mediating the TGF-β–dependent differentiation of the cells to monocytic end points.
Targeting p38 MAPK inhibits multiple myeloma cell growth in the bone marrow milieu
Authors: Hideshima Teru , Akiyama Masaharu , Hayashi Toshiaki , Richardson Paul , Schlossman Robert , Chauhan Dharminder , Anderson Kenneth C.
Published: December 2002

p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK family which is activated by cytokines and growth factors, but its role in pathogenesis of multiple myeloma (MM) is unknown. In this study, we demonstrate that the specific p38 MAPK inhibitor VX-745 inhibits interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in bone marrow stromal cells (BMSCs), without affecting their viability. Tumor necrosis factor alpha (TNF-α)–induced IL-6 secretion in BMSCs is also inhibited by VX-745. Importantly, VX-745 inhibits both MM cell proliferation and IL-6 secretion in BMSCs triggered by adherence of MM cells to BMSCs, suggesting that it can inhibit paracrine MM cell growth in the BM milieu and overcome cell adhesion–related drug resistance. These studies therefore identify p38 MAPK as a novel therapeutic target to overcome drug resistance and improve patient outcome in MM.
Gene therapy of apolipoprotein E–deficient mice using a novel macrophage-specific retroviral vector
Authors: Gough Peter J. , Raines Elaine W.
Published: December 2002

The use of retroviral gene transfer into hematopoietic stem cells for human gene therapy has been hampered by the absence of retroviral vectors that can generate long-lasting, lineage-specific gene expression. We developed self-inactivating retroviral vectors that incorporate gene-regulatory elements from the macrophage-restricted human CD68 gene. Through the transplantation of transduced murine hematopoietic stem cells (HSCs), we show that a vector incorporating a 342–base pair (bp) fragment of 5′ flanking sequence from the CD68 gene, in addition to the CD68 first intron, was able to direct macrophage-specific expression of an enhanced green fluorescent protein (EGFP) reporter gene in inflammatory cell exudates and lymphoid organs in vivo. Levels of EGFP expression generated by this vector were greater than those generated by a standard Moloney murine leukemia retroviral vector, and they were stable for at least a year after transplantation of transduced HSCs. To evaluate the ability of this vector to generate therapeutically useful levels of gene expression, we transplanted apolipoprotein E (ApoE)–deficient HSCs transduced with a virus encoding ApoE into ApoE-deficient mice. Macrophages from these mice expressed levels of ApoE that were comparable to those from wild-type mice, and vector-driven expression of ApoE in macrophages was sufficient to reverse both hypercholesterolemia and atherosclerotic lesion development. The future application of this retroviral vector should provide a powerful tool to further elucidate macrophage function and for human gene therapy.
Early identification of individuals at risk for antisocial personality disorder
Authors: Hill Jonathan
Published: December 2002

Background

Antisocial personality disorder is usually preceded by serious and persistent conduct problems starting in early childhood, and so there is little difficulty in identifying an at-risk group.

Aims

To address six key areas concerning the relationship between early conduct problems and antisocial personality disorder.

Method

Review of recent research into early identification of and intervention in child conduct problems, following up to possible adult antisocial behaviour.

Results

Conduct problems are predictive of antisocial personality disorder independently of the associated adverse family and social factors. Prediction could be aided through identification of subtypes of conduct problems. There is limited evidence on which children have problems that are likely to persist and which will improve; children who desist from early conduct problems and those with onset in adolescence are also vulnerable as adults.

Conclusions

The predictive power of the childhood precursors of antisocial personality disorder provides ample justification for early intervention. Greater understanding of subgroups within the broad category of antisocial children and adults should assist with devising and targeting interventions.
Problems encountered when testing for LSD in a regional medium secure unit
Authors: Acosta-Armas A. J.
Published: December 2002

Aims and Method

Between 1998 and 2000, a surprisingly high number of positive results was noticed in our regional medium secure unit when testing for D-lysergic acid diethylamide (LSD). This led to an investigation of possible factors involved. It was felt that the testing protocol, particularly the use of a single, non-isotopic homogeneous immunoassay without routine further confirmatory testing, was largely to blame for what seemed to be a high incidence of false positives. On two different occasions, samples from each patient were sent, on the same day, to two different laboratories. At the first laboratory, only one test method was used and at the second one test plus two confirmatory tests were carried out.

