bluuebunny/crossref_metadata_2025 huggingface dataset:
Agreement Between Ambulance Paramedic- and Physician-Recorded Neurological Signs With Face Arm Speech Test (FAST) in Acute Stroke Patients
Authors: Nor A. Mohd , McAllister C. , Louw S.J. , Dyker A.G. , Davis M. , Jenkinson D. , Ford G.A.
Published: May 2004

Background and Purpose—

Patients with suspected stroke first assessed by ambulance paramedics require early recognition to facilitate appropriate triage and early treatment. We determined paramedic’s accuracy in detecting acute stroke signs by comparing agreement between neurological signs recorded in the Face Arm Speech Test (FAST), a stroke recognition instrument, by paramedics on the scene and by stroke physicians after admission.

Methods—

Suspected stroke patients admitted by ambulance paramedics directly to an acute stroke unit through a rapid ambulance protocol were examined by a trainee stroke neurologist or admitting stroke physician over a 1-year period. Recorded neurological signs (facial weakness, arm weakness, speech disturbance) in confirmed acute stroke/transient ischemic attack (TIA) cases were compared between paramedics and the stroke neurologist/physician.

Results—

Ambulance crews referred 278 suspected stroke patients of whom 217 (78%) had confirmed stroke (n=189) or TIA (n=28); 95% were examined by the stroke neurologist (median 18 hours after paramedic assessment). Recorded signs and agreement between paramedics and stroke physicians in confirmed stroke group were: facial weakness, 68% versus 70% (κ=0.49; 95% CI: 0.36 to 0.62); arm weakness, 96% versus 95% (κ=0.77; 95% CI: 0.55 to 0.99); and speech disturbance, 79% versus 77% (κ=0.69; 95% CI: 0.56 to 0.82). Interrater agreement was complete for arm weakness in 98% cases.

Conclusions—

Recognition of neurological deficits by ambulance paramedics using FAST shows good agreement with physician assessment, even allowing for temporal evolution of deficits. The high prevalence and good agreement for arm weakness suggest that this sign may have the greatest usefulness for prehospital ambulance triage and paramedic-based neuroprotective trials.
The etiology of severe anemia in a village and a periurban area in Mali
Authors: Dicko Alassane , Klion Amy D. , Théra Mahamadou A. , Sagara Issaka , Yalcouyé Daniel , Niambele Mohamed B. , Sogoba Moussa , Dolo Guimogo , Dao Adama , Diallo Dapa A. , Doumbo Ogobara K. , Miller Louis H.
Published: May 2004

Severe anemia is one of the major complications of malaria in Africa. We studied 2 populations, one in a village and the second in a periurban area in Mali, to understand the preventable factors in the disease. The 2 correlates of disease were parasitemia above 100 000 parasitized red blood cells per microliter (0.1 × 1012/L) and a low baseline hemoglobin level. All cases of moderate to severe anemia occurred in children under 3.2 years of age. Raising the baseline hemoglobin level and lowering peak parasitemia in infants and young children may reduce the incidence of severe anemia resulting from malarial infection. (Blood. 2004;104: 1198-1200)
Tuning the volume of the immune response: strength and persistence of stimulation determine migration and cytokine secretion of dendritic cells
Authors: Luft Thomas , Maraskovsky Eugene , Schnurr Max , Knebel Katja , Kirsch Michael , Görner Martin , Skoda Radek , Ho Anthony D. , Nawroth Peter , Bierhaus Angelika
Published: May 2004

Migration to lymph nodes and secretion of cytokines are critical functions of mature dendritic cells (DCs); however, these 2 functions are not necessarily linked. This is the first report showing that quantitative differences in identical signaling pathways determine DC migration and cytokine secretion. Using different polymerized forms of CD40 ligand, we demonstrate that the strength and persistence of CD40 signaling can induce either function. Induction of monocyte-derived DC (MoDC) migration required a weak and transient CD40 signal, whereas strong and persistent CD40 signaling blocked migration and biased toward cytokine secretion. In contrast to MoDCs, CD40 activation of CD1c+ peripheral blood DCs (PBDCs) induced a nonpersistent, intracellular signaling profile resulting in migratory-type DCs unable to secrete interleukin-12p70 (IL-12p70). Extracellular signal-regulated kinase 1/2 (ERK1/2) and p38K activation synergistically mediated cytokine secretion, whereas migration was enhanced by p38K activation but reduced by persistent ERK1/2 activity. This model of signal strength and persistence also applied when stimulating DCs with intact microbes. Thus, a novel concept emerges in which the type of immune response induced by DCs is tuned by the strength and persistence of DC activating signals.
Pleiotropic consequences of Bruton tyrosine kinase deficiency in myeloid lineages lead to poor inflammatory responses
Authors: Mangla Anita , Khare Anupriya , Vineeth Varanasi , Panday Nagesh Narayan , Mukhopadhyay Asok , Ravindran Balachandran , Bal Vineeta , George Anna , Rath Satyajit
Published: May 2004

