bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolonged Opportunity for Ischemic Neuroprotection with Selective κ-Opioid Receptor Agonist in Rats
Authors: Chen Tsung-Ying , Goyagi Toru , Toung Thomas J.K. , Kirsch Jeffrey R. , Hurn Patricia D. , Koehler Raymond C. , Bhardwaj Anish
Published: March 2004

Background and Purpose—

We have previously demonstrated that pretreatment with selective κ-opioid agonist BRL 52537 hydrochloride [(±)-1-(3,4-dichlorophenyl) acetyl-2-(1-pyrrolidinyl) methylpiperidine], provides ischemic neuroprotection following transient focal ischemia in rats. The present study was undertaken to a) define “therapeutic opportunity” for ischemic neuroprotection with BRL 52537, and b) determine if BRL 52537 attenuates ischemia-evoked efflux of dopamine and its metabolites in the striatum in vivo following transient focal ischemia.

Methods—

Using the intraluminal filament technique, halothane-anesthetized male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO). In a blinded, randomized fashion, rats were treated with saline (vehicle) or 1 mg/Kg/hr BRL 52537 infusion for 22 hours, initiated at onset, 2, 4, or 6 hours of reperfusion (Rep). In a separate set of experiments utilizing in vivo microdialysis, extracellular levels of dopamine and its metabolites were determined in the striatum during 2 hours of MCAO and 3 hours of reperfusion.

Results—

Infarct volume (% of contralateral structure; mean ±SEM) in cortex was significantly attenuated when BRL 52537 was administered at reperfusion (22±6%), 2 hours (21±6%), and 4 hours (18±5%) compared with controls (39±5%). In striatum, infarct volume was significantly attenuated when BRL 52537 was administered at reperfusion (38±9%), 2 hours (40±8%), 4 hours (50±8%), and 6 hours (46±9%) as compared with controls (70±4%). A 6- to 8-fold increase in dopamine in microdialysates occurred within 40 minutes of MCAO. Pretreatment with BRL 52537 did not alter microdialysate levels of dopamine or its metabolites in the striatum during MCAO and early reperfusion, as compared with saline controls.

Conclusions—

These data demonstrate that BRL 52537 provides robust ischemic neurprotection with a long therapeutic opportunity (at least 6 hours) without altering ischemia-evoked efflux of dopamine (DA) and its metabolites in striatum during ischemia and early reperfusion.
Trial Application of a Model of Resource Utilization, Costs, and Outcomes for Stroke (MORUCOS) to Assist Priority Setting in Stroke
Authors: Moodie Marjory L. , Carter Robert , Mihalopoulos Cathrine , Thrift Amanda G. , Chambers Brian R. , Donnan Geoffrey A. , Dewey Helen M.
Published: March 2004

Background and Purpose—

Cost-effectiveness data for stroke interventions are limited, and comparisons between studies are confounded by methodological inconsistencies. The aim of this study was to trial the use of the intervention module of the economic model, a Model of Resource Utilization, Costs, and Outcomes for Stroke (MORUCOS) to facilitate evaluation and ranking of the options.

Methods—

The approach involves using an economic model together with added secondary considerations. A consistent approach was taken using standard economic evaluation methods. Data from the North East Melbourne Stroke Incidence Study (NEMESIS) were used to model “current practice” (base case), against which 2 interventions were compared. A 2-stage process was used to measure benefit: health gains (expressed in disability-adjusted life years [DALYs]) and filter analysis. Incremental cost-effectiveness ratios (ICERs) were calculated, and probabilistic uncertainty analysis was undertaken.

Results—

Aspirin, a low-cost intervention applicable to a large number of stroke patients (9153 first-ever cases), resulted in modest health benefits (946 DALYs saved) and a mean ICER (based on incidence costs) of US $1421 per DALY saved. Although the health gains from recombinant tissue-type plasminogen activator (rtPA) were less (155 DALYs saved), these results were impressive given the small number of persons (256) eligible for treatment. rtPA dominates current practice because it is more effective and cost-saving.

Conclusions—

If used to assess interventions across the stroke care continuum, MORUCOS offers enormous capacity to support decision-making in the prioritising of stroke services.
Orienting Attention to Locations in Perceptual Versus Mental Representations
Authors: Nobre A. C. , Coull J. T. , Maquet P. , Frith C. D. , Vandenberghe R. , Mesulam M. M.
Published: March 2004

Extensive clinical and imaging research has characterized the neural networks mediating the adaptive distribution of spatial attention. In everyday behavior, the distribution of attention is guided not only by extrapersonal targets but also by mental representations of their spatial layout. We used eventrelated functional magnetic resonance imaging to identify the neural system involved in directing attention to locations in arrays held as mental representations, and to compare it with the system for directing spatial attention to locations in the external world. We found that these two crucial aspects of spatial cognition are subserved by extensively overlapping networks. However, we also found that a region of right parietal cortex selectively participated in orienting attention to the extrapersonal space, whereas several frontal lobe regions selectively participated in orienting attention within on-line mental representations.
Neural Correlates of Mental State Decoding in Human Adults: An Event-related Potential Study
Authors: Sabbagh Mark A. , Moulson Margaret C. , Harkness Kate L.
Published: March 2004

