bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional deactivations: Change with age and dementia of the Alzheimer type
Authors: Lustig Cindy , Snyder Abraham Z. , Bhakta Mehul , O'Brien Katherine C. , McAvoy Mark , Raichle Marcus E. , Morris John C. , Buckner Randy L.
Published: November 2003

Young adults typically deactivate specific brain regions during active task performance. Deactivated regions overlap with those that show reduced resting metabolic activity in aging and dementia, raising the possibility of a relation. Here, the magnitude and dynamic temporal properties of these typically deactivated regions were explored in aging by using functional MRI in 82 participants. Young adults (
n
= 32), older adults without dementia (
n
= 27), and older adults with early-stage dementia of the Alzheimer type (DAT) (
n
= 23) were imaged while alternating between blocks of an active semantic classification task and a passive fixation baseline. Deactivation in lateral parietal regions was equivalent across groups; in medial frontal regions, it was reduced by aging but was not reduced further by DAT. Of greatest interest, a medial parietal/ posterior cingulate region showed differences between young adults and older adults without dementia and an even more marked difference with DAT. The temporal profile of the medial parietal/posterior cingulate response suggested that it was initially activated by all three groups, but the response in young adults quickly reversed sign, whereas DAT individuals maintained activation throughout the task block. Exploratory whole-brain analyses confirmed the importance of medial parietal/posterior cingulate cortex differences in aging and DAT. These results introduce important opportunities to explore the functional properties of regions showing deactivations, how their dynamic functional properties relate to their baseline metabolic rates, and how they change with age and dementia.
IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Authors: Song Sonya W. , Fuller Gregory N. , Khan Asadullah , Kong Shouming , Shen Weiping , Taylor Ellen , Ramdas Latha , Lang Frederick F. , Zhang Wei
Published: November 2003

Our previous studies have shown that insulin-like growth factor binding protein 2 (IGFBP-2) is frequently overexpressed in the highly invasive glioblastoma multiforme (GBM). By using a yeast two-hybrid system, we identified a gene, invasion inhibitory protein 45 (
IIp45
), whose protein product bound to IGFBP-2 through the thyroglobulin-RGD region of the C terminus of IGFBP-2. The
IIp45
gene is located on chromosome 1p36 and has nine exons. The IIp45 protein has three SEG (segment of low compositional complexity) domains and an integrin-binding RGD motif. The IIp45 protein was not expressed in some GBMs. Functional studies showed that IIp45 inhibited GBM cell invasion both
in vitro
and in xenograft model. Gene expression profiling studies showed that IIp45 consistently inhibited the expression of cell invasion-associated genes, such as the transcriptional NFκB, and its downstream target gene, intercellular adhesion molecule 1. Thus, we report here the isolation and characterization of a gene,
IIp45
, whose protein product binds to IGFBP-2 and inhibits glioma cell invasion.
Measurement of unidirectional Pito ATP flux in human visual cortex at 7 T by usingin vivo31P magnetic resonance spectroscopy
Authors: Lei Hao , Ugurbil Kamil , Chen Wei
Published: November 2003

Taking advantage of the high NMR detection sensitivity and the large chemical shift dispersion offered by ultra-high field strength of 7 T, the effect of magnetization transfer on inorganic phosphate (Pi) resonance during saturation of γ-ATP resonance, mediated by the ATP synthesis reaction, was observed noninvasively in the human primary visual cortex by usingin vivo31P magnetic resonance spectroscopy. The unidirectional flux from Pito ATP was measured by using progressive saturation transfer experiments. The cerebral ATP synthesis rate in the human primary visual cortex measured by31P magnetic resonance spectroscopy in this study was 12.1 ± 2.8 μmol ATP/g per min, which agreed well with the value that was calculated indirectly from the cerebral metabolic rate of glucose consumption reported previously.
Computational evidence for a rivalry hierarchy in vision
Authors: Wilson Hugh R.
Published: November 2003

Cortical-form vision comprises multiple, hierarchically arranged areas with feedforward and feedback interconnections. This complex architecture poses difficulties for attempts to link perceptual phenomena to activity at a particular level of the system. This difficulty has been especially salient in studies of binocular rivalry alternations, where there is seemingly conflicting evidence for a locus in primary visual cortex or alternatively in higher cortical areas devoted to object perception. Here, I use a competitive neural model to demonstrate that the data require at least two hierarchic rivalry stages for their explanation. This model demonstrates that competitive inhibition in the first rivalry stage can be eliminated by using suitable stimulus dynamics, thereby revealing properties of a later stage, a result obtained with both spike-rate and conductance-based model neurons. This result provides a synthesis of competing rivalry theories and suggests that neural competition may be a general characteristic throughout the form-vision hierarchy.
X-ray structure of glutathione
S
-transferase from the malarial parasite
Plasmodium falciparum
Authors: Fritz-Wolf Karin , Becker Andreas , Rahlfs Stefan , Harwaldt Petra , Schirmer R. Heiner , Kabsch Wolfgang , Becker Katja
Published: November 2003

