bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoid receptor pathways are involved in the inhibition of astrocyte proliferation
Authors: Crossin Kathryn L. , Tai Ming-Hong , Krushel Leslie A. , Mauro Vincent P. , Edelman Gerald M.
Published: July 2002

In earlier studies, the neural cell adhesion molecule, N-CAM, was found to inhibit the proliferation of rat astrocytes bothin vitroandin vivo. To identify the gene targets involved, we used subtractive hybridization to examine changes in gene expression that occur after astrocytes are exposed to N-CAMin vitro. While the mRNA levels for N-CAM decreased after such treatment, the levels of mRNAs for glutamine synthetase and calreticulin increased. Since glutamine synthetase and calreticulin are known to be involved in glucocorticoid receptor pathways, we tested a number of steroids for their effects on astrocyte proliferation. Dexamethasone, corticosterone, and aldosterone were all found to inhibit rat cortical astrocyte proliferation in culture in a dose-dependent manner. RU-486, a potent glucocorticoid antagonist, reversed the inhibitory effects of dexamethasone. These observations prompted the hypothesis that the inhibition of proliferation by N-CAM might be mediated through the glucocorticoid receptor pathway. Consistent with this hypothesis, the inhibition of astrocyte proliferation by N-CAM was reversed in part by a number of glucocorticoid antagonists, including RU-486, dehydroepiandrosterone, and progesterone. In transfection experiments with cultured astrocytes, N-CAM treatment increased the expression of a luciferase reporter gene under the control of a minimal promoter linked to a glucocorticoid response element. The enhanced activity of this construct stimulated by N-CAM was abolished in the presence of RU-486. The combined data suggest that astrocyte proliferation is in part regulated by alterations in glucocorticoid receptor pathways.
The rate of telomere sequence loss in human leukocytes varies with age
Authors: Frenck Robert W. , Blackburn Elizabeth H. , Shannon Kevin M.
Published: July 2002

A gradual loss of telomeric repeat sequences with aging previously has been noted in normal adult tissues, and this process has been implicated in cell senescence. No data exist that address the rate of telomere shortening in normal human cells within families or early in life. To address these questions, we measured telomere lengths in peripheral blood leukocytes (PBLs) from 75 members of 12 families and in a group of unrelated healthy children who were 5–48 months old. Here we report the surprising observation that rates of telomere attrition vary markedly at different ages. Telomeric repeats are lost rapidly (at a rate of >1 kilobase per year) from the PBLs of young children, followed by an apparent plateau between age 4 and young adulthood, and by gradual attrition later in life. These data suggest that the loss of telomeric repeats in hematopoietic cells is a dynamic process that is differentially regulated in young children and adults. Our results have implications for current models of how telomeric sequences are lost in normal somatic cells and suggest that PBLs are an excellent tissue to investigate how this process is controlled.
Folding funnels and frustration in off-lattice minimalist protein landscapes
Authors: Nymeyer Hugh , García Angel E. , Onuchic José Nelson
Published: July 2002

A full quantitative understanding of the protein folding problem is
now becoming possible with the help of the energy landscape theory and
the protein folding funnel concept. Good folding sequences have a
landscape that resembles a rough funnel where the energy bias towards
the native state is larger than its ruggedness. Such a landscape leads
not only to fast folding and stable native conformations but, more
importantly, to sequences that are robust to variations in the protein
environment and to sequence mutations. In this paper, an off-lattice
model of sequences that fold into a β-barrel native structure is used
to describe a framework that can quantitatively distinguish good and
bad folders. The two sequences analyzed have the same native structure,
but one of them is minimally frustrated whereas the other one exhibits
a high degree of frustration.
Silicatein α: Cathepsin L-like protein in sponge biosilica
Authors: Shimizu Katsuhiko , Cha Jennifer , Stucky Galen D. , Morse Daniel E.
Published: July 2002