Successful negotiation of human social interactions rests on having a theory of mind—an understanding of how others' behaviors can be understood in terms of internal mental states, such as beliefs, desires, intentions, and emotions. A core theory-of-mind skill is the ability to decode others' mental states on the basis of observable information, such as facial expressions. Although several recent studies have focused on the neural correlates of reasoning about mental states, no research has addressed the question of what neural systems underlie mental state decoding. We used dense-array eventrelated potentials (ERP) to show that decoding mental states from pictures of eyes is associated with an N270–400 component over inferior frontal and anterior temporal regions of the right hemisphere. Source estimation procedures suggest that orbitofrontal and medial temporal regions may underlie this ERP effect. These findings suggest that different components of everyday theory-of-mind skills may rely on dissociable neural mechanisms.
Impaired Judgments of Sadness But Not Happiness Following Bilateral Amygdala Damage
Authors: Adolphs Ralph , Tranel Daniel
Published: March 2004

Although the amygdala's role in processing facial expressions of fear has been well established, its role in the processing of other emotions is unclear. In particular, evidence for the amygdala's involvement in processing expressions of happiness and sadness remains controversial. To clarify this issue, we constructed a series of morphed stimuli whose emotional expression varied gradually from very faint to more pronounced. Five morphs each of sadness and happiness, as well as neutral faces, were shown to 27 subjects with unilateral amygdala damage and 5 with complete bilateral amygdala damage, whose data were compared to those from 12 braindamaged and 26 normal controls. Subjects were asked to rate the intensity and to label the stimuli. Subjects with unilateral amygdala damage performed very comparably to controls. By contrast, subjects with bilateral amygdala damage showed a specific impairment in rating sad faces, but performed normally in rating happy faces. Furthermore, subjects with right unilateral amygdala damage performed somewhat worse than subjects with left unilateral amygdala damage. The findings suggest that the amygdala's role in processing of emotional facial expressions encompasses multiple negatively valenced emotions, including fear and sadness.
A Temporal Dissociation of Subliminal versus Supraliminal Fear Perception: An Event-related Potential Study
Authors: Liddell Belinda J. , Williams Leanne M. , Rathjen Jennifer , Shevrin Howard , Gordon Evian
Published: March 2004

Current theories of emotion suggest that threat-related stimuli are first processed via an automatically engaged neural mechanism, which occurs outside conscious awareness. This mechanism operates in conjunction with a slower and more comprehensive process that allows a detailed evaluation of the potentially harmful stimulus (LeDoux, 1998). We drew on the Halgren and Marinkovic (1995) model to examine these processes using event-related potentials (ERPs) within a backward masking paradigm. Stimuli used were faces with fear and neutral (as baseline control) expressions, presented above (supraliminal) and below (subliminal) the threshold for conscious detection. ERP data revealed a double dissociation for the supraliminal versus subliminal perception of fear. In the subliminal condition, responses to the perception of fear stimuli were enhanced relative to neutral for the N2 “excitatory” component, which is thought to represent orienting and automatic aspects of face processing. By contrast, supraliminal perception of fear was associated with relatively enhanced responses for the late P3 “inhibitory” component, implicated in the integration of emotional processes. These findings provide evidence in support of Halgren and Marinkovic's temporal model of emotion processing, and indicate that the neural mechanisms for appraising signals of threat may be initiated, not only automatically, but also without the need for conscious detection of these signals.
Insulin-like growth factor binding protein-3 antagonizes the effects of retinoids in myeloid leukemia cells
Authors: Ikezoe Takayuki , Tanosaki Sakae , Krug Utz , Liu Bingrong , Cohen Pinchas , Taguchi Hirokuni , Koeffler H. Phillip
Published: March 2004

Insulin-like growth factor binding protein-3 (IGFBP-3) can cause growth suppressive and proapoptotic effects on retinoids in many types of cancer cells. However, the expression and effects of IGFBP-3 in myeloid leukemia cells have not been elucidated. In this study, we found no IGFBP-3 expression in the human myeloid leukemia cell lines either at baseline or after stimulation with all-trans retinoic acid (ATRA). Human recombinant IGFBP-3 induced growth arrest and apoptosis of HL-60 and NB4 cells. We have previously identified RXRα as a nuclear receptor for IGFBP-3 and have proceeded to examine further the role of this interaction in leukemia cell lines. In signaling assays, IGFBP-3 potently suppressed RAR- and VDR-mediated signaling while enhancing RXR signaling. Interestingly, when IGFBP-3 was administered to these cells in combination with an RAR-selective ligand, the ability of these retinoids to induce differentiation was blunted. On the other hand, IGFBP-3 enhanced the effect of an RXR-selective ligand to induce differentiation of HL-60 and NB4 cells. Further studies showed that IGFBP-3 down-regulated (at the transcriptional level) the retinoid-induced expression of C/EBPϵ in NB4 cells. Taken together, these results indicate that IGFBP-3 has antiproliferative activity against myeloid leukemia cells; while it enhances signaling through RXR/RXR, it blunts signaling by activated RAR/RXR.
Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen–specific cytotoxic T lymphocytes
Authors: Oosten Liesbeth E. M. , Blokland Els , van Halteren Astrid G. S. , Curtsinger Julie , Mescher Matthew F. , Falkenburg J. H. Frederik , Mutis Tuna , Goulmy Els
Published: March 2004