GSTs catalyze the conjugation of glutathione with a wide variety of hydrophobic compounds, generally resulting in nontoxic products that can be readily eliminated. In contrast to many other organisms, the malarial parasite
Plasmodium falciparum
possesses only one GST isoenzyme (PfGST). This GST is highly abundant in the parasite, its activity was found to be increased in chloroquine-resistant cells, and it has been shown to act as a ligandin for parasitotoxic hemin. Thus, the enzyme represents a promising target for antimalarial drug development. We now have solved the crystal structure of PfGST at a resolution of 1.9 Å. The homodimeric protein of 26 kDa per subunit represents a GST form that cannot be assigned to any of the known GST classes. In comparison to other GSTs, and, in particular, to the human isoforms, PfGST possesses a shorter C-terminal section resulting in a more solvent-accessible binding site for the hydrophobic and amphiphilic substrates. The structure furthermore reveals features in this region that could be exploited for the design of specific PfGST inhibitors.
Base pair analogs in the gas phase
Authors: Roscioli Joseph R. , Pratt David W.
Published: November 2003

A rotationally resolved electronic spectrum of the gas-phase dimer 2-aminopyridine·2-pyridone, an analog of the adenine·thymine base pair, has been observed and assigned, leading to precise measurements of its moments of inertia and preliminary determinations of its structure. A Watson–Crick configuration results, with N···H—N and N—H···O hydrogen bond lengths of 2.898 and 2.810 Å, respectively. The two bases are found not to be coplanar; a dihedral angle of 6.1° between the base planes is also estimated from the measured moments of inertia. Possible chemical and biological implications of these results are discussed.
Spatial memory performances of aged rats in the water maze predict levels of hippocampal neurogenesis
Authors: Drapeau Elodie , Mayo Willy , Aurousseau Catherine , Le Moal Michel , Piazza Pier-Vincenzo , Abrous Djoher Nora
Published: November 2003

Neurogenesis occurs within the adult dentate gyrus of the hippocampal formation and it has been proposed that the newly born neurons, recruited into the preexistent neuronal circuits, might be involved in hippocampal-dependent learning processes. Age-dependent spatial memory impairments have been related to an alteration in hippocampal plasticity. The aim of the current study was to examine whether cognitive functions in aged rats are quantitatively correlated with hippocampal neurogenesis. To this end, we took advantage of the existence of spontaneous individual differences observed in aged subjects in a hippocampal-dependent task, the water maze. We expected that the spatial memory capabilities of aged rats would be related to the levels of hippocampal neurogenesis. Old rats were trained in the water maze, and, 3 weeks after training, rats were injected with 5-bromo-2′-deoxyuridine (BrdUrd, 50 or 150 mg/kg) to label dividing cells. Cell proliferation was examined one day after the last BrdUrd injection, whereas cell survival and differentiation were determined 3 weeks later. It is shown that a quantitative relationship exists between learning and the number of newly generated neurons. Animals with preserved spatial memory, i.e., the aged-unimpaired rats, exhibited a higher level of cell proliferation and a higher number of new neurons in comparison with rats with spatial memory impairments, i.e., the aged-impaired rats. In conclusion, the extent of memory dysfunction in aged rats is quantitatively related to the hippocampal neurogenesis. These data reinforce the assumption that neurogenesis is involved in memory processes and aged-related cognitive alterations.
Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: Manifestation of a dual activation threshold
Authors: Faroudi Mustapha , Utzny Clemens , Salio Mariolina , Cerundolo Vincenzo , Guiraud Martine , Müller Sabina , Valitutti Salvatore
Published: November 2003

Activation of biological functions in T lymphocytes is determined by the molecular dynamics occurring at the T cell/opposing cell interface. In the present study, a central question of cytotoxic T lymphocyte (CTL) biology was studied at the single-cell level: can two distinct activation thresholds for cytotoxicity and cytokine production be explained by intercellular molecular dynamics between CTLs and targets? In this study, we combine morphological approaches with numerical analysis, which allows us to associate specific patterns of calcium mobilization with different biological responses. We show that CTLs selectively activated to cytotoxicity lack a mature immunological synapse while exhibiting a low threshold polarized secretion of lytic granules and spike-like patterns of calcium mobilization. This finding is contrasted by fully activated CTLs, which exhibit a mature immunological synapse and smooth and sustained calcium mobilization. Our results indicate that intercellular molecular dynamics and signaling characteristics allow the definition of two activation thresholds in individual CTLs: one for polarized granule secretion (lytic synapse formation) and the other for cytokine production (stimulatory synapse formation).
Myosin XVa localizes to the tips of inner ear sensory cell stereocilia and is essential for staircase formation of the hair bundle
Authors: Belyantseva Inna A. , Boger Erich T. , Friedman Thomas B.
Published: November 2003

Mutations of the gene encoding unconventional myosin XVa are associated with sensorineural deafness in humans (
DFNB3
) and shaker (