Earth’s biota produces vast quantities of polymerized silica at ambient temperatures and pressures by mechanisms that are not understood. Silica spicules constitute 75% of the dry weight of the sponge
Tethya aurantia,
making this organism uniquely tractable for analyses of the proteins intimately associated with the biosilica. Each spicule contains a central protein filament, shown by x-ray diffraction to exhibit a highly regular, repeating structure. The protein filaments can be dissociated to yield three similar subunits, named silicatein α, β, and γ. The molecular weights and amino acid compositions of the three silicateins are similar, suggesting that they are members of a single protein family. The cDNA sequence of silicatein α, the most abundant of these subunits, reveals that this protein is highly similar to members of the cathepsin L and papain family of proteases. The cysteine at the active site in the proteases is replaced by serine in silicatein α, although the six cysteines that form disulfide bridges in the proteases are conserved. Silicatein α also contains unique tandem arrays of multiple hydroxyls. These structural features may help explain the mechanism of biosilicification and the recently discovered activity of the silicateins in promoting the condensation of silica and organically modified siloxane polymers (silicones) from the corresponding silicon alkoxides. They suggest the possibility of a dynamic role of the silicateins in silicification of the sponge spicule and offer the prospect of a new synthetic route to silica and siloxane polymers at low temperature and pressure and neutral pH.
A physiological role of the adenosine A
3
receptor: Sustained cardioprotection
Authors: Liang Bruce T. , Jacobson Kenneth A.
Published: July 2002

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart. A newly recognized adenosine receptor, the A
3
subtype, is expressed on the cardiac ventricular cell, and its activation protects the ventricular heart cell against injury during a subsequent exposure to ischemia. A cultured chicken ventricular myocyte model was used to investigate the cardioprotective role of a novel adenosine A
3
receptor. The protection mediated by prior activation of A
3
receptors exhibits a significantly longer duration than that produced by activation of the adenosine A
1
receptor. Prior exposure of the myocytes to brief ischemia also protected them against injury sustained during a subsequent exposure to prolonged ischemia. The adenosine A
3
receptor-selective antagonist 3-ethyl 5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191) caused a biphasic inhibition of the protective effect of the brief ischemia. The concomitant presence of the A
1
receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) converted the MRS1191-induced dose inhibition curve to a monophasic one. The combined presence of both antagonists abolished the protective effect induced by the brief ischemia. Thus, activation of both A
1
and A
3
receptors is required to mediate the cardioprotective effect of the brief ischemia. Cardiac atrial cells lack native A
3
receptors and exhibit a shorter duration of cardioprotection than do ventricular cells. Transfection of atrial cells with cDNA encoding the human adenosine A
3
receptor causes a sustained A
3
agonist-mediated cardioprotection. The study indicates that cardiac adenosine A
3
receptor mediates a sustained cardioprotective function and represents a new cardiac therapeutic target.
Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers
Authors: Türeci Özlem , Sahin Ugur , Vollmar Evi , Siemer Stefan , Göttert Elisabeth , Seitz Gerhard , Parkkila Anna-Kaisa , Shah Gul N. , Grubb Jeffrey H. , Pfreundschuh Michael , Sly William S.
Published: July 2002

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30–42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence
in situ
hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.
Activation of ventrolateral preoptic neurons by the somnogen prostaglandin D
2
Authors: Scammell Thomas , Gerashchenko Dmitry , Urade Yoshihiro , Onoe Hirotaka , Saper Clifford , Hayaishi Osamu
Published: July 2002

Prostaglandin D
2
(PGD
2
) is an extensively studied sleep-promoting substance, but the neuroanatomical basis of PGD
2
-induced sleep is only partially understood. To determine potential regions involved in this response, we used Fos immunohistochemistry to identify neurons activated by infusion of PGD
2
into the subarachnoid space below the rostral basal forebrain. PGD
2
increased nonrapid eye movement sleep and induced striking expression of Fos in the ventrolateral preoptic area (VLPO), a cluster of neurons that may promote sleep by inhibiting the tuberomammillary nucleus, the source of the ascending histaminergic arousal system. Fos expression in the VLPO was positively correlated with the preceding amount of sleep and negatively correlated with Fos expression in the tuberomammillary nucleus. PGD
2
also increased Fos immunoreactivity in the basal leptomeninges and several regions implicated in autonomic regulation. These observations suggest that PGD
2
may induce sleep via leptomeningeal PGD
2
receptors with subsequent activation of the VLPO.
The evolution of primate malaria parasites based on the gene encoding cytochromebfrom the linear mitochondrial genome
Authors: Escalante Ananias A. , Freeland Denise E. , Collins William E. , Lal Altaf A.
Published: July 2002