Cytotoxic T lymphocytes (CTLs) specific for hematopoietic-restricted minor histocompatibility antigens (mHags) are important reagents for adoptive immunotherapy of relapsed leukemia after allogeneic stem cell transplantation. However, expansion of these CTLs to therapeutic numbers is often hampered by the limited supply of antigen-presenting cells (APCs). Therefore, we evaluated whether cell-sized latex beads coated with HLA/mHag complexes HLA-A2/HA-1 or HLA-A2/HA-2 and recombinant CD80 and CD54 molecules can replace professional APCs. The artificial antigen-presenting constructs (aAPCs) effectively stimulated HA-1– and HA-2–specific CTL clones as shown by ligand-specific expansion, cytokine production, and maintenance of cytotoxic activity, without alteration of CTL phenotype. Furthermore, HA-1–specific polyclonal CTL lines were enriched as efficiently by aAPCs as by autologous HA-1 peptide-pulsed dendritic cells. Thus, aAPCs coated with HLA/mHag complexes, CD80, and CD54 may serve as tools for in vitro enrichment of immunotherapeutic mHag-specific CTL lines.
Treatment with arsenic trioxide (ATO) and MEK1 inhibitor activates the p73-p53AIP1 apoptotic pathway in leukemia cells
Authors: Lunghi Paolo , Costanzo Antonio , Levrero Massimo , Bonati Antonio
Published: March 2004

Arsenic trioxide (ATO) induces differentiation and apoptosis of malignant cells in vitro and in vivo and has been used in the treatment of a variety of hematologic malignancies. We found that in NB4 acute promyelocytic and in K562 erythroleukemia cell lines treatment with the MEK1 inhibitors PD98059 and PD184352 greatly enhances apoptotic cell death induced by ATO alone. Combined treatment results in the induction of the p53AIP1 (p53-regulated apoptosis-inducing protein 1) gene in both cell lines. Because NB4 and K562 cell lines carry an inactive p53, we investigated the possible role of p73, a p53 paralogue that has been shown to regulate several p53 target genes including p21, Bax, and p53AIP1. We found that MEK1 inhibitors reduce the levels of dominant-negative (ΔN) p73 proteins and promote the accumulation of endogenous p73α through its transcriptional activation and its tyrosine phosphorylation, resulting in p21 up-regulation and significant inhibition of cell growth. ATO reduces ΔNp73 levels and promotes a p300-mediated acetylation of endogenous p73, thus favoring cell cycle arrest and apoptosis. Finally, the combined treatment with MEK1 inhibitors and ATO enhances the affinity of phosphoacetylated p73 for the p53AIP1 promoter in vivo, as determined by chromatin immunoprecipitation experiments, leading to p53AIP1 up-regulation and increased apoptosis. (Blood. 2004; 104:519-525)
Blood mononuclear cell gene expression profiles characterize the oxidant, hemolytic, and inflammatory stress of sickle cell disease
Authors: Jison Maria L. , Munson Peter J. , Barb Jennifer J. , Suffredini Anthony F. , Talwar Shefali , Logun Carolea , Raghavachari Nalini , Beigel John H. , Shelhamer James H. , Danner Robert L. , Gladwin Mark T.
Published: March 2004

In sickle cell disease, deoxygenation of intra-erythrocytic hemoglobin S leads to hemoglobin polymerization, erythrocyte rigidity, hemolysis, and microvascular occlusion. Ischemia-reperfusion injury, plasma hemoglobin-mediated nitric oxide consumption, and free radical generation activate systemic inflammatory responses. To characterize the role of circulating leukocytes in sickle cell pathogenesis we performed global transcriptional analysis of blood mononuclear cells from 27 patients in steady-state sickle cell disease (10 patients treated and 17 patients untreated with hydroxyurea) compared with 13 control subjects. We used gender-specific gene expression to validate human microarray experiments. Patients with sickle cell disease demonstrated differential gene expression of 112 genes involved in heme metabolism, cell-cycle regulation, antioxidant and stress responses, inflammation, and angiogenesis. Inducible heme oxygenase-1 and downstream proteins biliverdin reductase and p21, a cyclin-dependent kinase, were up-regulated, potentially contributing to phenotypic heterogeneity and absence of atherosclerosis in patients with sickle cell disease despite endothelial dysfunction and vascular inflammation. Hydroxyurea therapy did not significantly affect leukocyte gene expression, suggesting that such therapy has limited direct anti-inflammatory activity beyond leukoreduction. Global transcriptional analysis of circulating leukocytes highlights the intense oxidant and inflammatory nature of steady-state sickle cell disease and provides insight into the broad compensatory responses to vascular injury.
Altered leukocyte response to CXCL12 in patients with warts hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome
Authors: Gulino Anna Virginia , Moratto Daniele , Sozzani Silvano , Cavadini Patrizia , Otero Karel , Tassone Laura , Imberti Luisa , Pirovano Silvia , Notarangelo Lucia D. , Soresina Roberta , Mazzolari Evelina , Nelson David L. , Notarangelo Luigi D. , Badolato Raffaele
Published: March 2004

The chemokine receptor CXCR4 and its functional ligand, CXCL12, are essential regulators of development and homeostasis of hematopoietic and lymphoid organs. Heterozygous truncating mutations in the CXCR4 intracellular tail cause a rare genetic disease known as WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), whose pathophysiology remains unclear. We report CXCR4 function in 3 patients with WHIM syndrome carrying heterozygous truncating mutations of CXCR4. We show that CXCR4 gene mutations in WHIM patients do not affect cell surface expression of the chemokine receptor and its internalization upon stimulation with CXCL12. Moreover, no significant differences in calcium mobilization in response to CXCL12 are found. However, the chemotactic response of both polymorphonuclear cells and T lymphocytes in response to CXCL12 is increased. Furthermore, immunophenotypic analysis of circulating T and B lymphocytes reveals a decreased number of memory B cells and of naive T cells and an accumulation of effector memory T cells associated with a restricted T-cell repertoire. Based on our results, we suggest that the altered leukocyte response to CXCL12 may account for the pathologic retention of mature polymorphonuclear cells in the bone marrow (myelokathexis) and for an altered lymphocyte trafficking, which may cause the immunophenotyping abnormalities observed in WHIM patients. (Blood. 2004;104:444-452)
The contribution of inherited and acquired thrombophilic defects, alone or combined with antiphospholipid antibodies, to venous and arterial thromboembolism in patients with systemic lupus erythematosus
Authors: Brouwer Jan-Leendert P. , Bijl Marc , Veeger Nic J. G. M. , Kluin-Nelemans Hanneke C. , van der Meer Jan
Published: March 2004