Bruton tyrosine kinase (Btk), a non-receptor-associated tyrosine kinase of the Tec family, appears to participate in many myeloid cell functions. We show that macrophages from X-linked immunodeficient (XID) mice lacking functional Btk cannot generate efficient bursts of reactive oxygen intermediates (ROIs). The induction of apoptotic cell death by inflammatory stimuli is also enhanced in XID macrophages. Phagocytosis of bacterial particles is only marginally affected in them. In vivo, XID mice show reduced severity of inflammatory diseases in models of experimental autoimmune encephalomyelitis (EAE), dextran sulfate sodium (DSS)-induced colitis, and carrageenan-induced acute edema. Also, polymorphonuclear neutrophil granulocytes (PMNs) in XID mice show poor ROI and nitric oxide (NO) induction, along with a reduction in PMN recruitment to peritoneal inflammation. XID mice show reduction in PMN numbers in peripheral blood, and their bone marrow shows a reduction in the numbers of both monocytic and granulocytic lineages, extending to the earliest progenitor populations. Thus, Btk is likely to play a significant role at multiple points during the development and functioning of the myeloid lineages, affecting the outcome of many infectious as well as noninfectious inflammatory events in vivo. (Blood. 2004;104:1191-1197)
Risk factors for syngeneic graft-versus-host disease after adult hematopoietic cell transplantation
Authors: Adams Kristina M. , Holmberg Leona A. , Leisenring Wendy , Fefer Alexander , Guthrie Katherine A. , Tylee Tracy S. , McDonald George B. , Bensinger William I. , Nelson J. Lee
Published: May 2004

Syngeneic graft-versus-host disease (sGVHD) has been described after hematopoietic cell transplantation (HCT) but remains poorly defined. We retrospectively reviewed adult syngeneic HCTs at our center (1980-2002) for sGVHD to investigate incidence, morbidity, and risk factors with a primary focus on parity. Among 119 transplantations, there were 21 cases of biopsy-proven sGVHD. The cumulative incidence was 18%, with multiorgan involvement in 6 cases and 1 death. sGVHD was more frequent when the donor was parous (32%) than nulliparous (9%) or male (13%; P = .03) and when the recipient was parous (31%) than nulliparous (7%) or male (13%; P = .02). Other univariable risk factors included older age (P < .01), busulfan/melphalan/thiotepa conditioning (P < .01), interleukin-2 (P = .02), HLA-A26 (P = .03), and more recent transplantation year (P < .01). Overall, risk factors were similar to those described in GVHD. Although an independent effect of parity could not be completely separated from other factors, donor and recipient pregnancy history merits further investigation.
Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules
Authors: Bonaparte Matthew I. , Barker Edward
Published: May 2004

In the current study, we evaluated whether the capacity of HIV to modulate major histocompatibility complex (MHC) class I molecules has an impact on the ability of autologous natural killer (NK) cells to kill the HIV-infected cells. Analysis of HIV-infected T-cell blasts revealed that the decrease in MHC class I molecules on the infected cell surface was selective. HLA-A and -B were decreased on cells infected with HIV strains that could decrease MHC class I molecules, whereas HLA-C and -E remained on the surface. Blocking the interaction between HLA-C and -E and their corresponding inhibitory receptors increased NK cell killing of T-cell blasts infected with HIV strains that reduced MHC class I molecules. Moreover, we demonstrate that NK cells lacking HLA-C and -E inhibitory receptors kill T-cell blasts infected with HIV strains that decrease MHC class I molecules. In contrast, NK cells are incapable of destroying T-cell blasts infected with HIV strains that were unable to reduce MHC class I molecules. These findings suggest that NK cells lacking inhibitory receptors to HLA-C and -E kill HIV-infected CD4+ T cells, and they indicate that the capacity of NK cells to destroy HIV-infected cells depends on the ability of the virus to modulate MHC class I molecules. (Blood. 2004;104:2087-2094)
Continuous in vivo infusion of interferon-gamma (IFN-γ) preferentially reduces myeloid progenitor numbers and enhances engraftment of syngeneic wild-type cells in Fancc-/- mice
Authors: Li Xiaxin , Yang Yanzhu , Yuan Jin , Hong Ping , Freie Brian , Orazi Attilio , Haneline Laura S. , Clapp D. Wade
Published: May 2004

Fanconi anemia (FA) is characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of many FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. Previous studies in FA murine models and in a phase 1 clinical trial suggest that myelopreparation is required for significant engraftment of exogenous, genetically corrected stem cells. Since myeloid progenitors from Fancc-/- mice and human Fanconi anemia group C protein (FANCC) patients have increased apoptosis in response to interferon γ (IFN-γ) in vitro, we hypothesized that IFN-γ may be useful as a nongenotoxic, myelopreparative conditioning agent. To test this hypothesis, IFN-γ was administered as a continuous infusion to Fancc-/- and wild-type (WT) mice for 1 week. Primitive and mature myeloid lineages were preferentially reduced in IFN-γ-treated Fancc-/- mice. Further, IFN-γ conditioning of Fancc-/- recipients was sufficient as a myelopreparative regimen to allow consistent engraftment of isogenic WT repopulating stem cells. Collectively, these data demonstrate that Fancc-/- hematopoietic cell populations have increased hypersensitivity to IFN-γ in vivo and that IFN-γ conditioning may be useful as a nongenotoxic strategy for myelopreparation in this disorder. (Blood. 2004;104:1204-1209)
CD1d-restricted “NKT” cells and myeloid IL-12 production: an immunological crossroads leading to promotion or suppression of effective anti-tumor immune responses?
Authors: Gumperz Jenny E
Published: May 2004