We report a phylogenetic analysis of primate malaria parasites based on the gene encoding the cytochromebprotein from the mitochondrial genome. We have studied 17 species ofPlasmodium, including 14 parasitic in primates. In our analysis, four species were used for rooting thePlasmodiumphylogenetic tree: two from closely related genera (Hepatocystissp. andHaemoproteus columbae)and two other Apicomplexa (Toxoplasma gondiiandTheileria parva). We found that primate malaria parasites form a monophyletic group, with the only exception being thePlasmodium falciparum–Plasmodium reichenowilineage. Phylogenetic analyses that include two species of non-PlasmodiumHaemosporina suggest that the genusPlasmodiumis polyphyletic. We conclude that the biologic traits, such as periodicity and the capacity to relapse, have limited value for assessing the phylogenetic relationships amongPlasmodiumspecies. For instance, we found no evidence that would link virulence with the age of the host–parasite association. Our studies also reveal that the primate malaria parasites originated in Africa, which contradicts the presently held opinion of Southeast Asia as their center of origin. We propose that the radiation of Asian monkey parasites is a recent event where several life history traits, like differences in periodicity, appearedde novo.
Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials
Authors: Koester Helmut J. , Sakmann Bert
Published: July 2002

We compared the transient increase of Ca
2+
in single spines on basal dendrites of rat neocortical layer 5 pyramidal neurons evoked by subthreshold excitatory postsynaptic potentials (EPSPs) and back-propagating action potentials (APs) by using calcium fluorescence imaging. AP-evoked Ca
2+
transients were detected in both the spines and in the adjacent dendritic shaft, whereas Ca
2+
transients evoked by single EPSPs were largely restricted to a single active spine head. Calcium transients elicited in the active spines by a single AP or EPSP, in spines up to 80 μm for the soma, were of comparable amplitude. The Ca
2+
transient in an active spine evoked by pairing an EPSP and a back-propagating AP separated by a time interval of 50 ms was larger if the AP followed the EPSP than if it preceded it. This difference reflected supra- and sublinear summation of Ca
2+
transients, respectively. A comparable dependence of spinous Ca
2+
transients on relative timing was observed also when short bursts of APs and EPSPs were paired. These results indicate that the amplitude of the spinous Ca
2+
transients during coincident pre- and postsynaptic activity depended critically on the relative order of subthreshold EPSPs and back-propagating APs. Thus, in neocortical neurons the amplitude of spinous Ca
2+
transients could encode small time differences between pre- and postsynaptic activity.
Molecular markers for cell types of the inner ear and candidate genes for hearing disorders
Authors: Heller Stefan , Sheane Charlotte A. , Javed Zarqa , Hudspeth A. J.
Published: July 2002

To identify genes expressed in the vertebrate inner ear, we have established an assay that allows rapid analysis of the differential expression pattern of mRNAs derived from an auditory epithelium-specific cDNA library. We performed subtractive hybridization to create an enriched probe, which then was used to screen the cDNA library. After digoxigenin-labeled antisense cRNAs had been transcribed from hybridization-positive clones, we conducted
in situ
hybridization on slides bearing cryosections of late embryonic chicken heads, bodies, and cochleae. One hundred and twenty of the 196 clones analyzed encode 12 proteins whose mRNAs are specifically or highly expressed in the chicken’s inner ear; the remainder encode proteins that occur more widely. We identified proteins that have been described previously as expressed in the inner ear, such as β-tectorin, calbindin, and type II collagen. A second group of proteins abundant in the inner ear includes five additional types of collagens. A third group, including Coch-5B2 and an ear-specific connexin, comprises proteins whose human equivalents are candidates to account for hearing disorders. This group also includes proteins expressed in two unique cell types of the inner ear, homogene cells and cells of the tegmentum vasculosum.
Experience-dependent corticofugal adjustment of midbrain frequency map in bat auditory system
Authors: Gao Enquan , Suga Nobuo
Published: July 2002