Results

Out of a total of 23 patients tested on two separate occasions, the first laboratory gave three positive results the first time and three positive results the second, while the second laboratory gave only one positive result on the second occasion that samples were sent and none on the first. This reinforces the belief that, without adequate confirmatory analysis, many psychiatric and non-psychiatric prescribed drugs can give false positives.

Clinical Implications

Positive LSD results should be confirmed by at least one, preferably chromatographic, alternative method. A protocol for testing and reporting LSD in psychiatric patients should be considered in order to minimise the risk of obtaining false-positive results which have negative clinical, legal and psychological repercussions.
Acute mental health admissions in inner London: changes in patient characteristics and clinical admission thresholds between 1988 and 1998
Authors: Fitzpatrick N. K. , Thompson C. J. , Hemingway H. , Barnes T. R. E. , Higgitt A. , Molloy C. , Hargreaves S.
Published: December 2002

Aims and Method

We undertook a retrospective case-note review of three cohorts of mental health admissions to determine the extent to which patient and service characteristics changed between 1988 and 1998. Changes in clinical admission thresholds were investigated by a psychiatrists' review of handwritten medical admission assessments.

Results

Patients admitted in 1998 were demographically less stable and clinically more complex than those admitted 10 years earlier. Clinical admission thresholds remained consistent.

Clinical Implications

Our findings suggest that the perceived increase in pressure on psychiatric services over this period was a response to a change in population need. This study highlights important questions about the clinical decision-making process leading to use of alternatives to admission and the appropriateness of acute admissions.
Programmed cell death in the developing inner ear is balanced by nerve growth factor and insulin-like growth factor I
Authors: Frago Laura M. , Cañón Susana , de la Rosa Enrique J. , León Yolanda , Varela-Nieto Isabel
Published: December 2002

Nerve growth factor induces cell death in organotypic cultures of otic vesicle explants. This cell death has a restricted pattern that reproduces the in vivo pattern of apoptosis occurring during inner ear development. In this study, we show that binding of nerve growth factor to its low affinity p75 neurotrophin receptor is essential to achieve the apoptotic response. Blockage of binding to p75 receptor neutralized nerve-growth-factor-induced cell death,as measured by immunoassays detecting the presence of cytosolic oligonucleosomes and by TUNEL assay to visualize DNA fragmentation. Nerve growth factor also induced a number of cell-death-related intracellular events including ceramide generation, caspase activation and poly-(ADP ribose)polymerase cleavage. Again, p75 receptor blockade completely abolished all of these effects. Concerning the intracellular pathway, ceramide increase depended on initiator caspases, whereas its actions depended on both initiator and effector caspases, as shown by using site-specific caspase inhibitors. Conversely, insulin-like growth factor I, which promotes cell growth and survival in the inner ear, abolished apoptosis induced by nerve growth factor. Insulin-like growth factor cytoprotective actions were accomplished, at least in part, by decreasing endogenous ceramide levels and activating Akt. Taken together, these results strongly suggest that regulation of nerve-growth-factor-induced apoptosis in the otocysts occurs via p75 receptor binding and is strictly controlled by the interaction with survival signalling pathways.
Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis
Authors: Düßmann Heiko , Rehm Markus , Kögel Donat , Prehn Jochen H. M.
Published: December 2002