Myo15
sh2

) mice. In deaf

Myo15
sh2/sh2

mice, stereocilia are short, nearly equal in length, and lack myosin XVa immunoreactivity. We previously reported that myosin XVa mRNA and protein are expressed in cochlear hair cells. We now show that in the mouse, rat, and guinea pig, endogenous myosin XVa localizes to the tips of the stereocilia of the cochlear and vestibular hair cells. Myosin XVa localization overlaps with the barbed ends of actin filaments and extends to the apical plasma membrane of the stereocilia. Gene gun-mediated transfection of mouse inner ear sensory epithelia explants shows selective accumulation of myosin XVa-GFP at the tips of stereocilia, confirming the localization of native myosin XVa. Expression in COS7 cells also reveals targeting of myosin XVa-GFP to the dynamic actin region at the tips of filopodia. In a wild-type mouse, during auditory and vestibular hair cell development, myosin XVa appears at the tips of stereocilia at the time when the hair bundle begins to develop its characteristic staircase pattern. We propose that myosin XVa is essential for the graded elongation of stereocilia during their functional maturation.
Cell size as a link between noncoding DNA and metabolic rate scaling
Authors: Kozłowski J. , Konarzewski M. , Gawelczyk A. T.
Published: November 2003

Accumulation of noncoding DNA and therefore genome size (C-value) may be under strong selection toward increase of body size accompanied by low metabolic costs. C-value directly affects cell size and specific metabolic rate indirectly. Body size can enlarge through increase of cell size and/or cell number, with small cells having higher metabolic rates. We argue that scaling exponents of interspecific allometries of metabolic rates are by-products of evolutionary diversification of C-values within narrow taxonomic groups, which underlines the participation of cell size and cell number in body size optimization. This optimization leads to an inverse relation between slopes of interspecific allometries of metabolic rates and C-value. To test this prediction we extracted literature data on basal metabolic rate (BMR), body mass, and C-value of mammals and birds representing six and eight orders, respectively. Analysis of covariance revealed significant heterogeneity of the allometric slopes of BMR and C-value in both mammals and birds. As we predicted, the correlation between allometric exponents of BMR and C-value was negative and statistically significant among mammalian and avian orders.
B7h is required for T cell activation, differentiation, and effector function
Authors: Nurieva Roza I. , Mai Xoi Moui , Forbush Katherine , Bevan Michael J. , Dong Chen
Published: November 2003

T helper (Th) cell activation, differentiation, and immune function are regulated by costimulatory molecules. Inducible costimulator (ICOS) is a recently identified costimulatory receptor expressed on activated T cells. A ligand for ICOS, B7h, is expressed on B cells and other types of antigen-presenting cells (APC). Although ICOS has been shown to be essential in T cell activation and differentiation, the regulatory roles of B7h at different stages of T cell immune responses have not been examined genetically. In this study, we generated and analyzed B7h-deficient mice. We present evidence that B7h is the only ligand for ICOS, and ICOS, its only corresponding receptor. Th cells, when activated with B7h-deficient APC, exhibited reduced proliferation and IL-2 production. In addition, Th cells produced significantly reduced amounts of IL-4 and -13 after differentiation at the presence of B7h
–/–
APC. This cytokine defect was associated with a deficiency in c-Maf expression and could be rescued completely by c-Maf overexpression in T cells. Furthermore, we showed that effector T cells, when restimulated in the presence of B7h-deficient APC, exhibited reduced Th2 cytokine production. Therefore, B7h is required for proper Th cell activation, differentiation, and effector cytokine expression.
Regulation of zinc homeostasis by inducible NO synthase-derived NO: Nuclear metallothionein translocation and intranuclear Zn2+release
Authors: Spahl Daniela U. , Berendji-Grün Denise , Suschek Christoph V. , Kolb-Bachofen Victoria , Kröncke Klaus-D.
Published: November 2003

Zn2+is critical for the functional and structural integrity of cells and contributes to a number of important processes including gene expression. It has been shown that NO exogenously applied via NO donors resulting in nitrosative stress leads to cytoplasmic Zn2+release from the zinc storing protein metallothionein (MT) and probably other proteins that complex Zn2+via cysteine thiols. We show here that, in cytokine-activated murine aortic endothelial cells, NO derived from the inducible NO synthase (iNOS) induces a transient nuclear release of Zn2+. This nuclear Zn2+release depends on the presence of MT as shown by the lack of this effect in activated endothelial cells from MT-deficient mice and temporally correlates with nuclear MT translocation. Data also show that NO is an essential but not sufficient signal for MT-mediated Zn2+trafficking from the cytoplasm into the nucleus. In addition, we found that, endogenously via iNOS, synthesized NO increases the constitutive mRNA expression of both MT-1 and MT-2 genes and that nitrosative stress exogenously applied via an NO donor increases constitutive MT mRNA expression via intracellular Zn2+release. In conclusion, we here provide evidence for a signaling mechanism based on iNOS-derived NO through the regulation of intracellular Zn2+trafficking and homeostasis.
Quantitative evolutionary design of glucose 6-phosphate dehydrogenase expression in human erythrocytes
Authors: Salvador Armindo , Savageau Michael A.
Published: November 2003