CD1d-restricted T cells are remarkable for their unusual ability to respond to self-antigens and to contribute to both immunostimulatory and immunosuppressive responses. Their effects in different cancer models have appeared contradictory; in some cases, they are linked to the generation of effective tumor clearance, and in others, they seem to contribute to suppression of anti-tumor responses. Recent results suggest CD1d-restricted T cells are involved in critical interactions with myeloid dendritic cells (DCs) that can affect the subsequent course of the immune response, and that factors such as the strength of the antigenic signal and the presence or absence of proinflammatory cytokines may determine the outcome of these interactions. In the presence of a strong antigenic signal, CD1d-restricted T cells induced myeloid DCs to secrete interleukin (IL)-12, and these DCs in turn activated naive T cells to secrete Th1 cytokines. When exposed to the weak antigenic stimulus of self-antigens, CD1d-restricted T cells induced DCs to secrete IL-10 but not IL-12, and these DCs failed to stimulate Th1 cytokine production by naive T cells. In contrast, CD1d-restricted T cells that were stimulated by self-antigens in the presence of IL-12 potently secreted interferon-γ (IFN-γ) and were among the first lymphocytes to become activated in vivo. Hence, CD1d-restricted T cells may promote or prevent effective anti-tumor responses that are mediated by other lymphocytic effector cells by influencing IL-12 production by myeloid DCs and by their own production of early IFN-γ in response to IL-12.
IFN-γ inhibits the proliferation of allergen-activated T lymphocytes from atopic, asthmatic patients by inducing Fas/FasL-mediated apoptosis
Authors: De Rose Virginia , Cappello Paola , Sorbello Valentina , Ceccarini Barbara , Gani Federica , Bosticardo Marita , Fassio Stefania , Novelli Francesco
Published: May 2004

The defect in interferon-γ (IFN-γ) production that results in a T helper cell type 2-dominated response may be responsible for a decrease in the apoptosis of allergen-activated T cells in asthma. We investigated the effect of recombinant IFN-γ on proliferation, Fas/Fas ligand (FasL) expression, and apoptosis in allergen-stimulated peripheral blood mononuclear cells obtained from atopic, asthmatic patients and nonatopic, control subjects. The addition of IFN-γ at the start of cultures markedly inhibited the proliferative response to a specific allergen in cells from all asthmatic patients, whereas no change was observed in cells from nonatopic, control subjects. IFN-γ induced an increase in the expression of Fas and FasL by allergen-stimulated CD4+ T cells from asthmatic patients and caused the apoptosis of these cells. A Fas-blocking monoclonal antibody prevented the inhibitory effect of IFN-γ on allergen-induced proliferation. These results suggest that IFN-γ inhibits the proliferation of allergen-stimulated CD4+ T cells from atopic, asthmatic patients by inducing the surface expression of Fas and FasL, which in turn triggers their apoptotic program. The defect in IFN-γ production involved in the allergic, immune response may therefore be responsible for a decrease in apoptosis of allergen-activated T lymphocytes in the airways of atopic, asthmatic patients.
Interleukin-15 enhances human neutrophil phagocytosis by a Syk-dependent mechanism: importance of the IL-15Rα chain
Authors: Ratthé Claude , Girard Denis
Published: May 2004

Interleukin-15 (IL-15) is a cytokine that possesses interesting, potential therapeutic properties. However, based on several parameters including activation of neutrophils, it is also recognized as a proinflammatory cytokine. The mechanisms by which IL-15 activates human neutrophil functions are not fully understood. Although these cells express a functional IL-15 receptor (IL-15R) composed of IL-15Rα, IL-2/15Rβ (CD122), and γc (CD132) subunits, the role of each receptor component has not been investigated in IL-15-induced human neutrophil responses. In the present study, fluorescein-activated cell sorter analysis revealed that the ability of IL-15 to enhance neutrophil phagocytosis is not a result of increased expression of IL-15Rα, CD122, or CD132 on the neutrophil cell surface. Pretreatment of neutrophils with specific antibodies to IL-15Rα, CD122, or CD132 was found to inhibit phagocytosis of opsonized-sheep red blood cells by nearly 40%, 21%, and 27%, respectively. As expected, pretreatment of neutrophils with anti-IL-2Rα (CD25) had no effect. Pretreatment of cells with the Syk inhibitor piceatannol was found to significantly inhibit the ability of IL-15 to enhance phagocytosis. In addition, IL-15 was found to induce tyrosine phosphorylation of Syk that was largely inhibited by pretreating cells with piceatannol. Moreover, we found that Syk kinase is physically associated with IL-15Rα. We conclude that IL-15R enhances neutrophil phagocytosis by a Syk-dependent mechanism and that the IL-15Rα chain plays a key role in mediating this response, at least by interacting with Syk kinase.
Cytosolic phospholipase A2 is responsible for prostaglandin E2 and leukotriene B4 formation in phagocyte-like PLB-985 cells: studies of differentiated cPLA2-deficient PLB-985 cells
Authors: Liberty I Furstenberg , Raichel L , Hazan-Eitan Z , Pessach I , Hadad N , Schlaeffer F , Levy R
Published: May 2004