Little is known about the temporal relationship between mitochondrial and plasma membrane potential changes and outer mitochondrial membrane permeabilization during apoptosis. Confocal imaging of breast carcinoma and HeLa cells stably transfected with cytochrome-C-GFP demonstrated that mitochondria rapidly depolarized after the release of the fusion protein into the cytosol. Of note, mitochondria did not completely depolarize but established a new steady-state level that could be further dissipated by treatment with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. Treatment with the FOF1-ATP-synthase inhibitor oligomycin likewise induced a collapse of this steady-state level, suggesting that FOF1-ATP-synthase reversal maintained mitochondrial potential after outer mitochondrial membrane permeabilization. Treatment with a broad spectrum caspase inhibitor failed to inhibit the partial depolarization of mitochondria during apoptosis, yet potently abolished the activation of effector caspases detected by fluorescence resonance energy transfer analysis in the same experiment. Interestingly, the onset of mitochondrial depolarization was always coupled with a depolarization of the plasma membrane potential. This was associated with the degradation of the regulatory Na+/K+-ATPase β-subunit, and both events were blocked by caspase inhibition. Our results demonstrate that outer mitochondrial membrane permeabilization coordinates the depolarization of both membrane potentials during apoptosis.
Functional interaction of megalin with the megalinbinding protein(MegBP), a novel tetratrico peptide repeat-containing adaptor molecule
Authors: Petersen Helle Heibroch , Hilpert Jan , Militz Daniel , Zandler Valerie , Jacobsen Christian , Roebroek Anton J. M. , Willnow Thomas E.
Published: December 2002

Megalin is a member of the LDL receptor gene family that plays an important role in forebrain development and in cellular vitamin D metabolism through endocytic uptake of vitamin D metabolites. Similar to other receptors in this gene family, megalin is believed to functionally interact with intracellular proteins through adaptors that bind to the receptor tail and regulate its endocytic and signal transducing activities. Using yeast two-hybrid screens,we identified a novel scaffold protein with tetratrico peptide repeats, the megalin-binding protein (MegBP) that associates with the receptor. The binding site of MegBP was mapped to an N-terminal region on the receptor tail harboring a proline-rich peptide element. MegBP binding did not block the endocytic activity of the receptor; however, overexpression resulted in cellular lethality. In further screens, we identified proteins that bound to MegBP and thus might be recruited to the megalin tail. MegBP-interacting partners included several transcriptional regulators such as the SKI-interacting protein (SKIP), a co-activator of the vitamin D receptor. These finding suggest a model whereby megalin directly participates in transcriptional regulation through controlled sequestration or release of transcription factors via MegBP.
The multi-adaptor proto-oncoprotein Cbl is a key regulator of Rac and actin assembly
Authors: Scaife Robin M. , Courtneidge Sara A. , Langdon Wallace Y.
Published: December 2002

The induction of protein tyrosine kinase signaling pathways is a principal mechanism for promoting cellular activation. Biochemical and genetic analyses have implicated the multi-adaptor proto-oncogene protein Cbl as a key negative regulator of activated protein tyrosine kinases. By inhibiting the function of Cbl as a multi-domain adaptor protein, through expression of a truncated form(480-Cbl), we demonstrate that Cbl is a potent negative regulator of actin assembly in response to receptor tyrosine kinase (RTK) activation. Expression of 480-Cbl dramatically enhances RTK-dependent induction of actin dorsal ruffles, which correlates with a pronounced increase in Rac activation. By contrast, mitogenic signaling by RTK targets, such as PI 3-kinase and MAP kinases, as well as RTK-mediated tyrosine phosphorylation do not appear to be affected by 480-Cbl expression. Further, we determined that Cbl undergoes a striking RTK-activation-dependent translocation to sites of active actin dorsal ruffle nucleation. Hence, the selective regulation of RTK signaling to the actin cytoskeleton appears to result from recruitment of signaling proteins on a Cbl template bound to the actin cytoskeleton.
Nuclear oscillations and nuclear filament formation accompany single-strand annealing repair of a dicentric chromosome in Saccharomyces cerevisiae
Authors: Thrower Douglas A. , Stemple Jennifer , Yeh Elaine , Bloom Kerry
Published: December 2002