Why do the activities of some enzymes greatly exceed the flux capacity of the embedding pathways? This is a puzzling open problem in quantitative evolutionary design. In this work we investigate reasons for high expression of a thoroughly characterized enzyme: glucose 6-phosphate dehydrogenase (G6PD) in human erythrocytes. G6PD catalyses the first step of the pathway that supplies NADPH for antioxidant defense mechanisms. Normal G6PD activity far exceeds the capacity of human erythrocytes for a steady NADPH supply, which is limited upstream of G6PD. However, the distribution of erythrocyte G6PD activity in human populations reveals a selective pressure for maintaining high activity. To clarify the nature of this selective pressure, we studied how G6PD activity and other parameters in a model of the NADPH redox cycle affect metabolic performance. Our analysis indicates that normal G6PD activity is sufficient but not superfluous to avoid NADPH depletion and ensure timely adaptation of the NADPH supply during pulses of oxidative load such as those that occur during adherence of erythrocytes to phagocytes. These results suggest that large excess capacities found in some biochemical and physiological systems, rather than representing large safety factors, may reflect a close match of system design to unscrutinized performance requirements. Understanding quantitative evolutionary design thus calls for careful consideration of the various performance specifications that biological components/processes must meet in order for the organism to be fit. The biochemical systems framework used in this paper is generally applicable for such a detailed examination of the quantitative evolutionary design of gene expression levels in other systems.
Molecular evolution of the insect chemoreceptor gene superfamily in
Drosophila melanogaster
Authors: Robertson Hugh M. , Warr Coral G. , Carlson John R.
Published: November 2003

The insect chemoreceptor superfamily in
Drosophila melanogaster
is predicted to consist of 62 odorant receptor (Or) and 68 gustatory receptor (Gr) proteins, encoded by families of 60
Or
and 60
Gr
genes through alternative splicing. We include two previously undescribed
Or
genes and two previously undescribed
Gr
genes; two previously predicted
Or
genes are shown to be alternative splice forms. Three polymorphic pseudogenes and one highly defective pseudogene are recognized. Phylogenetic analysis reveals deep branches connecting multiple highly divergent clades within the
Gr
family, and the
Or
family appears to be a single highly expanded lineage within the superfamily. The genes are spread throughout the
Drosophila
genome, with some relatively recently diverged genes still clustered in the genome. The
Gr5a
gene on the X chromosome, which encodes a receptor for the sugar trehalose, has transposed from one such tandem cluster of six genes at cytological location 64, as has
Gr61a
, and all eight of these receptors might bind sugars. Analysis of intron evolution suggests that the common ancestor consisted of a long N-terminal exon encoding transmembrane domains 1-5 followed by three exons encoding transmembrane domains 6-7. As many as 57 additional introns have been acquired idiosyncratically during the evolution of the superfamily, whereas the ancestral introns and some of the older idiosyncratic introns have been lost at least 48 times independently. Altogether, these patterns of molecular evolution suggest that this is an ancient superfamily of chemoreceptors, probably dating back at least to the origin of the arthropods.
Ciliary neurotrophic factor improves diabetic parameters and hepatic steatosis and increases basal metabolic rate in
db/db
mice
Authors: Sleeman M. W. , Garcia K. , Liu R. , Murray J. D. , Malinova L. , Moncrieffe M. , Yancopoulos G. D. , Wiegand S. J.
Published: November 2003

Obesity plays a central role in the development of insulin resistance and type 2 diabetes. We therefore examined the effects of a modified form of ciliary neurotrophic factor [Axokine, which is hereafter referred to as ciliary neurotrophic factor (CNTF)
Ax15
], which uses a leptin-like mechanism to reduce body weight, in the
db
/
db
murine model of type 2 diabetes. In previous studies, weight loss produced by CNTF treatment could largely be attributed to its effects on food intake. In contrast, CNTF
Ax15
treatment of
db
/
db
mice caused significantly greater weight loss and marked improvements in diabetic parameters (e.g., levels of glucose, insulin, triglyceride, cholesterol, and nonesterified free fatty acids) than could be accounted for by reduced caloric intake alone. These beneficial effects, above and beyond those seen in animals controlled for either food restriction or body weight, correlated with the ability of CNTF
Ax15
to increase metabolic rate and energy expenditure and reduce hepatic steatosis while enhancing hepatic responsiveness to insulin. The hepatic effects were linked to rapid alterations in hepatic gene expression, most notably reduced expression of stearoyl-CoA desaturase 1, a rate-limiting enzyme in the synthesis of complex lipids that is also markedly suppressed by leptin in
ob
/
ob
mice. These observations further link the mechanisms of CNTF and leptin action, and they suggest important, beneficial effects for CNTF in diabetes that may be distinct from its ability to decrease food intake; instead, these effects may be more related to its influence on energy expenditure and hepatic gene expression.
Translationally controlled tumor protein acts as a guanine nucleotide dissociation inhibitor on the translation elongation factor eEF1A
Authors: Cans Christophe , Passer Brent J. , Shalak Vyacheslav , Nancy-Portebois Vanessa , Crible Virginie , Amzallag Nathalie , Allanic David , Tufino Rowena , Argentini Manuela , Moras Dino , Fiucci Giusy , Goud Bruno , Mirande Marc , Amson Robert , Telerman Adam
Published: November 2003