Our previously established model of cytosolic phospholipase A2 (cPLA2)-deficient, differentiated PLB-985 cells (PLB-D cells) was used to determine the physiological role of cPLA2 in eicosanoid production. Parent PLB-985 (PLB) cells and PLB-D cells were differentiated toward the monocyte or granulocyte lineages using 5 × 10−8 M 1,25 dihydroxyvitamin D3 or 1.25% dimethyl sulfoxide, respectively. Parent monocyte- or granulocyte-like PLB cells released prostaglandin E2 (PGE2) when stimulated by ionomycin, A23187, opsonized zymosan, phorbol 12-myristate 13-acetate, or formyl-Met-Leu-Phe (fMLP), and monocyte- or granulocyte-like PLB-D cells did not release PGE2 with any of the agonists. The kinetics of cPLA2 translocation to nuclear fractions in monocyte-like PLB cells stimulated with fMLP or ionomycin was in correlation with the kinetics of PGE2 production. Granulocyte-like PLB cells, but not granulocyte-like PLB-D cells, secreted leukotriene B4 (LTB4) after stimulation with ionomycin or A23187. Preincubation of monocyte-like parent PLB cells with 100 ng/ml lipopolysaccharide (LPS) for 16 h enhanced stimulated PGE2 production, which is in correlation with the increased levels of cPLA2 detected in these cells. LPS preincubation was less potent in increasing PGE2 and LTB4 secretion and did not affect cPLA2 expression in granulocyte-like PLB cells, which may be a result of their lower levels of surface LPS receptor expression. LPS had no effect on monocyte- or granulocyte-like PLB-D cells. The lack of eicosanoid formation in stimulated, differentiated cPLA2-deficient PLB cells indicates that cPLA2 contributes to stimulated eicosanoid formation in monocyte- and granulocyte-like PLB cells.
Regulation of interleukin-12 gene expression and its anti-tumor activities by prostaglandin E2 derived from mammary carcinomas
Authors: Mitsuhashi Maki , Liu Jianguo , Cao Shanjin , Shi Xiaoyan , Ma Xiaojing
Published: May 2004

Interleukin-12 (IL-12)-mediated immune responses are critical for the control of malignant development. Tumors can actively resist detrimental immunity of the host via many routes. Prostaglandin E2 (PGE2) is one of the major immune-suppressive factors derived from many types of tumors. Here, we show that systemic administration of recombinant IL-12 could therapeutically control the growth of aggressive TS/A and 4T1 mouse mammary carcinomas. However, PGE2 produced by tumors potently inhibits the production of endogenous IL-12 at the level of protein secretion, mRNA synthesis, and transcription of the constituent p40 and p35 genes. The inhibition can be reversed by NS-398, a selective inhibitor of the enzymatic activity of cyclooxygenase 2 in PGE2 synthesis. Moreover, PGE2-mediated inhibition of IL-12 production requires the functional cooperation of AP-1 and AP-1 strongly suppresses IL-12 p40 transcription. Blocking PGE2 production in vivo results in a marked reduction in lung metastasis of 4T1 tumors, accompanied by enhanced ability of peritoneal macrophages to produce IL-12 and spleen lymphocytes to produce interferon-γ. This study contributes to the elucidation of the molecular mechanisms underlying the interaction between a progressive malignancy and the immune defense apparatus.
Non-pharmacological interventions in dementia
Authors: Douglas Simon , James Ian , Ballard Clive
Published: May 2004

It is increasingly recognised that pharmacological treatments for dementia should be used as a second-line approach and that non-pharmacological options should, in best practice, be pursued first. This review examines current non-pharmacological approaches. It highlights the more traditional treatments such as behavioural therapy, reality orientation and validation therapy, and also examines the potential of interesting new alternative options such as cognitive therapy, aromatherapy and multisensory therapies. The current literature is explored with particular reference to recent research, especially randomised controlled trials in the area. Although many non-pharmacological treatments have reported benefits in multiple research studies, there is a need for further reliable and valid data before the efficacy of these approaches is more widely recognised.
Doctor to doctor: getting on with colleagues
Authors: Garelick Antony , Fagin Leonard
Published: May 2004

Doctor-to-doctor relationships are explored. The impact of the employing organisation on working relationships is considered and methods of analysing problems at the organisational and individual level are described. The application of psychodynamic and systems theory in deepening understanding is discussed, illustrated by vignettes from the work situation. The impact of changes in the work environment are considered and potential sources of support listed.
Exposure of Melanoma Cells to Dacarbazine Results in Enhanced Tumor Growth and Metastasis In Vivo
Authors: Lev Dina Chelouche , Onn Amir , Melinkova Vladislava O. , Miller Claudia , Stone Valerie , Ruiz Maribelis , McGary Eric C. , Ananthaswamy Honnavara N. , Price Janet E. , Bar-Eli Menashe
Published: May 2004

Purpose

In recent years, the incidence of cutaneous melanoma has increased more than that of any other cancer. Dacarbazine is considered the gold standard for treatment, having a response rate of 15% to 20%, but most responses are not sustained. Previously, we have shown that short exposure of primary cutaneous melanoma cells to dacarbazine resulted in the upregulation of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). The purpose of the present study was to determine how long-term exposure of melanoma cells to dacarbazine would affect their tumorigenic and metastatic potential in vivo.