Dicentric chromosomes undergo breakage during mitosis as a result of the attachment of two centromeres on one sister chromatid to opposite spindle poles. Studies utilizing a conditional dicentric chromosome III in Saccharomyces cerevisiae have shown that dicentric chromosome repair occurs primarily by deletion of one centromere via a RAD52-dependent recombination pathway. We report that dicentric chromosome resolution requires RAD1, a gene involved in the single-strand annealing DNA repair pathway. We additionally show that single-strand annealing repair of a dicentric chromosome can occur in the absence of RAD52. RAD52-independent repair requires the adaptation-defective cdc5-ad allele of the yeast polo kinase and the DNA damage checkpoint gene RAD9. Dicentric chromosome breakage in cdc5-ad rad52mutant cells is associated with a prolonged mitotic arrest, during which nuclei undergo microtubule-dependent oscillations, accompanied by dynamic changes in nuclear morphology. We further demonstrate that the frequency of spontaneous direct repeat recombination is suppressed in yeast cells treated with benomyl, a drug that perturbs microtubules. Our findings indicate that microtubule-dependent processes facilitate recombination.
Phosphorylation of FcγRIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts
Authors: Kwiatkowska Katarzyna , Frey Jürgen , Sobota Andrzej
Published: December 2002

Activation of Fcγ receptor II (FcγRII) induces rearrangement of the actin-based cytoskeleton that serves as a driving force for FcγRII-mediated phagocytosis and FcγRII capping. To get insight into the signaling events that lead to the actin reorganization we investigated the role of raft-associated Src family tyrosine kinases in capping of FcγRII in U937 cells. After crosslinking, FcγRII was found to be recruited to detergent-resistant membrane domains (DRMs), rafts,where it coexisted with Lyn kinase and underwent tyrosine phosphorylation. Lyn was displaced from DRMs under the influence of DL-α-hydroxymyristic acid and 2-bromopalmitic acid, agents blocking N-terminal myristoylation and palmitoylation of proteins, respectively, and after disruption of DRM integrity by depletion of plasma membrane cholesterol withβ-cyclodextrin. Under these conditions, phosphorylation of the crosslinked FcγRII was diminished and assembly of FcγRII caps was blocked. The similar reduction of FcγRII cap formation correlated with inhibition of receptor phosphorylation was achieved with the use of PP1 and herbimycin A, specific inhibitors of Src family tyrosine kinases. Phosphorylation of FcγRIIA expressed in BHK cells, lacking endogenous FcγRs, was abolished by substitution of tyrosine 298 by phenylalanine in the ITAM of the receptor. The mutant receptor did not undergo translocation towards cap-like structures and failed to promote the receptor-mediated spreading of the cells, as compared to BHK cells transfected with the wild-type FcγRIIA. On the basis of these data, we suggest that tyrosine phosphorylation of activated FcγRIIA by raft-residing tyrosine kinases of the Src family triggers signaling pathways that control the rearrangement of the actin cytoskeleton required for FcγRII-mediated motility.
Perfluorocyclobutane‐Based Arylamine Hole‐Transporting Materials for Organic and Polymer Light‐Emitting Diodes
Authors: Jiang X.Z. , Liu S. , Liu M.S. , Herguth P. , Jen A.K.‐Y. , Fong H. , Sarikaya M.
Published: December 2002

A series of in‐situ thermally polymerizable hole‐transporting materials based on the triarylamine‐containing perfluorocyclobutanes (PFCBs) have been developed and characterized. Electrochemical studies reveal that the energy level of the highest occupied molecular orbital (HOMO) for these materials is between –5.1 and –5.3 eV, which is a good match for the work function of indium tin oxide (ITO), a commonly used anode for organic and polymeric light‐emitting diodes. UV‐vis absorption, photo‐ and electro‐luminescence spectral studies indicate that aggregates are formed during the thermal polymerization. The PFCB‐based materials are very robust after polymerization, which enables the fabrication of multilayer polymer light‐emitting diodes. Highly efficient organic and polymer LEDs are demonstrated using these materials as the hole‐transporting layer, illustrating the superb performance of the PFCB‐based polymers.
A Novel Family of Boron‐Containing Hole‐Blocking Amorphous Molecular Materials for Blue‐ and Blue–Violet‐Emitting Organic Electroluminescent Devices
Authors: Kinoshita M. , Kita H. , Shirota Y.
Published: December 2002