Recently, we demonstrated that the expression levels of the translationally controlled tumor protein (TCTP) were strongly down-regulated at the mRNA and protein levels during tumor reversion/suppression and by the activation of p53 and Siah-1. To better characterize the function of TCTP, a yeast two-hybrid hunt was performed. Subsequent analysis identified the translation elongation factor, eEF1A, and its guanine nucleotide exchange factor, eEF1Bβ, as TCTP-interacting partners.
In vitro
and
in vivo
studies confirmed that TCTP bound specifically eEF1Bβ and eEF1A. Additionally, MS analysis also identified eEF1A as a TCTP interactor. Because eEF1A is a GTPase, we investigated the role of TCTP on the nucleotide exchange reaction of eEF1A. Our results show that TCTP preferentially stabilized the GDP form of eEF1A, and, furthermore, impaired the GDP exchange reaction promoted by eEF1Bβ. These data suggest that TCTP has guanine nucleotide dissociation inhibitor activity, and, moreover, implicate TCTP in the elongation step of protein synthesis.
Transgenic planarian lines obtained by electroporation using transposon-derived vectors and an eye-specific GFP marker
Authors: González-Estévez C. , Momose T. , Gehring W. J. , Saló E.
Published: November 2003

To generate transgenic planarians we used a set of versatile vectors for animal transgenesis based on the promiscuous transposons,
mariner
,
Hermes
and
piggyBac,
and a universal enhanced GFP (EGFP) marker system with three Pax6 dimeric binding sites, the 3xP3-EGFP developed by Berghammer
et al.
[Berghammer, A. J., Klinger, M. & Wimmer, E. A. (1999)
Nature
402, 370–371]. This marker is expressed specifically in the eyes of various arthropod taxa. Upon microinjection into the parenchyma of adult planarians and subsequent electroporation, these vectors transpose efficiently into the planarian genome. One of the cell types transformed are the totipotent “neoblast” stem cells present in the adults, representing 30% of total cells. The neoblast represents a unique cell type with the capacity to proliferate and to differentiate into all somatic cell types as well as into germ cells. All three transposon vectors have high transformation efficiency, but only
Hermes
and
piggyBac
show stable integration. The
mariner
vector is frequently lost presumably because of the presence of active
mariner
-type transposons in the genome of the
Girardia tigrina
. Transformed animals are mosaics containing both transformed and untransformed neoblasts. These differentiate to form EGFP-positive and -negative photoreceptor cells. Such mosaicism is maintained through several cycles of regeneration induced by decapitation or asexual reproduction. Transformed neoblasts also contribute to the germ line, and can give rise to pure transgenic planarian lines in which EGFP is expressed in all photoreceptor cells after sexual reproduction. The presence of the transgenes was confirmed by PCR, plasmid rescue assay, inverse PCR, and Southern blotting. Our results with the 3xP3-EGFP marker confirm the presence of Pax6 activity in the differentiated photoreceptor cells of planarian eyes. Transgenesis will be an important tool to dissect developmental molecular mechanisms in planarian regeneration, development and stem cell biology, and may also be an entry point to analyze the biology of parasitic Platyhelminthes.
In vitro
assay for neutralizing antibody to hepatitis C virus: Evidence for broadly conserved neutralization epitopes
Authors: Bartosch Birke , Bukh Jens , Meunier Jean-Christophe , Granier Christelle , Engle Ronald E. , Blackwelder William C. , Emerson Suzanne U. , Cosset François-Loïc , Purcell Robert H.
Published: November 2003

Our understanding of the humoral immune response to hepatitis C virus (HCV) is limited because the virus can be studied only in humans and chimpanzees and because previously described neutralization assays have not been robust or simple to perform. Nevertheless, epidemiologic and laboratory studies suggested that neutralizing Ab to HCV might be important in preventing infection. We have recently described a neutralization assay based on the neutralization of pseudotyped murine retrovirus constructs bearing HCV envelope glycoproteins on their surface. We have applied the assay to well characterized clinical samples from HCV-infected patients and chimpanzees, confirmed the existence of neutralizing Ab to HCV, and validated most previously reported neutralizations of the virus. We did not find neutralizing anti-HCV in resolving infections but did find relatively high titers (>1:320) of such Ab in chronic infections. Neutralizing Ab was directed not only to epitope(s) in the hypervariable region of the E2 envelope protein but also to one or more epitopes elsewhere in the envelope of the virus. Neutralizing Ab was broadly reactive and could neutralize pseudotype particles bearing the envelope glycoproteins of two different subgenotypes (1a and 1b). The ability to assay neutralizing anti-HCV should permit an assessment of the prospects for successful Ab-mediated passive and active immunoprophylaxis against hepatitis C.
Shade is the
Drosophila
P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone
Authors: Petryk Anna , Warren James T. , Marqués Guillermo , Jarcho Michael P. , Gilbert Lawrence I. , Kahler Jonathan , Parvy Jean-Philippe , Li Yutai , Dauphin-Villemant Chantal , O'Connor Michael B.
Published: November 2003

The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any insect. We show here that the
Drosophila
E-20-monooxygenase (E20MO) is the product of the
shade
(
shd
) locus (cytochrome p450, CYP314a1). When
shd
is transfected into
Drosophila
S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous
shd
embryos, no E20MO activity is apparent either
in vivo
or
in vitro
. Mutations in
shd
lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in
disembodied
(
dib
) and
shadow
(
sad
) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike
dib
and
sad
,
shd
is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of insects. However, the tissue in which
shd
is expressed does not appear to be important for developmental function because misexpression of
shd
in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which
shd
is expressed, rescues embryonic lethality.
Target-specific utilization of transcriptional regulatory surfaces by the glucocorticoid receptor
Authors: Rogatsky Inez , Wang Jen-Chywan , Derynck Mika K. , Nonaka Daisuke F. , Khodabakhsh Daniel B. , Haqq Christopher M. , Darimont Beatrice D. , Garabedian Michael J. , Yamamoto Keith R.
Published: November 2003