Materials and Methods

The primary cutaneous melanoma cell lines SB2 and MeWo were repeatedly exposed in vitro to increasing concentrations of dacarbazine, and dacarbazine-resistant cell lines SB2-D and MeWo-D were selected and examined for their ability to grow and metastasize in nude mice.

Results

The dacarbazine-resistant cell lines SB2-D and MeWo-D exhibited increased tumor growth and metastatic behavior in vivo. This increase could be explained by the activation of RAF, MEK, and ERK, which led to the upregulation of IL-8 and VEGF. More IL-8, VEGF, matrix metalloproteinase-2 (MMP-2), and microvessel density (CD-31) were found in tumors produced by SB2-D and MeWo-D in vivo than in those produced by their parental counterparts. No mutations were observed in BRAF.

Conclusion

Our results have significant clinical implications. Treatment of melanoma patients with dacarbazine could select for a more aggressive melanoma phenotype. We propose that combination treatment with anti-VEGF/IL-8 or MEK inhibitors may potentiate the therapeutic effects of dacarbazine.
Fate of Internalized Thyrotropin-Releasing Hormone Receptors Monitored with a Timer Fusion Protein
Authors: Cook Laurie B. , Hinkle Patricia M.
Published: May 2004

Trafficking of TRH receptors was studied in a stable HEK293 cell line expressing receptor fused to a Timer protein (TRHR-Timer) that spontaneously changes from green to red over 10 h. Cells expressing TRHR-Timer responded to TRH with an 11-fold increase in inositol phosphate formation, increased intracellular free calcium, and internalization of 75% of bound [3H][N3-methyl-His2]TRH within 10 min. After a 20-min exposure to TRH at 37 C, 75–80% of surface binding sites disappeared as receptors internalized. When TRH was removed and cells incubated in hormone-free medium, approximately 75% of [3H][N3-methyl-His2]TRH binding sites reappeared at the surface over the next 2 h with or without cycloheximide. Trafficking of TRHR-Timer was monitored microscopically after addition and withdrawal of TRH. In untreated cells, both new (green) and old (red) receptors were seen at the plasma membrane, and TRH caused rapid movement of young and old receptors into cytoplasmic vesicles. When TRH was withdrawn, some TRHR-Timer reappeared at the plasma membrane after several hours, but much of the internalized receptor remained intracellular in vesicles that condensed to larger structures in perinuclear regions deeper within the cell. Strikingly, receptors that moved to the plasma membrane were generally younger (more green) than those that underwent endocytosis. There was no change in the red to green ratio over the course of the experiment in cells exposed to vehicle. The results indicate that, after agonist-driven receptor internalization, the plasma membrane is replenished with younger receptors, arising either from an intracellular pool or preferential recycling of younger receptors.
Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element
Authors: Lee Leo Tsz-On , Tan-Un Kian-Cheng , Pang Ronald Ting-Kai , Lam David Tai-Wai , Chow Billy Kwok-Chong
Published: May 2004

To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (−11 to −341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (−336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5′-Aza-2′ deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.
Hypomorphic expression ofDkk1in thedoubleridgemouse: dose dependence and compensatory interactions withLrp6
Authors: MacDonald Bryan T. , Adamska Maja , Meisler Miriam H.
Published: May 2004

doubleridge is a transgene-induced mouse mutation displaying forelimb postaxial polysyndactyly. We have cloned the doubleridgetransgene insertion site and demonstrate that doubleridge acts in cis from a distance of 150 kb to reduce the expression of dickkopf 1(Dkk1), the secreted Wnt antagonist. Expression of Dkk1 from the doubleridge allele ranges from 35% of wild-type level in E7.0 head to <1% of wild type in E13.5 tail. doubleridge homozygotes and doubleridge/null compound heterozygotes are viable. An allelic series combining the wild-type, doubleridge and null alleles of Dkk1 demonstrates the effect of varying Dkk1 concentration on development of limb, head and vertebrae. Decreasing expression of Dkk1 results in hemivertebral fusions in progressively more anterior positions, with severity increasing from tail kinks to spinal curvature. We demonstrated interaction between Dkk1 and the Wnt coreceptors Lrp5 and Lrp6 by analysis of several types of double mutants. The polydactyly of Dkk1d/d mice was corrected by reduced expression of Lrp5 or Lrp6. The posterior digit loss and axial truncation characteristic of Lrp6 null mice was partially corrected by reduction of Dkk1. Similarly, the anterior head truncation characteristic of Dkk1 null mice was rescued by reduction of Lrp6. These compensatory interactions between Dkk1 and Lrp6 demonstrate the importance of correctly balancing positive and negative regulation of Wnt signaling during mammalian development.
The ascidianMespgene specifies heart precursor cells
Authors: Satou Yutaka , Imai Kaoru S. , Satoh Nori
Published: May 2004