A novel family of amorphous molecular materials that function as hole blockers in organic electroluminescent (EL) devices, tris(2,3,5,6‐tetramethylphenyl)borane (TPhB), tris(2,3,5,6‐tetramethylbiphenyl‐4‐yl)borane (TBPhB), tris(2,3,5,6‐tetramethyl‐1,1′;4′,1″‐terphenyl‐4‐yl)borane (TTPhB), and tris[4‐(1,1′;3′,1″‐terphenyl‐5′‐yl)‐2,3,5,6‐tetramethylphenyl]borane (TTPhPhB), have been designed and synthesized. They readily form stable amorphous glasses with high glass‐transition temperatures, and are characterized by reversible cathodic reduction and relatively large HOMO–LUMO energy gaps. High‐performance blue‐ and blue–violet‐emitting organic EL devices have been developed using TBPhB, TTPhB, and TTPhPhB as hole blockers and N,N′‐di(1‐naphthyl)‐N,N′‐diphenyl[1,1′‐biphenyl]‐4,4′‐diamine, tri(p‐terphenyl‐4‐yl)amine, and N,N′‐bis(3‐methylphenyl)‐N,N′‐diphenyl‐[1,1′‐biphenyl]‐4,4′‐diamine as emitters.
Soft Contact Deposition onto Molecularly Modified GaAs. Thin Metal Film Flotation: Principles and Electrical Effects
Authors: Vilan A. , Cahen D.
Published: December 2002

We describe and analyze a process to position a ∼ 1 nm thick molecular layer between two solid surfaces without damage to the molecules. The method is used to deposit a metal film in a soft, gentle manner on a semiconductor, yielding functional semiconductor/molecule/metal junctions. It is a combination of the lift‐off procedure, known from, for example, lithography, and the bonding process, known from, for example, wafer bonding. The combined method may find application also outside the area described here. We point out its major difficulties as well as solutions to overcome them. For this we rely on concepts from the physics of liquid and solid surfaces and interfaces. Conditions are found, in terms of choice of solvents, under which the method will be effective. The efficacy of floatation as a soft contacting procedure is demonstrated by the preparation of Au and Al contacts on GaAs single crystal surfaces, modified by a self‐assembled monolayer of small organic molecules. The resulting electrical properties of the contacts depend crucially on how the molecular interface with the contacting metal is formed. This type of wet contacting procedure to make dry devices may be advantageous especially if biomolecules are used.
Incorporation of Proteins into Polymer Materials by a Novel Supercritical Fluid Processing Method
Authors: Watson M.S. , Whitaker M.J. , Howdle S.M. , Shakesheff K.M.
Published: December 2002

A very clean route to porous materials, utilizing the combination of conventional solution and supercritical fluid processing, is exploited to prepare porous tissue engineering scaffolds that contain biologically active material for controlled release. This allows accurate dosing of the biological agent, whilst retaining the advantages of supercritical fluid technology. The retention of biological activity throughout the processing is also demonstrated. A wide range of alternative morphologies, including microparticles, are possible simply via control of the method of depressurization.
Low‐Temperature Self‐Assembly of Novel Encapsulated Compound Nanowires
Authors: Hofmann S. , Ducati C. , Robertson J.
Published: December 2002

Nickel sulfide nanowires and compound‐filled MoS2 nanotubes have been grown by controlled annealing of patterned fullerene and nickel thin films on molybdenum substrates. The versatile synthesis route gives highly crystalline nanostructures at temperatures below 550 °C with a high degree of positional control. The Figure shows a portion of an as‐grown nickel sulfide nanowire 19 nm in diameter (scale bar 2 nm).
Synthesis, Structural Evaluation, and Estrogen Receptor Interaction of 4, 5‐Bis(4‐hydroxyphenyl)imidazoles
Authors: Gust Ronald , Keilitz Roland , Schmidt Kathrin , von Rauch Moriz
Published: December 2002