Recent studies of corticofugal modulation of auditory information processing indicate that cortical neurons mediate both a highly focused positive feedback to subcortical neurons “matched” in tuning to a particular acoustic parameter and a widespread lateral inhibition to “unmatched” subcortical neurons. This cortical function for the adjustment and improvement of subcortical information processing is called egocentric selection. Egocentric selection enhances the neural representation of frequently occurring signals in the central auditory system. For our present studies performed with the big brown bat (
Eptesicus fuscus
), we hypothesized that egocentric selection adjusts the frequency map of the inferior colliculus (IC) according to auditory experience based on associative learning. To test this hypothesis, we delivered acoustic stimuli paired with electric leg stimulation to the bat, because such paired stimuli allowed the animal to learn that the acoustic stimulus was behaviorally important and to make behavioral and neural adjustments based on the acquired importance of the acoustic stimulus. We found that acoustic stimulation alone evokes a change in the frequency map of the IC; that this change in the IC becomes greater when the acoustic stimulation is made behaviorally relevant by pairing it with electrical stimulation; that the collicular change is mediated by the corticofugal system; and that the IC itself can sustain the change evoked by the corticofugal system for some time. Our data support the hypothesis.
Memory-enhancing effects of secreted forms of the β-amyloid precursor protein in normal and amnestic mice
Authors: Meziane H. , Dodart J.-C. , Mathis C. , Little S. , Clemens J. , Paul S. M. , Ungerer A.
Published: July 2002

When administered intracerebroventricularly to mice performing various learning tasks involving either short-term or long-term memory, secreted forms of the β-amyloid precursor protein (APP
s
751
and APP
s
695
) have potent memory-enhancing effects and block learning deficits induced by scopolamine. The memory-enhancing effects of APP
s
were observed over a wide range of extremely low doses (0.05-5,000 pg intracerebroventricularly), blocked by anti-APP
s
antisera, and observed when APP
s
was administered either after the first training session in a visual discrimination or a lever-press learning task or before the acquisition trial in an object recognition task. APP
s
had no effect on motor performance or exploratory activity. APP
s
695
and APP
s
751
were equally effective in the object recognition task, suggesting that the memory-enhancing effect of APP
s
does not require the Kunitz protease inhibitor domain. These data suggest an important role for APP
s
s on memory processes.
NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase
Authors: Ralph John , Hatfield Ronald D. , Piquemal Joël , Yahiaoui Nabila , Pean Michel , Lapierre Catherine , Boudet Alain M.
Published: July 2002

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC
1.1.1.195
) and cinnamoyl-CoA reductase (CCR; EC
1.2.1.44
) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.
β
2
-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein
Authors: Premont Richard T. , Claing Audrey , Vitale Nicolas , Freeman Jennifer L. R. , Pitcher Julie A. , Patton Walter A. , Moss Joel , Vaughan Martha , Lefkowitz Robert J.
Published: July 2002

G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β
2
-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β
2
-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.
Serotonin receptor 1A knockout: An animal model of anxiety-related disorder
Authors: Ramboz Sylvie , Oosting Ronald , Amara Djamel Aït , Kung Hank F. , Blier Pierre , Mendelsohn Monica , Mann J. John , Brunner Dani , Hen René
Published: July 2002

To investigate the contribution of individual serotonin (5-hydroxytryptamine; 5-HT) receptors to mood control, we have used homologous recombination to generate mice lacking specific serotonergic receptor subtypes. In the present report, we demonstrate that mice without 5-HT1A receptors display decreased exploratory activity and increased fear of aversive environments (open or elevated spaces). 5-HT1A knockout mice also exhibited a decreased immobility in the forced swim test, an effect commonly associated with antidepressant treatment. Although 5-HT1A receptors are involved in controlling the activity of serotonergic neurons, 5-HT1A knockout mice had normal levels of 5-HT and 5-hydroxyindoleacetic acid, possibly because of an up-regulation of 5-HT1B autoreceptors. Heterozygote 5-HT1A mutants expressed approximately one-half of wild-type receptor density and displayed intermediate phenotypes in most behavioral tests. These results demonstrate that 5-HT1A receptors are involved in the modulation of exploratory and fear-related behaviors and suggest that reductions in 5-HT1A receptor density due to genetic defects or environmental stressors might result in heightened anxiety.
Rat heart: A site of oxytocin production and action
Authors: Jankowski Marek , Hajjar Fadi , Kawas Sausan Al , Mukaddam-Daher Suhayla , Hoffman Gloria , McCann Samuel M. , Gutkowska Jolanta
Published: July 2002