Systemic lupus erythematosus (SLE) is associated with an increased risk of venous (VTE) and arterial thromboembolism (ATE). Lupus anticoagulant (LA) and anticardiolipin antibodies (ACAs) are established risk factors. We assessed the contribution of deficiencies of antithrombin, protein C, total protein S, factor V Leiden, the prothrombin G20210A mutation and APC resistance, either alone or in various combinations with LA and/or ACAs, to the thrombotic risk in a cohort of 144 consecutive patients with SLE. Median follow-up was 12.7 years. VTE had occurred in 10% and ATE in 11% of patients. LA,ACAs, factor V Leiden, and the prothrombin mutation were identified as risk factors for VTE. Annual incidences of VTE were 2.01 (0.74-4.37) in patients with one of these disorders and 3.05 (0.63-8.93) in patients with 2 disorders. The risk of VTE was 20- and 30-fold higher, respectively, compared with the normal population. In contrast with LA and ACAs, thrombophilic disorders did not influence the risk of ATE. In conclusion, factor V Leiden and the prothrombin mutation contribute to the risk of VTE in patients with SLE, and potentiate this risk when one of these thrombophilic defects are combined with LA and/or ACAs.
Effects of inflammatory cytokines on the release and cleavage of the endothelial cell–derived ultralarge von Willebrand factor multimers under flow
Authors: Bernardo Aubrey , Ball Chalmette , Nolasco Leticia , Moake Joel F. , Dong Jing-fei
Published: March 2004

ADAMTS13 cleaves ultralarge and hyperreactive von Willebrand factor (ULVWF) freshly released from activated endothelial cells to smaller and less active forms. This process may be affected by the amount of ULVWF released and the processing capacity of ADAMTS13, contributing to the development of thrombotic diseases. We examined the effects of inflammatory cytokines on the release and cleavage of ULVWF to evaluate potential links between inflammation and thrombosis. Human umbilical vein endothelial cells were treated with interleukin 6 (IL-6), IL-8, or tumor necrosis factor α (TNF-α), and the formation of platelet-decorated ULVWF strings was quantitated. IL-8 and TNF-α significantly stimulated the release of ULVWF in a dose-dependent manner. IL-6 induced ULVWF release only when it was in complex with the soluble IL-6 receptor. IL-6, but not IL-8 nor TNF-α, inhibited the cleavage of ULVWF strings by ADAMTS13 under flowing, but not static, conditions. These results suggest that inflammatory cytokines may stimulate the ULVWF release (IL-8 and TNF-α) and inhibit the ULVWF cleavage (IL-6), resulting in the accumulation of hyperreactive ULVWF in plasma and on the surface of endothelial cells to induce platelet aggregation and adhesion on the vascular endothelium. The findings describe a potential linkage between inflammation and thrombosis that may be of therapeutic importance.
In vitro–expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendritic cell–stimulated MLR cultures
Authors: Godfrey Wayne R. , Ge Ying G. , Spoden Darrin J. , Levine Bruce L. , June Carl H. , Blazar Bruce R. , Porter Stephen B.
Published: March 2004

CD4+CD25+ T-regulatory (Treg) cells have been shown to critically regulate self- and allograft tolerance in several model systems. Studies of human Treg cells have been restricted by the small number present in peripheral blood and their naturally hypoproliferative state. To better characterize Treg suppressor cell function, we determined methods for the isolation and expansion of these cells. Stringent magnetic microbead-based purification was required for potent suppressor cell line generation. Culture stimulation with cell-sized Dynabeads coated with anti-CD3 and anti-CD28 monoclonal antibodies, CD4+ feeder cells, and interleukin 2, provided for marked expansion in cell number (100-fold), with retention and enhancement of suppressor function. The potent Treg cell lines suppressed proliferation in dendritic cell-driven allo-mixed lymphocyte reaction (MLR) cultures by more than 90%. The Treg-derived suppressor cells functioned early in allo-MLR because expression of activation antigens and accumulation of cytokines was nearly completely prevented. Importantly, cultured Treg cells also suppressed activated and matured dendritic cell-driven responses. These results demonstrate that short-term suppressor cell lines can be generated, and they can express a very potent suppressive activity. This approach will enable more detailed biologic studies of Treg cells and facilitate the evaluation of cultured Treg cells as a novel form of immunosuppressive therapy. (Blood. 2004;104:453-461)
Antigen-induced regulatory T cells
Authors: Vigouroux Stephane , Yvon Eric , Biagi Ettore , Brenner Malcolm K.
Published: March 2004

Regulatory T cells participate in immunologic homeostasis by active suppression of inappropriate immune responses. Regulatory T lymphocytes expressing CD4 and CD25 antigens and naturally present in the peripheral blood were the first to be phenotypically characterized. However, their small number and antigen nonspecific suppression has prompted efforts to identify and dissect antigen-specific regulatory T cells. In this review we discuss how antigen-specific regulatory T cells can be identified, the cellular and molecular mechanisms underlying their induction and activity, and the challenges facing their potential clinical application.
Does the Type II Glucocorticoid Receptor Mediate Cortisol-Induced Suppression in Pituitary Responsiveness to Gonadotropin-Releasing Hormone?
Authors: Breen Kellie M. , Stackpole Catherine A. , Clarke Iain J. , Pytiak Andrew V. , Tilbrook Alan J. , Wagenmaker Elizabeth R. , Young Elizabeth A. , Karsch Fred J.
Published: March 2004