4, 5‐Bis(4‐hydroxyphenyl)imidazoles with 2, 2′‐H (1), 2, 2′‐F (2), 2, 2′‐Cl (3), and 2, 2′6‐Cl (4) substituents in the aromatic rings were synthesized by oxidation of the respective methoxy‐substituted (R, S)/(S, R)‐4, 5‐diaryl‐2‐imidazolines with MnO2 and subsequent ether cleavage with BBr3. N‐alkylation of 1 and 3 with ethyl iodide yielded the compounds 5 and 6. The imidazoles were characterized by NMR spectroscopy and tested for estrogen receptor binding in a competition experiment with [3H]estradiol using calf uterine cytosol. Gene activation was verified in a luciferase assay using estrogen receptor positive MCF‐7‐2a cells stably transfected with the plasmid EREwtcluc. All halide substituted imidazoles competed with estradiol for the binding site at the estrogen receptor. The N‐ethyl derivative 6 showed the highest relative binding affinity of 1.26 %. Treatment of MCF‐7‐2a cells, however, did not lead to gene activation. The relative activation of 6 amounted only to 10 % at 1μM compared to E2 (100 %).
[60]Fullerene: A Versatile Photoactive Core for Dendrimer Chemistry
Authors: Nierengarten Jean‐François , Armaroli Nicola , Accorsi Gianluca , Rio Yannick , Eckert Jean‐François
Published: December 2002

Owing to their special photophysical properties, fullerene derivatives are good candidates to demonstrate dendritic effects. In particular, the triplet lifetimes of a C60 core can be used to evaluate its degree of isolation from external contacts. On the other hand, the fullerene core can act as a terminal energy receptor in dendrimer‐based light‐harvesting systems. When a fullerodendrimer is further functionalized with a suitable electron donor, it may exhibit the essential features of a multicomponent artificial photosynthetic system in which photoinduced energy transfer from the antenna to the C60 core is followed by electron transfer.
Modular Synthesis of Heparin Oligosaccharides
Authors: Orgueira Hernán A. , Bartolozzi Alessandra , Schell Peter , Litjens Remy E. J. N. , Palmacci Emma R. , Seeberger Peter H.
Published: December 2002

A general, modular strategy for the first completely stereoselective synthesis of defined heparin oligosaccharides is described. Six monosaccharide building blocks (four differentially protected glucosamines, one glucuronic and one iduronic acid) were utilized to prepare di‐ and trisaccharide modules in a fully selective fashion. Installation of the α‐glucosamine linkage was controlled by placing a conformational constraint on the uronic acid glycosyl acceptors thereby establishing a new concept for stereochemical control. Combination of disaccharide modules to form trans‐uronic acid linkages was completely selective by virtue of C2 participating groups. Coupling reactions between disaccharide modules exhibited sequence dependence. While the union of many glucosamine uronic acid disaccharide modules did not meet any problems, certain sequences proved not accessible. Elaboration of glucosamine uronic acid disaccharide building blocks to trisaccharide modules by addition of either one additional glucosamine or uronic acid allowed for stereoselective access to oligosaccharides as demonstrated on the example of a hexasaccharide resembling the ATIII‐binding sequence. Final deprotection and sulfation yielded the fully synthetic heparin oligosaccharides.
Formation and Chemical Reactivities of a New Type of Double‐Butterfly [{Fe2(μ‐CO)(CO)6}2(μ‐SZS‐μ)]2−: Synthetic and Structural Studies on Novel Linear and Macrocyclic Butterfly Fe/E (E=S, Se) Cluster Complexes
Authors: Song Li‐Cheng , Fan Hong‐Tao , Hu Qing‐Mei , Yang Zhi‐Yong , Sun Yi , Gong Feng‐Hua
Published: December 2002