Understanding the molecular basis of heart development is an important research area, because malformation of the cardiovascular system is among the most frequent inborn defects. Although recent research has identified molecules responsible for heart morphogenesis in vertebrates, the initial specification of heart progenitors has not been well characterized. Ascidians provide an appropriate experimental system for exploring this specification mechanism, because the lineage for the juvenile heart is well characterized,with B7.5 cells at the 110-cell stage giving rise to embryonic trunk ventral cells (TVCs) or the juvenile heart progenitors. Here, we show that Cs-Mesp, the sole ortholog of vertebrate Mesp genes in the ascidian Ciona savignyi, is specifically and transiently expressed in the embryonic heart progenitor cells (B7.5 cells). Cs-Mesp is essential for the specification of heart precursor cells, in which Nkx, HAND and HAND-like (NoTrlc) genes are expressed. As a result, knockdown of Cs-Mesp with specific morpholino antisense oligonucleotides causes failure of the development of the juvenile heart. Together with previous evidence obtained in mice, the present results suggest that a mechanism for heart specification beginning with Mespthrough Nkx and HAND is conserved among chordates.
Complexin I regulates glucose-induced secretion in pancreatic β-cells
Authors: Abderrahmani Amar , Niederhauser Guy , Plaisance Valérie , Roehrich Marc-Estienne , Lenain Vincent , Coppola Thierry , Regazzi Romano , Waeber Gérard
Published: May 2004

The neuronal-specific protein complexin I (CPX I) plays an important role in controlling the Ca2+-dependent neurotransmitter release. Since insulin exocytosis and neurotransmitter release rely on similar molecular mechanisms and that pancreatic β-cells and neuronal cells share the expression of many restricted genes, we investigated the potential role of CPX I in insulin-secreting cells. We found that pancreatic islets and several insulin-secreting cell lines express high levels of CPX I. The β-cell expression of CPX I is mediated by the presence of a neuron restrictive silencer element located within the regulatory region of the gene. This element bound the transcriptional repressor REST, which is found in most cell types with the exception of mature neuronal cells and β-cells. Overexpression of CPX I or silencing of the CPX I gene (Cplx1) by RNA interference led to strong impairment in β-cell secretion in response to nutrients such as glucose, leucine and KCl. This effect was detected both in the early and the sustained secretory phases but was much more pronounced in the early phase. We conclude that CPX I plays a critical role in β-cells in the control of the stimulated-exocytosis of insulin.
Processing and activation of latent heparanase occurs in lysosomes
Authors: Zetser Anna , Levy-Adam Flonia , Kaplan Victoria , Gingis-Velitski Svetlana , Bashenko Yulia , Schubert Shay , Flugelman Moshe Y. , Vlodavsky Israel , Ilan Neta
Published: May 2004

Heparanase is a heparan sulfate degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Heparanase is synthesized as a 65 kDa non-active precursor that subsequently undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The protease responsible for heparanase processing is currently unknown, as is the sub-cellular processing site. In this study, we characterize an antibody (733) that preferentially recognizes the active 50 kDa heparanase form as compared to the non-active 65 kDa heparanase precursor. We have utilized this and other anti-heparanase antibodies to study the cellular localization of the latent 65 kDa and active 50 kDa heparanase forms during uptake and processing of exogenously added heparanase. Interestingly, not only the processed 50 kDa, but also the 65 kDa heparanase precursor was localized to perinuclear vesicles, suggesting that heparanase processing occurs in lysosomes. Indeed, heparanase processing was completely inhibited by chloroquine and bafilomycin A1, inhibitors of lysosome proteases. Similarly, processing of membrane-targeted heparanase was also chloroquine-sensitive, further ruling out the plasma membrane as the heparanase processing site. Finally, we provide evidence that antibody 733 partially neutralizes the enzymatic activity of heparanase, suggesting that the N-terminal region of the molecule is involved in assuming an active conformation. Monoclonal antibodies directed to this region are likely to provide specific heparanase inhibitors and hence assist in resolving heparanase functions under normal and pathological conditions.
A novel isoform of sarcolemmal membrane-associated protein (SLMAP) is a component of the microtubule organizing centre
Authors: Guzzo Rosa M. , Sevinc Serdal , Salih Maysoon , Tuana Balwant S.
Published: May 2004

The microtubule organizing centre (MTOC) or the centrosome serves a crucial role in the establishment of cellular polarity, organization of interphase microtubules and the formation of the bipolar mitotic spindle. We have elucidated the genomic structure of a gene encoding the sarcolemmal membrane-associated protein (SLMAP), which encodes a 91 kDa polypeptide with a previously uncharacterized N-terminal sequence encompassing a forkhead-associated (FHA) domain that resides at the centrosome. Anti-peptide antibodies directed against SLMAP N-terminal sequences showed colocalization with γ-tubulin at the centrosomes at all phases of the cell cycle. Agents that specifically disrupt microtubules did not affect SLMAP association with centrosomes. Furthermore, SLMAP sequences directed a reporter green fluorescent protein (GFP) to the centrosome, and deletions of the newly identified N-terminal sequence from SLMAP prevented the centrosomal targeting. Deletion-mutant analysis concluded that overall, structural determinants in SLMAP were responsible for centrosomal targeting. Elevated levels of centrosomal SLMAP were found to be lethal, whereas mutants that lacked centrosomal targeting inhibited cell growth accompanied by an accumulation of cells at the G2/M phase of the cell cycle.
Efficient endosome-to-Golgi transport of Shiga toxin is dependent on dynamin and clathrin
Authors: Lauvrak Silje U. , Torgersen Maria L. , Sandvig Kirsten
Published: May 2004