Stress-like elevations in plasma cortisol suppress LH pulse amplitude in ovariectomized ewes by inhibiting pituitary responsiveness to GnRH. Here we sought to identify the receptor mediating this effect. In a preliminary experiment GnRH and LH pulses were monitored in ovariectomized ewes treated with cortisol plus spironolactone, which antagonizes the type I mineralocorticoid receptor (MR), or with cortisol plus RU486, which antagonizes both the type II glucocorticoid receptor (GR) and the progesterone receptor (PR). Cortisol alone reduced LH pulse amplitude, but not pulsatile GnRH secretion, indicating that it reduced pituitary responsiveness to endogenous GnRH. RU486, but not spironolactone, reversed this suppression. We next tested whether RU486 reverses the inhibitory effect of cortisol on pituitary responsiveness to exogenous GnRH pulses of fixed amplitude, frequency, and duration. Hourly GnRH pulses were delivered to ovariectomized ewes in which endogenous GnRH pulses were blocked by estradiol during seasonal anestrus. Cortisol alone reduced the amplitude of LH pulses driven by the exogenous GnRH pulses. RU486, but not an antagonist of PR (Organon 31710), prevented this suppression. Thus, the efficacy of RU486 in blocking the suppressive effect of cortisol is attributed to antagonism of GR, not PR. Together, these observations imply that the type II GR mediates cortisolinduced suppression of pituitary responsiveness to GnRH.
EGF receptor signalling protects smooth-cuticle cells from apoptosis duringDrosophilaventral epidermis development
Authors: Urban Sinisa , Brown Gemma , Freeman Matthew
Published: March 2004

Patterning of the Drosophila ventral epidermis is a tractable model for understanding the role of signalling pathways in development. Interplay between Wingless and EGFR signalling determines the segmentally repeated pattern of alternating denticle belts and smooth cuticle: spitz group genes, which encode factors that stimulate EGFR signalling, induce the denticle fate, while Wingless signalling antagonizes the effect of EGFR signalling, allowing cells to adopt the smooth-cuticle fate. Medial fusion of denticle belts is also a hallmark of spitzgroup genes, yet its underlying cause is unknown. We have studied this phenotype and discovered a new function for EGFR signalling in epidermal patterning. Smooth-cuticle cells, which are receiving Wingless signalling, are nevertheless dependent on EGFR signalling for survival. Reducing EGFR signalling results in apoptosis of smooth-cuticle cells between stages 12 and 14, bringing adjacent denticle regions together to result in denticle belt fusions by stage 15. Multiple factors stimulate EGFR signalling to promote smooth-cuticle cell survival: in addition to the spitz group genes,Rhomboid-3/roughoid, but not Rhomboid-2 or -4, and the neuregulin-like ligand Vein also function in survival signalling. Pointed mutants display the lowest frequency of fusions, suggesting that EGFR signalling may inhibit apoptosis primarily at the post-translational level. All ventral epidermal cells therefore require some level of EGFR signalling; high levels specify the denticle fate, while lower levels maintain smooth-cuticle cell survival. This strategy might guard against developmental errors, and may be conserved in mammalian epidermal patterning.
Specification of the otic placode depends on Sox9 function inXenopus
Authors: Saint-Germain Natasha , Lee Young-Hoon , Zhang Yanhui , Sargent Thomas D. , Saint-Jeannet Jean-Pierre
Published: March 2004

The vertebrate inner ear develops from a thickening of the embryonic ectoderm, adjacent to the hindbrain, known as the otic placode. All components of the inner ear derive from the embryonic otic placode. Sox proteins form a large class of transcriptional regulators implicated in the control of a variety of developmental processes. One member of this family, Sox9, is expressed in the developing inner ear, but little is known about the early function of Sox9 in this tissue. We report the functional analysis of Sox9 during development of Xenopus inner ear. Sox9 otic expression is initiated shortly after gastrulation in the sensory layer of the ectoderm, in a bilateral patch of cells immediately adjacent to the cranial neural crest. In the otic placode, Sox9 colocalizes with Pax8 one of the earliest gene expressed in response to otic placode inducing signals. Depletion of Sox9 protein in whole embryos using morpholino antisense oligonucleotides causes a dramatic loss of the early otic placode markers Pax8 and Tbx2. Later in embryogenesis, Sox9 morpholino-injected embryos lack a morphologically recognizable otic vesicle and fail to express late otic markers (Tbx2, Bmp4,Otx2 and Wnt3a) that normally exhibit regionalized expression pattern throughout the otocyst. Using a hormone inducible inhibitory mutant of Sox9,we demonstrate that Sox9 function is required for otic placode specification but not for its subsequent patterning. We propose that Sox9 is one of the key regulators of inner ear specification in Xenopus.
Suppression of neural fate and control of inner ear morphogenesis byTbx1
Authors: Raft Steven , Nowotschin Sonja , Liao Jun , Morrow Bernice E.
Published: March 2004