The glucocorticoid receptor (GR) activates or represses transcription depending on the sequence and architecture of the glucocorticoid response elements in target genes and the availability and activity of interacting cofactors. Numerous GR cofactors have been identified, but they alone are insufficient to dictate the specificity of GR action. Furthermore, the role of different functional surfaces on the receptor itself in regulating its targets is unclear, due in part to the paucity of known target genes. Using DNA microarrays and real-time quantitative PCR, we identified genes transcriptionally activated by GR, in a translation-independent manner, in two human cell lines. We then assessed in U2OS osteosarcoma cells the consequences of individually disrupting three GR domains, the N-terminal activation function (AF) 1, the C-terminal AF2, or the dimer interface, on activation of these genes. We found that GR targets differed in their requirements for AF1 or AF2, and that the dimer interface was dispensable for activation of some genes in each class. Thus, in a single cell type, different GR surfaces were used in a gene-specific manner. These findings have strong implications for the nature of gene response element signaling, the composition and structure of regulatory complexes, and the mechanisms of context-specific transcriptional regulation.
IL-18 cDNA vaccination protects mice from spontaneous lupus-like autoimmune disease
Authors: Bossù Paola , Neumann Detlef , Del Giudice Elda , Ciaramella Antonio , Gloaguen Isabelle , Fantuzzi Giamila , Dinarello Charles A. , Di Carlo Emma , Musiani Piero , Meroni Pier Luigi , Caselli Gianfranco , Ruggiero Paolo , Boraschi Diana
Published: November 2003

The lupus-like autoimmune syndrome of MRL/Mp-Tnfrsf6lpr(lpr) mice is characterized by progressive lymphadenopathy and autoantibody production, leading to early death from renal failure. Activation of T helper lymphocytes is one of the events in the pathogenesis of the disease in these mice and likely in human systemic lupus erythematosus. Among T helper lymphocytedependent cytokines, IFN-γ plays a pivotal role in the abnormal cell activation and the fatal development of thelprdisease. IL-18, an inducer of IFN-γ in T lymphocytes and natural killer cells, may contribute to the disease because cells fromlprmice are hypersensitive to IL-18 and express high levels of IL-18. To assess the contribution of IL-18 to the pathogenesis in the animal model,in vivoinhibition of IL-18 was attempted. Younglprmice were vaccinated against autologous IL-18 by repeated administration of a cDNA coding for the murine IL-18 precursor. Vaccinated mice produced autoantibodies to murine IL-18 and exhibited a significant reduction in spontaneous lymphoproliferation and IFN-γ production as well as less glomerulonephritis and renal damage. Moreover, mortality was significantly delayed in anti-IL-18-vaccinated mice. These studies support the concept that IL-18 plays a major role in the pathogenesis of the autoimmune syndrome oflprmice and that a reduction in IL-18 activity could be a therapeutic strategy in autoimmune diseases.
Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin
Authors: Kwik Jeanne , Boyle Sarah , Fooksman David , Margolis Leonid , Sheetz Michael P. , Edidin Michael
Published: November 2003

Responses to cholesterol depletion are often taken as evidence of a role for lipid rafts in cell function. Here, we show that depletion of cell cholesterol has global effects on cell and plasma membrane architecture and function. The lateral mobility of membrane proteins is reduced when cell cholesterol is chronically or acutely depleted. The change in mobility is a consequence of the reorganization of the cell actin. Binding of a GFP-tagged pleckstrin homology domain specific for phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to the plasma membrane is reduced after cholesterol depletion. This result implies that the reorganization of cytoskeleton depends on the loss or redistribution of plasma membrane PI(4,5)P2. Consistent with this observation, agents that sequester plasma membrane PI(4,5)P2 mimic the effects of cholesterol depletion on actin organization and on lateral mobility.
Wnk1 kinase deficiency lowers blood pressure in mice: A gene-trap screen to identify potential targets for therapeutic intervention
Authors: Zambrowicz Brian P. , Abuin Alejandro , Ramirez-Solis Ramiro , Richter Lizabeth J. , Piggott James , BeltrandelRio Hector , Buxton Eric C. , Edwards Joel , Finch Rick A. , Friddle Carl J. , Gupta Anupma , Hansen Gwenn , Hu Yi , Huang Wenhu , Jaing Crystal , Key Billie Wayne , Kipp Peter , Kohlhauff Buckley , Ma Zhi-Qing , Markesich Diane , Payne Robert , Potter David G. , Qian Ny , Shaw Joseph , Schrick Jeff , Shi Zheng-Zheng , Sparks Mary Jean , Van Sligtenhorst Isaac , Vogel Peter , Walke Wade , Xu Nianhua , Zhu Qichao , Person Christophe , Sands Arthur T.
Published: November 2003