A new type of double‐butterfly [{Fe2(μ‐CO)(CO)6}2(μ‐SZS‐μ)]2− (3), a dianion that has two μ‐CO ligands, has been synthesized from dithiol HSZSH (Z=(CH2)4, CH2(CH2OCH2)1–3CH2), [Fe3(CO)12], and Et3N in a molar ratio of 1:2:2 at room temperature. Interestingly, the in situ reactions of dianions 3 with various electrophiles affords a series of novel linear and macrocyclic butterfly Fe/E (E=S, Se) cluster complexes. For instance, while reactions of 3 with PhC(O)Cl and Ph2PCl give linear clusters [{Fe2(μ‐PhCO)(CO)6}2(μ‐SZS‐μ)] (4 a,b: Z=CH2(CH2OCH2)2,3CH2) and [{Fe2(μ‐Ph2P)(CO)6}2(μ‐SZS‐μ)] (5 a,b: Z=CH2(CH2OCH2)2,3CH2), reactions with CS2 followed by treatment with monohalides RX or dihalides XYX give both linear clusters [{Fe2(μ‐RCS2)(CO)6}2(μ‐SZS‐μ)] (6 a–e: Z=CH2(CH2OCH2)1,2CH2; R=Me, PhCH2, FeCp(CO)2) and macrocyclic clusters [{Fe2(CO)6}2(μ‐SZS‐μ)(μ‐CS2YCS2‐μ)] (7 a–e: Z=(CH2)4, CH2(CH2OCH2)1–3CH2; Y=(CH2)2–4, 1,3,5‐Me(CH2)2C6H3, 1,4‐(CH2)2C6H4). In addition, reactions of dianions 3 with [Fe2(μ‐S2)(CO)6] followed by treatment with RX or XYX give linear clusters [{[Fe2(CO)6]2(μ‐RS)(μ4‐S)}2(μ‐SZS‐μ)] (8 a–c: Z=CH2(CH2OCH2)1,2CH2; R=Me, PhCH2) and macrocyclic clusters [{[Fe2(CO)6]2(μ4‐S)}2(μ‐SYS‐μ)(μ‐SZS‐μ)] (9 a,b: Z=CH2(CH2OCH2)2,3CH2; Y=(CH2)4), and reactions with SeCl2 afford macrocycles [{Fe2(CO)6}2(μ4‐Se)(μ‐SZS‐μ)] (10 d: Z=CH2(CH2OCH2)3CH2) and [{[Fe2(CO)6]2(μ4‐Se)}2(μ‐SZS‐μ)2] (11 a–d: Z=(CH2)4, CH2(CH2OCH2)1–3CH2). Production pathways have been suggested; these involve initial nucleophilic attacks by the Fe‐centered dianions 3 at the corresponding electrophiles. All the products are new and have been characterized by combustion analysis and spectroscopy, and by X‐ray diffraction techniques for 6 c, 7 d, 9 b, 10 d, and 11 c in particular. X‐ray diffraction analyses revealed that the double‐butterfly cluster core Fe4S2Se in 10 d is severely distorted in comparison to that in 11 c. In view of the Z chains in 10 a–c being shorter than the chain in 10 d, the double cluster core Fe4S2Se in 10 a–c would be expected to be even more severely distorted, a possible reason for why 10 a–c could not be formed.
Metal Catalysis inside Polymer Frameworks: Evaluation of Catalyst Stability and Reusability
Authors: Králik Milan , Kratky Vieroslav , De Rosso Mirco , Tonelli Massimo , Lora Silvano , Corain Benedetto
Published: December 2002

The polymer framework of a resin‐based catalyst built up with Pd nanoclusters (ca. 3 nm) dispersed inside the nanoporous domains of a thermally stable gel‐type polyacrylic resin exhibits a good chemical stability under 5 bar H2 at 40 °C for reasonable contact times. Chemical and physico‐chemical integrity of the polymer framework are checked with a variety of instrumental analytical methods. Catalyst reusability turns out to be quite good.