Inner ear sensory organs and VIIIth cranial ganglion neurons of the auditory/vestibular pathway derive from an ectodermal placode that invaginates to form an otocyst. We show that in the mouse otocyst epithelium, Tbx1 suppresses neurogenin 1-mediated neural fate determination and is required for induction or proper patterning of gene expression related to sensory organ morphogenesis (Otx1 and Bmp4, respectively). Tbx1 loss-of-function causes dysregulation of neural competence in otocyst regions linked to the formation of either mechanosensory or structural sensory organ epithelia. Subsequently, VIIIth ganglion rudiment form is duplicated posteriorly, while the inner ear is hypoplastic and shows neither a vestibular apparatus nor a coiled cochlear duct. We propose that Tbx1acts in the manner of a selector gene to control neural and sensory organ fate specification in the otocyst.
Intercellular signaling of reproductive development by theC. elegansDAF-9 cytochrome P450
Authors: Mak Ho Yi , Ruvkun Gary
Published: March 2004

Parallel pathways control C. elegans reproductive development in response to environmental cues. Attenuation of daf-2 insulin-like or daf-7 TGFβ-like signaling pathways cause developmental arrest at the stress resistant and long-lived dauer stage. Loss-of-function mutations in the cytochrome P450 gene daf-9 also cause dauer arrest and defects in cell migration. A rescuing daf-9::GFP fusion gene driven by the daf-9 promoter is expressed in two head cells at all stages, in the hypodermis from mid-second larval stage (L2) to the fourth larval stage (L4),and in the spermatheca of the adult hermaphrodite. Although the level of daf-9::GFP expression in the head cells and spermatheca is constant,hypodermal daf-9::GFP expression is modulated by multiple inputs. In particular, daf-9::GFP expression in the hypodermis is absolutely dependent on daf-12, the nuclear receptor that is negatively regulated by daf-9 gene activity, suggesting feedback control between daf-9 and daf-12 in this tissue. daf-9 expression exclusively in the hypodermis is sufficient to restore reproductive development in daf-9 mutant animals, suggesting that daf-9functions in a cell nonautonomous manner. Furthermore, constitutive expression of daf-9 in the hypodermis suppresses dauer arrest of daf-7mutant animals and inhibits dauer remodelling of some tissues in daf-2 mutant animals. Thus, daf-9 may integrate outputs from daf-2 and daf-7 signaling pathways to relay neuroendocrine signals through synthesis of a lipophilic hormone.
BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice
Authors: Yuhki Munehiro , Yamada Masahisa , Kawano Masako , Iwasato Takuji , Itohara Shigeyoshi , Yoshida Hisahiro , Ogawa Masaharu , Mishina Yuji
Published: March 2004

Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, we disrupted Bmpr1a, which encodes the BMP receptor type IA (BMPR1A) in an HF cell-specific manner, using the Cre/loxP system. We found that the differentiation of inner root sheath, but not outer root sheath, was severely impaired in mutant mice. The number of HFs was reduced in the dermis and subcutaneous tissue, and cycling epithelial cells were reduced in mutant mice HFs. Our results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin.
Both mitogen activated protein kinase and the mammalian target of rapamycin modulate the development of functional renal proximal tubules in matrigel
Authors: Han Ho Jae , Sigurdson Wade J. , Nickerson Peter A. , Taub Mary
Published: March 2004

Tubules may arise during branching morphogenesis through several mechanisms including wrapping, budding, cavitation and cord hollowing. In this report we present evidence that is consistent with renal proximal tubule formation through a process of cord hollowing (a process that requires the concomitant establishment of apicobasal polarity and lumen formation). Pockets of lumen filled with Lucifer Yellow were observed within developing cords of rabbit renal proximal tubule cells in matrigel. The observation of Lucifer Yellow accumulation suggests functional polarization. In the renal proximal tubule Lucifer Yellow is initially transported intracellularly by means of a basolaterally oriented p-aminohippurate transport system, followed by apical secretion into the lumen of the nephron. Consistent with such polarization in developing tubules, Triticum vulgare was observed to bind to the lumenal membranes within pockets of Lucifer Yellow-filled lumens. As this lectin binds apically in the rabbit renal proximal tubule, T. vulgare binding is indicative of the emergence of an apical domain before the formation of a contiguous lumen. Both epidermal growth factor and hepatocyte growth factor stimulated the formation of transporting tubules. The stimulatory effect of both epidermal growth factor and hepatocyte growth factor on tubulogenesis was inhibited by PD98059, a mitogen activated protein kinase kinase inhibitor, rather than by wortmannin, an inhibitor of phosphoinositide 3-kinase. Nevertheless, Lucifer Yellow-filled lumens were observed in tubules that formed in the presence of PD98059 as well as with wortmannin, indicating that these drugs did not prevent the process of cavitation. By contrast, rapamycin, an inhibitor of the mammalian target of rapamycin, prevented the process of cavitation without affecting the frequency of formation of developing cords. Multicellular cysts were observed to form in 8-bromocyclic AMP-treated cultures. As these cysts did not similarly accumulate Lucifer Yellow lumenally, it is very likely that processes other than organic anion accumulation are involved in the process of cystogenesis, including the Na,K-ATPase.
IIGP, a member of the IFN inducible and microbial defense mediating 47 kDa GTPase family, interacts with the microtubule binding protein hook3
Authors: Kaiser Frank , Kaufmann Stefan H. E. , Zerrahn Jens
Published: March 2004