The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in ≈60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the
Wnk1
kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in
WNK1
lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K
+
and H
+
excretion. Consistent with the human genetic studies,
Wnk1
heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the
Wnk1
mutation died during embryonic development before day 13 of gestation. These results demonstrate that
Wnk1
is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.
Neurotrophin 3 activation of TrkC induces Schwann cell migration through the c-Jun N-terminal kinase pathway
Authors: Yamauchi Junji , Chan Jonah R. , Shooter Eric M.
Published: November 2003

During development and nerve injury, complex interactions between glial cells and neurons are essential for establishing proper nerve function. Neurotrophins play multiple roles in the developing nervous system, including cell survival, growth, and differentiation. Here we show that migration of Schwann cells, isolated from sciatic nerves, is significantly enhanced by neurotrophin 3, but not by nerve growth factor or brain-derived neurotrophic factor. The neurotrophin-3-induced cell migration was also observed in Schwann cells isolated from sciatic nerves of p75
NTR-/-
mice, indicating that neurotrophin 3 enhances cell migration through TrkC. This effect was blocked by K252a, an inhibitor of the Trk receptor family. Additionally, the neurotrophin-3-induced cell migration depended on Rho GTPases (Rac1 and Cdc42) and c-Jun N-terminal kinase. We obtained the same results with Cos-7 cells expressing TrkC. Taken together, these results suggest that neurotrophin 3 activation of TrkC induces Schwann cell migration through the c-Jun N-terminal kinase signaling pathway.
Glatiramer acetate-specific T cells in the brain express T helper 2/3 cytokines and brain-derived neurotrophic factorin situ
Authors: Aharoni Rina , Kayhan Basak , Eilam Raya , Sela Michael , Arnon Ruth
Published: November 2003

The ability of a remedy to modulate the pathological process in the target organ is crucial for its therapeutic activity. Glatiramer acetate (GA, Copaxone, Copolymer 1), a drug approved for the treatment of multiple sclerosis, induces regulatory T helper 2/3 cells that penetrate the CNS. Here we investigated whether these GA-specific T cells can function as suppressor cells with therapeutic potential in the target organ byin situexpression of T helper 2/3 cytokines and neurotrophic factors. GA-specific cells and theirin situexpression were detected on the level of whole-brain tissue by using a two-stage double-labeling system: (i) labeling of the GA-specific T cells, followed by their adoptive transfer, and (ii) detection of the secreted factors in the brain by immunohistological methods. GA-specific T cells in the CNS demonstrated intense expression of the brain-derived neurotrophic factor and of two antiinflammatory cytokines, IL-10 and transforming growth factor β. No expression of the inflammatory cytokine IFN-γ was observed. This pattern of expression was manifested in brains of normal and experimental autoimmune encephalomyelitis-induced mice to which GA-specific cells were adoptively transferred, but not in control mice. Furthermore, infiltration of GA-induced cells to the brain resulted in bystander expression of IL-10 and transforming growth factor β by resident astrocytes and microglia. The ability of infiltrating GA-specific cells to express antiinflammatory cytokines and neurotrophic factor in the organ in which the pathological processes occur correlates directly with the therapeutic activity of GA in experimental autoimmune encephalomyelitis/multiple sclerosis.
Shutdown of an acute T cell immune response to viral infection is mediated by the proapoptotic Bcl-2 homology 3-only protein Bim
Authors: Pellegrini Marc , Belz Gabrielle , Bouillet Philippe , Strasser Andreas
Published: November 2003

We used mutant Fas-deficient (lpr) or Bim-deficient mice to investigate the role of the death receptor and Bcl-2-regulated apoptotic pathways in terminating a physiological T cell response to herpes simplex virus infection. In WT andlprmice CD8+antigen-specific T cells were deleted after viral clearance. In contrast, the immune response was not terminated in Bim-deficient mice despite viral clearance, and CD8+antigen-specific T cells accumulated in the spleen. Thus, Bim is dispensable for viral clearance but is necessary for the death of activated T cells when immune responses are terminated. These findings have implications for the therapeutic manipulation of immune responses to infections and immunization.
Identification, characterization, and gene expression profiling of endotoxin-induced myocarditis
Authors: Wong Ma-Li , O'Kirwan Fiona , Khan Nadia , Hannestad Jonas , Wu Kwok H. , Elashoff David , Lawson Gregory , Gold Philip W. , McCann Samuel M. , Licinio Julio
Published: November 2003

In septic shock, reversible cardiac dysfunction starts within 24 h. Myocardial depressant factors are thought to cause myocyte dysfunction, resulting in alterations of intrinsic cardiac function. Nitric oxide is a myocardial depressant factor candidate. Here we identify endotoxin-induced myocarditis (EIM) a previously uncharacterized pathophysiological entity. Features of EIM include differential patterns of inducible NO synthase (NOS2) mRNA induction in the left (LV) and right (RV) ventricles during the systemic response inflammatory syndrome (SIRS) and the presence of myocarditis with focal areas of aseptic necrosis in the RV 24 h after SIRS induction. Even though clinical data lead to the presumption of myocardial injury in sepsis, the underlying pathophysiological mechanisms have not been previously elucidated. Gene expression profiling was used to test the hypothesis of differential LV and RV responses in EIM, and revealed novel patterns of qualitative and quantitative expansion of transcription. Those genes are novel targets for drug development in SIRS and sepsis. Our results demonstrate spatial and temporal heterogeneity of myocardial responses in EIM. These findings justify the design of treatments to ameliorate tissue injury in the RV. Because the complexity of the inflammatory response increases substantially as time elapses, we suggest a stepwise and multitarget therapeutic approach for SIRS and sepsis. Our findings can help identify innate immune pathways that could become targets for immunotherapy in the treatment of disease caused by potential bioterrorism agents.
Site- and sequence-selective ultrafast hydration of DNA
Authors: Pal Samir Kumar , Zhao Liang , Xia Tianbing , Zewail Ahmed H.
Published: November 2003