It has previously been shown that Shiga toxin, despite being bound to a glycolipid receptor, can be efficiently endocytosed from clathrin-coated pits. However, clathrin-independent endocytosis is also responsible for a proportion of the toxin uptake in some cells. After endocytosis the toxin can be transported in retrograde fashion to the Golgi apparatus and the endoplasmic reticulum, and then to the cytosol, where it exerts its toxic effect by inactivating ribosomes. In order to investigate the role of dynamin and clathrin in endosome-to-Golgi transport of Shiga toxin, we have used HeLa dynK44A and BHK antisense clathrin heavy chain (CHC) cells that, in an inducible manner, express mutant dynamin or CHC antisense RNA, respectively. In these cell lines, one can study the role of dynamin and clathrin on endosome-to-Golgi transport because they, as shown here, still internalize Shiga toxin when dynamin- and clathrin-dependent endocytosis is blocked. Butyric acid has been shown to sensitize A431 cells to Shiga toxin by increasing the proportion of cell-associated toxin that is transported to the Golgi apparatus and the endoplasmic reticulum. Here, we find that, in HeLa and BHK cells also, butyric acid also increased toxin transport to the Golgi apparatus and sensitized the cells to Shiga toxin. We have therefore studied the role of dynamin and clathrin in both untreated and butyric-acid-treated cells by measuring the sulfation of a modified Shiga B fragment. Our results indicate that endosome-to-Golgi transport of Shiga toxin is dependent on functional dynamin in both untreated cells and in cells treated with butyric acid. Interestingly, the regulation of Shiga toxin transport in untreated and butyric-acid-treated cells differs when it comes to the role of clathrin, because only cells that are sensitized to Shiga toxin with butyric acid need functional clathrin for endosome-to-Golgi transport.
Calcium transients induce spatially coordinated increases in traction force during the movement of fish keratocytes
Authors: Doyle Andrew , Marganski William , Lee Juliet
Published: May 2004

The coordination of protrusion with retraction is essential for continuous cell movement. In fish keratocytes the activation of stretch-activated calcium channels, and the resulting increase in intracellular calcium, trigger release of the rear cell margin when forward movement is impeded. Although it is likely that retraction involves a calcium-dependent increase in cytoskeletal contractility, it is not known how the timing, magnitude and localization of contractile forces are organized during retraction. We have addressed this question using a new gelatin traction force assay in combination with calcium imaging to determine what changes in cytoskeletal force production accompany calcium-induced retraction. We find that individual calcium transients are followed within seconds by a rapid increase in traction stress that is maintained, or increases in a stepwise manner, until retraction occurs. Increases in traction stress are accompanied by a distinct sequence of changes in the spatial distribution of large traction stresses. Regions of increased traction stress enlarge at the lateral cell margins and expand forward along the cell margin. In particular, rearward facing propulsive' tractions at the leading edge of the cell, which are normally very low, increase several fold. Following retraction, a precipitous drop in traction stress is observed. Such distinct variations in traction stress are not observed in cells when calcium transients are absent. These results suggest a mechanism by which global increases in intracellular calcium can locally regulate contractile force production, in order to maintain a rapid highly directed mode of movement.
Scrapie-like prion protein is translocated to the nuclei of infected cells independently of proteasome inhibition and interacts with chromatin
Authors: Mangé Alain , Crozet Carole , Lehmann Sylvain , Béranger Florence
Published: May 2004

Prion diseases are fatal transmissible neurodegenerative disorders characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrPC) denoted PrPSc. Recently, wild-type and pathogenic PrP mutants have been shown to be degraded by the endoplasmic reticulum-associated degradation proteasome pathway after translocation into the cytosol. We show here that a protease resistant form of PrP accumulated in the nuclei of prion-infected cells independently of proteasome activity, and that this nuclear translocation required an intact microtubule network. Moreover, our results show for the first time that nuclear PrP interacts with chromatin in vivo, which may have physiopathological consequences in prion diseases
Nedd4.1-mediated ubiquitination and subsequent recruitment of Tsg101 ensure HTLV-1 Gag trafficking towards the multivesicular body pathway prior to virus budding
Authors: Blot Vincent , Perugi Fabien , Gay Bernard , Prévost Marie-Christine , Briant Laurence , Tangy Frédéric , Abriel Hugues , Staub Olivier , Dokhélar Marie-Christine , Pique Claudine
Published: May 2004