Innate immunity against intracellular pathogens is critically determined by an as yet unknown interferon (IFN)-inducible mechanism exerted by members of the 47 kDa GTPase family. The association of IGTP and IIGP with membranous compartments, the endoplasmic reticulum and, in addition in case of IIGP, the Golgi, implicate these GTPases in intracellular membrane trafficking or processing. We identified the cytoplasmic linker molecule hook3 as an interactor for IIGP by yeast two-hybrid screening. The physical complex between these molecules was present in lysates of IFNγ-stimulated macrophages as demonstrated by co-immunoprecipitation. Only a minor subfraction of total cellular IIGP or hook3 was co-purified, indicating that this interaction is either transient and/or involves distinct subpopulations of the total cellular pools of these molecules. Binding of IIGP to hook3 depends on a GTP-bound conformation. Hook3 is a microtubule-binding protein which participates in the organization of the cis-Golgi compartment. Both proteins were detected in the Golgi-membrane-enriched fraction upon subcellular fractionation. Apart from the Golgi localization of both proteins, hook3 was detected in perinuclear regions in close spatial proximity to IIGP, associated with the endoplasmic reticulum. Our experiments identify hook3 as the first cooperation partner of a member of the 47 kDa GTPase protein family and indicate that hook3 links in an IFNγ-inducible fashion to cytoskeleton-based membrane trafficking.
MEK5 and ERK5 are localized in the nuclei of resting as well as stimulated cells, while MEKK2 translocates from the cytosol to the nucleus upon stimulation
Authors: Raviv Ziv , Kalie Eyal , Seger Rony
Published: March 2004

The ERK5 signaling cascade acts through sequential activation of MEKK2/3, MEK5 and ERK5 and transmits signals to a variety of stress and mitogenic related targets. In this study we examined the subcellular localization of the components of the ERK5 cascade and found that in resting, as well as in EGF-stimulated HeLa and Rat-1 cells, endogenous ERK5 is localized mainly in the nucleus. This location is different from the previously described location of exogenous ERK5, in the cytosol of resting cells, which is confirmed in this study. The reason for the different localization could be a saturation of anchoring moieties by the endogenous ERK5. Indeed, in situ detergent extraction analysis using Nonidet P-40, revealed that ERK5 is bound to detergent resistant moieties in the nucleus, while the exogenous protein fails to interact with those anchors. The upstream activator MEK5 is also localized in the nucleus both before and after EGF stimulation and is resistant to NP-40 extraction in resting cells. ERK5 remains bound to these nuclear moieties even after stimulation, while MEK5 is detached from the anchors but remains localized in the nucleus. Unlike ERK5 and MEK5, their upstream activator MEKK2 is localized mainly in the cytosol of resting cells, and translocates into the nucleus upon EGF stimulation, allowing transmission of signals to the nuclear MEK5. The nuclear localization of MEK5 and ERK5 is different from that of ERK1/2 and MEK1/2 in resting cells, indicating that each MAPK cascade uses distinct mechanisms to transmit extracellular signals to their nuclear targets.
Deletion mutant of FGFR4 induces onion-like membrane structures in the nucleus
Authors: Sørensen Vigdis , Brech Andreas , Khnykin Denis , Kolpakova Elona , Citores Lucia , Olsnes Sjur
Published: March 2004

The expression of several deletion mutants of fibroblast growth factor receptor 4 (FGFR4) was studied in COS-1 cells. FGFR4-mutants lacking most of the extracellular region did not efficiently reach the plasma membrane but accumulated in the endoplasmic reticulum (ER) and Golgi body. A mutant FGFR4 lacking the kinase domain as well as most of the extracellular region (ΔExt/R4Tth) had a distinct intracellular distribution. It localized in part to the nucleus, where it exhibited a striking spotted pattern. Ultrastructural studies showed that the nuclear spots consisted of several layers of membrane that were folded into onion-like structures at the nucleoplasmic side of the nuclear envelope. These intranuclear structures did not contain nuclear pores but were positive for the ER proteins calreticulin and protein disulfide isomerase, in addition to abundant ΔExt/R4Tth. Formation of the intranuclear structures was sensitive to inhibition of protein kinase C. Live microscopy of a green-fluorescent-protein/ΔExt/R4Tth fusion protein showed that the intranuclear structures were stable and immobile, suggesting that they function as deposits of the overexpressed mutant and associated membrane. The ΔExt/R4Tth protein also induced formation of densely packed membrane stacks in the cytosol and we suggest a model were the intranuclear structures are formed by invagination of ER-derived membrane stacks into the nucleus.
Novel Acidic Ionic Liquids Catalytic Systems for Friedel–Crafts Alkylation of Aromatic Compounds with Alkenes
Authors: Qiao Kun , Yokoyama Chiaki
Published: March 2004

Novel acidic ionic liquids catalytic systems are developed for the Friedel–Crafts alkylation of aromatic compounds with alkenes.
Methanol Electrooxidation on Carbon-Supported Pt3Ru2Sn Ternary Catalyst
Authors: Kim Taeyoon , Takahashi Masashi , Nagai Masayuki , Kobayashi Koichi
Published: March 2004

The electrocatalytic methanol oxidation over Pt–Ru–Sn/C ternary alloy catalysts was investigated. The catalytic activity of Pt3Ru2Sn/C was significantly higher than that of PtRu/C. Sn in ternary alloy catalyst modified the Pt electronic structure without the large increase of Pt–Pt bond distance, which may enhance the catalytic activity for methanol oxidation by improving CO tolerance.
Application of Liquid Chromatography-Tandem Mass Spectrometry to the Analysis of Benzodiazepines in Blood
Authors: Rivera Hazel M. , Walker G. Stewart , Sims D. Noel , Stockham Peter C.
Published: March 2004