Water molecules in the DNA grooves are critical for maintaining structural integrity, conformational changes, and molecular recognition. Here we report studies of site- and sequence-specific hydration dynamics, using 2-aminopurine (Ap) as the intrinsic fluorescence probe and with femtosecond resolution. The dodecamer d[CGCA(Ap)ATTTGCG]
2
was investigated, and we also examined the effect of a specific minor groove-binding drug, pentamidine, on hydration dynamics. Two time scales were observed: ≈1 ps (bulk-like) and 10–12 ps (weakly bound type), consistent with layer hydration observed in proteins and DNA. However, for denatured DNA, the cosolvent condition of 40% formamide hydration is very different: it becomes that of bulk (in the presence of formamide). Well known electron transfer between Ap and nearby bases in stacked assemblies becomes inefficient in the single-stranded state. The rigidity of Ap in the single strands is significantly higher than that in bulk water and that attached to deoxyribose, suggesting a unique role for the dynamics of the phosphate-sugar-base in helix formation. The disparity in minor and major groove hydration is evident because of the site selection of Ap and in the time scale observed here (in the presence and absence of the drug), which is different by a factor of 2 from that observed in the minor groove–drug recognition.
A newborn lethal defect due to inactivation of retinaldehyde dehydrogenase type 3 is prevented by maternal retinoic acid treatment
Authors: Dupé Valérie , Matt Nicolas , Garnier Jean-Marie , Chambon Pierre , Mark Manuel , Ghyselinck Norbert B.
Published: November 2003

The retinoic acid (RA) signal, produced locally from vitamin A by retinaldehyde dehydrogenase (Raldh) and transduced by the nuclear receptors for retinoids (RA receptor and 9-
cis
-RA receptor), is indispensable for ontogenesis and homeostasis of numerous tissues. We demonstrate that
Raldh3
knockout in mouse suppresses RA synthesis and causes malformations restricted to ocular and nasal regions, which are similar to those observed in vitamin A-deficient fetuses and/or in retinoid receptor mutants.
Raldh3
knockout notably causes choanal atresia (CA), which is responsible for respiratory distress and death of
Raldh3
-null mutants at birth. CA is due to persistence of nasal fins, whose rupture normally allows the communication between nasal and oral cavities. This malformation, which is similar to isolated congenital CA in humans and may result from impaired RA-controlled down-regulation of
Fgf8
expression in nasal fins, can be prevented by a simple maternal treatment with RA.
The fragile X mental retardation protein is required for type-I metabotropic glutamate receptor-dependent translation of PSD-95
Authors: Todd Peter K. , Mack Kenneth J. , Malter James S.
Published: November 2003

Fragile X syndrome (FXS) is a common inherited cause of mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP is thought to regulate the translation of target mRNAs, including its own transcript. Here we show that the levels of FMRP are rapidly up-regulated in primary cortical neurons in response to the type-I metabotropic glutamate receptor (mGluR) agonist
S
-3,5-dihydrophenylglycine. These changes require new protein synthesis but not transcription and are specific to mGluR activation. We also demonstrate that the mRNA for PSD-95, a scaffolding protein involved in synaptic plasticity, contains a highly conserved canonical binding site for FMRP within its 3′ UTR. Furthermore, PSD-95 is rapidly translated in response to
S
-3,5-dihydrophenylglycine. Finally, we show that these mGluR-dependent changes in PSD-95 expression are lost in neurons derived from FMRP knockout mice, a model of FXS. Taken together, these studies suggest that FMRP is required for mGluR-dependent translation of PSD-95 and provide insights into the pathophysiology of FXS.
Intracellular formation of ”undisruptable” dimers of inducible nitric oxide synthase
Authors: Kolodziejski Pawel J. , Rashid Mohammad B. , Eissa N. Tony
Published: November 2003

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer. Dimerization of iNOS represents a potentially critical target for therapeutic intervention. In this study, we show that intracellular iNOS forms dimers that are ”undisruptable” by boiling, denaturants, or reducing agents. Undisruptable (UD) dimers are clearly distinguishable from the easily dissociated dimers formed by iNOSin vitro. UD dimers do not form inEscherichia coli-expressed iNOS and could not be assembledin vitro, which suggests that anin vivocellular process is required for their formation. iNOS UD dimers are not affected by intracellular depletion of H4B. However, the mutation of Cys-115 (critical for zinc binding) greatly affects the formation of UD dimers. This study reveals insight into the mechanisms ofin vivoiNOS dimer formation. UD dimers represent a class of iNOS dimers that had not been suspected. This unanticipated finding revises our understanding of the mechanisms of iNOS dimerization and lays the groundwork for future studies aimed at modulating iNOS activityin vivo.