One of the most exciting recent developments in the field of retroviruses is the finding that their Gag proteins hijack cellular proteins from the mutivesicular body (MVB) pathway during the budding process. The Gag proteins of oncoretroviruses possess a PPxY motif that recruits a ubiquitin ligase from the Nedd4 family, whereas those of the human immunodeficiency virus interact through a PTAP motif with Tsg101, a protein of the ESCRT-1 complex. It is currently assumed that Nedd4 and Tsg101 represent equivalent entry gates towards the same cellular process leading to budding, and that both partners are recruited to the plasma membrane where viral budding occurs. However, we report here that the budding of the human oncoretrovirus HTLV-1, the Gag proteins of which possess tandem PPPY/PTAP motifs, requires both Nedd4 and Tsg101. We show that Nedd4.1, but not Nedd4.2, is recruited by the PPPY motif of Gag and subsequently catalyzes Gag ubiquitination. We also demonstrate that Gag interacts first with Nedd4.1 at the plasma membrane and then with Tsg101 in late endosomes/MVBs. Consistently, we found that HTLV-1 particles mutated in the PPPY motif remain underneath the plasma membrane, blocked at an early step of the budding process, whereas PTAP-mutated viruses accumulate in intracellular vesicles, blocked at a later step. Our findings indicate that Nedd4.1 and Tsg101 act successively in the assembly process of HTLV-1 to ensure proper Gag trafficking through the endocytic pathway up to late endosomes where the late steps of retroviral release occur.
Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells
Authors: Kurz David J. , Decary Stephanie , Hong Ying , Trivier Elisabeth , Akhmedov Alexander , Erusalimsky Jorge D.
Published: May 2004

Replicative senescence and oxidative stress have been implicated in ageing, endothelial dysfunction and atherosclerosis. Replicative senescence is determined primarily by telomere integrity. In endothelial cells the glutathione redox-cycle plays a predominant role in the detoxification of peroxides. The aim of this study was to elucidate the role of the glutathione-dependent antioxidant system on the replicative capacity and telomere dynamics of cultured endothelial cells. Human umbilical vein endothelial cells were serially passaged while exposed to regular treatment with 0.1 μM tert-butyl hydroperoxide, a substrate of glutathione peroxidase, or 10 μM L-buthionine-[S,R]-sulphoximine, an inhibitor of glutathione synthesis. Both treatments induced intracellular oxidative stress but had no cytotoxic or cytostatic effects. Nonetheless, treated cultures entered senescence prematurely (30 versus 46 population doublings), as determined by senescence-associated β-galactosidase staining and a sharp decrease in cell density at confluence. In cultures subjected to oxidative stress terminal restriction fragment (TRF) analysis demonstrated faster telomere shortening (110 versus 55 bp/population doubling) and the appearance of distinct, long TRFs after more than 15-20 population doublings. Fluorescence in situ hybridisation analysis of metaphase spreads confirmed the presence of increased telomere length heterogeneity, and ruled out telomeric end-to-end fusions as the source of the long TRFs. The latter was also confirmed by Bal31 digestion of genomic DNA. Similarly, upregulation of telomerase could not account for the appearance of long TRFs, as oxidative stress induced a rapid and sustained decrease in this activity. These findings demonstrate a key role for glutathione-dependent redox homeostasis in the preservation of telomere function in endothelial cells and suggest that loss of telomere integrity is a major trigger for the onset of premature senescence under mild chronic oxidative stress.
The subapical compartment: a traffic center in membrane polarity development
Authors: Hoekstra Dick , Tyteca Donatienne , van IJzendoorn Sven C. D.
Published: May 2004

Spatially separated apical and basolateral plasma membrane domains that have distinct functions and molecular compositions are a characteristic feature of epithelial cell polarity. The subapical compartment (SAC), also known as the common endosome (CE), where endocytic pathways from both surfaces merge, plays a crucial role in the maintenance and probably the biogenesis of these distinct membrane domains. Although differences in morphology are apparent, the same principal features of a SAC can be distinguished in different types of epithelial cells. As polarity develops, the compartment acquires several distinct machineries that, in conjunction with the cytoskeleton, are necessary for polarized trafficking. Disrupting trafficking via the SAC and hence bypassing its sorting machinery, as occurs upon actin depolymerization, leads to mis-sorting of apical and basolateral molecules, thereby compromising the development of polarity. The structural and functional integrity of the compartment in part depends on microtubules. Moreover, the acquisition of a particular set of Rab proteins, including Rab11 and Rab3, appears to be crucial in regulating molecular sorting and vesicular transport relevant both to recycling to either plasma membrane domain and to de novo assembly of the apical domain. Furthermore, subcompartmentalization of the SAC appears to be key to its various functions.
Coronary Event Secondary Prevention with Statins Irrespective of LDL-Cholesterol
Authors: Kerst Leslie L , Mauro Vincent F
Published: May 2004

OBJECTIVE:

To review the evidence for statin secondary prevention of coronary artery disease in patients with near-optimal or optimal low-density lipoprotein cholesterol (LDL-C).

DATA SOURCES:

A MEDLINE search (1966-October 2003) was conducted using the search terms HMG-CoA reductase inhibitor, statins, coronary disease, post-myocardial infarction, and average cholesterol.

DATA SYNTHESIS:

Secondary prevention trials enrolling subjects with near-optimal (<130 mg/dL) or optimal (<100 mg/dL) baseline LDL-C were included. Early statin secondary prevention studies suggested attenuated benefit, but more recent trials challenge this finding.

CONCLUSIONS:

Statin secondary prevention of coronary artery disease in patients near goal LDL-C is controversial, but recent trial results show promise.