A sensitive and selective high-performance liquid chromatography/atmospheric pressure chemical ionisation tandem mass spectrometry (HPLC/APCI-MS/MS) method for the simultaneous detection of 18 benzodiazepines and metabolites in human blood is described. The procedure utilises butyl chloride extraction at alkaline pH followed by reversed-phase liquid chromatography. The technique is suitable for screening analyses and confirmation of identity of the benzodiazepines at their lowest reported therapeutic concentrations using 500 μL of blood. The method has been successfully applied in forensic cases involving low concentrations of benzodiazepines.
Time-of-Flight Secondary Ion Mass Spectrometry Analysis of Hair from Archaeological Remains
Authors: Kempson I.M. , Skinner W.M. , Kirkbride P.K. , Nelson A.J. , Martin R.R.
Published: March 2004

Hair from four individuals excavated from burial sites in Pacatnamu, Peru from the Moche (450–800 AD) and Lambayeque (900–1100 AD) periods was sectioned longitudinally and analysed with time-of-flight secondary ion mass spectrometry (ToF-SIMS). An attempt was made to distinguish biogenic and diagenetic contributions to the elemental concentrations in the hair samples. Significant contamination was observed to have penetrated the hair samples from the burial environment. Results from the analyses indicate that the burial environment plays an important role in the postmortem variation in elemental content of hair samples. Various elements demonstrated an ability to permeate through the hair matrix over time. In addition, NaCl and what are believed to be aluminosilicates and mineral sulphates, were observed to have accumulated on the surface of the samples. Degradation of the samples was also suspected due to the presence of molecular fragments, possibly resulting from oxidation of the keratin proteins. The results should assist in the identification of reliable elemental signals in the analysis of ancient hair samples and promote caution when considering elements that are abundant in the burial environment.
Structure Elucidation of Unsaturated Fatty Acids after Vicinal Hydroxylation of the Double Bonds by Negative-Ion Electrospray Ionisation Low-Energy Tandem Mass Spectrometry
Authors: Moe Morten K. , Jensen Einar
Published: March 2004

A method for determining the positions of double bonds in unsaturated fatty acids by use of negative-ion electrospray ionisation low-energy tandem mass spectrometry is described. First, a vicinal hydroxylation of the double bonds of mono- and polyunsaturated fatty acids was performed. Low-energy collision-activation dissociation of the deprotonated molecules produced structurally informative ions formed by α-cleavages relative to the hydroxyl groups. Abundant fragment ions that confirmed the positions of all hydroxyl groups, and thus the positions of the double bonds in the native fatty acids, were observed in the spectra of derivatised mono-, di- and tri-unsaturated fatty acids. Two types of ions were observed, called [α′n]− and [α′n]−. The letter n indicates the position of the hydroxyl groups. The structurally diagnostic ions [α′n]− were produced by cleavages distal to the hydroxyl groups with the charge retained on the carboxylate. [α′n]− ions originating from all hydroxyl groups were observed in the spectra of modified mono-, di- and tri-unsaturated fatty acids. Initial proton transfer of a hydroxyl proton to the carboxylate with subsequent cleavages proximal to the hydroxyl groups, relative to the carboxylate, resulted in the two structurally diagnostic [αn]− ions. In hydroxylated fatty acids having two or more double bonds in their native structure, [α′n]− ions originating only from the two final hydroxyl groups were observed. The formation of some of the ions of [αn]− and [αn]− type can be rationalised by a six-membered transition state. Hydroxylated deprotonated tetra-, penta- and hexa-unsaturated fatty acids also produced [α′n]− ions indicating the positions of most of the hydroxyl groups, whereas the [αn]− ions were observed as described above. The method described offers a simple approach to the determination of the positions of double bonds in unsaturated fatty acids and is an alternative to utilising di-lithiated fatty acids. The method should also be applicable when fatty acids are constituents of more complex molecules such as phospholipids.
The Coupling of Supercritical Fluid Chromatography and Field Ionization Time-of-Flight High-Resolution Mass Spectrometry for Rapid and Quantitative Analysis of Petroleum Middle Distillates
Authors: Qian Kuangnan , Diehl John W. , Dechert Gary J. , DiSanzo Frank P.
Published: March 2004

We report the first coupling of supercritical fluid chromatography (SFC) with field ionization time-of-flight high-resolution mass spectrometry (FI-ToF HRMS), in parallel with ultraviolet (UV) detection and flame ionization detection (FID), for rapid and quantitative analysis of petroleum middle distillates. SFC separates petroleum middle distillates into saturates and 1- to 3-ring aromatics. FI generates molecular ions for hydrocarbon species eluted from the SFC. The high resolution and exact mass measurements by ToF mass spectrometry provide elemental compositions of the molecules in the petroleum product. The amounts of saturates and aromatic ring types were quantified using the parallel SFC-FID assisted by SFC-UV. With a proper carbon–number calibration, the detailed composition of the petroleum middle distillate was rapidly determined.
The Ion Conveyor. An Ion Focusing and Conveying Device
Authors: Colburn Alex W. , Giannakopulos A.E. , Derrick Peter J.
Published: March 2004

The control and transmission of ions or small charged droplets in the intermediate to high-pressure regime is of primary importance in areas such as atmospheric pressure ionisation. Where small apertures separate differentially pumped vacuum regions in the inlet systems to mass spectrometers, a large proportion of the available ion current is lost to the surrounding electrode structures. A new ion-optical device, named the ion conveyor, incorporating electrodynamic focusing and conveying of charged entities is described. Results from ion-optical simulations are presented demonstrating the performance of the device in various operating modes and electrode configurations.