We report here that the rat heart is a site of oxytocin (OT) synthesis and release. Oxytocin was detected in all four chambers of the heart. The highest OT concentration was in the right atrium (2128 ± 114 pg/mg protein), which was 19-fold higher than in rat uterus but 3.3-fold lower than in the hypothalamus. OT concentrations were significantly greater in the right and left atria than in the corresponding ventricles. Furthermore, OT was released into the effluent of isolated, perfused rat heart (34.5 ± 4.7 pg/min) and into the medium of cultured atrial myocytes. Reverse-phase HPLC purification of the heart extracts and heart perfusates revealed a main peak identical with the retention time of synthetic OT. Southern blots of reverse transcription–PCR products from rat heart revealed gene expression of specific OT mRNA. OT immunostaining likewise was found in atrial myocytes and fibroblasts, and the intensity of positive stains from OT receptors paralleled the atrial natriuretic peptide stores. Our findings suggest that heart OT is structurally identical, and therefore derived from, the same gene as the OT that is primarily found in the hypothalamus. Thus, the heart synthesizes and processes a biologically active form of OT. The presence of OT and OT receptor in all of the heart’s chambers suggests an autocrine and/or paracrine role for the peptide. Our finding of abundant OT receptor in atrial myocytes supports our hypothesis that OT, directly and/or via atrial natriuretic peptide release, can regulate the force of cardiac contraction.
Altered ratios of alternatively spliced long and short γ2 subunit mRNAs of the γ-amino butyrate type A receptor in prefrontal cortex of schizophrenics
Authors: Huntsman Molly M. , Tran Bich-Van , Potkin Stephen G. , Bunney William E. , Jones Edward G.
Published: July 2002

The relative abundance of alternatively spliced long (γ2L) and short (γ2S) mRNAs of the γ2 subunit of the γ-amino butyrate type A (GABA
A
) receptor was examined in dorsolateral prefrontal cortex of schizophrenics and matched controls by using
in situ
hybridization histochemistry and semiquantitative reverse transcription–PCR (RT-PCR) amplification. A cRNA probe identifying both mRNAs showed that the transcripts are normally expressed at moderately high levels in the prefrontal cortex. Consistent with previous studies, overall levels of γ2 transcripts in prefrontal cortex of brains from schizophrenics were reduced by 28.0%, although this reduction did not reach statistical significance. RT-PCR, performed under nonsaturating conditions on total RNA from the same blocks of tissue used for
in situ
hybridization histochemistry, revealed a marked reduction in the relative proportion of γ2S transcripts in schizophrenic brains compared with controls. In schizophrenics, γ2S transcripts had fallen to 51.7% (±7.9% SE;
P
< 0.0001) relative to control levels. Levels of γ2L transcripts showed only a small and nonsignificant reduction of 16.9% (±12.0% SE,
P
> 0.05). These findings indicate differential transcriptional regulation of two functionally distinct isoforms of one of the major GABA
A
receptor subunits in the prefrontal cortex of schizophrenics. The specific reduction in relative abundance of γ2S mRNAs and the associated relative increase in γ2L mRNAs should result in functionally less active GABA
A
receptors and have severe consequences for cortical integrative function.
Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors
Authors: Marti Hugo H. , Risau Werner
Published: July 2002

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF
in vitro
. Here we demonstrate that VEGF is induced
in vivo
by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver.
In situ
hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia
in vivo
is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.
Displacement of insulin-like growth factors from their binding proteins as a potential treatment for stroke
Authors: Loddick Sarah A. , Liu Xin-Jun , Lu Zi-Xian , Liu Changlu , Behan Dominic P. , Chalmers Derek C. , Foster Alan C. , Vale Wylie W. , Ling Nicholas , De Souza Errol B.
Published: July 2002

Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role in normal growth and brain development and protect brain cells from several forms of injury. The effects of IGFs are mediated by type-I and type-II receptors and modulated by potentially six specific binding proteins that form high-affinity complexes with IGFs in blood and cerebrospinal fluid (CSF) and under most circumstances inactivate them. Because brain injury is commonly associated with increases in IGFs and their associated binding proteins, we hypothesized that displacement of this large “pool” of endogenous IGF from the binding proteins would elevate “free” IGF levels to elicit neuroprotective effects comparable to those produced by administration of exogenous IGF. A human IGF-I analog [(Leu
24, 59, 60
, Ala
31
)hIGF-I] with high affinity to IGF-binding proteins (
K
i
= 0.3–3.9 nM) and no biological activity at the IGF receptors (
K
i
= >10,000 nM) increased the levels of “free, bioavailable” IGF-I in the CSF. Intracerebroventricular administration of this analog up to 1h after an ischemic insult to the rat brain had a potent neuroprotective action comparable to IGF-I. This novel strategy for increasing “free” IGF levels in the brain may be useful for the treatment of stroke and other neurodegenerative diseases.
Overrepresentation of horizontal and vertical orientation preferences in developing ferret area 17
Authors: Chapman Barbara , Bonhoeffer Tobias
Published: July 2002

One of the fundamental principles of visual cortical organization is that neurons form a “map” in which neighboring cells have similar orientation preferences. Previous anatomical and imaging studies have shown that although the exact layouts of these orientation preference maps vary between individuals, features of iso-orientation domains such as width and spacing appear constant within a species. Using chronic optical imaging of intrinsic signals we now demonstrate that in ferret area 17 a larger proportion of cortical surface is dominated by responses to horizontal and vertical contours than to the two oblique orientations. This was true for all ferrets studied both during development and in adulthood. Interestingly, however, we found that the degree of the overrepresentation varied significantly between individual animals. In some young ferrets, responses to horizontal and vertical stimuli developed faster than responses to oblique stimuli, and a much larger percentage of the cortex responded preferentially to horizontal and vertical stimuli. In other individuals, responses to all stimuli developed at roughly the same rate, and there was relatively little overrepresentation of horizontal and vertical preferences.
Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression
Authors: Whang Young E. , Wu Xinyi , Suzuki Hiroyoshi , Reiter Robert E. , Tran Chris , Vessella Robert L. , Said Jonathan W. , Isaacs William B. , Sawyers Charles L.
Published: July 2002

The recently identified PTEN/MMAC1 gene is a candidate tumor suppressor implicated in multiple tumor types based on mutations or homozygous deletions of the gene in certain human cancers. No studies of PTEN/MMAC1 mRNA or protein expression in cancer cells have been reported, primarily because of significant numbers of normal cells contaminating most tumor samples and because of the lack of antibody reagents. We examined PTEN/MMAC1 in advanced prostate cancer for gene mutations or abnormalities in expression by using a series of recently derived xenografts free of normal human cells and a PTEN/MMAC1-specific antibody. Only 1 of 10 tumors contained a homozygous deletion of PTEN/MMAC1, and no mutations were detected in the entire coding region of the remaining nine xenografts. However, five of these showed reduced or absent PTEN/MMAC1 expression by Northern analysis and reverse transcription–PCR of mRNA. PTEN/MMAC1 mRNA expression was restored in nonexpressing prostate cancer cells by
in vitro
treatment with the demethylating agent 5-azadeoxycytidine. Alterations in PTEN/MMAC1 expression were confirmed at the protein level by immunoblot analysis, and immunohistochemical studies show that the endogenous wild-type PTEN/MMAC1 protein is localized exclusively in the cytoplasm. These results demonstrate that loss of PTEN/MMAC1 expression occurs frequently in advanced prostate cancer.
A change in actin conformation associated with filament instability after P
i
release
Authors: Belmont Lisa D. , Orlova Albina , Drubin David G. , Egelman Edward H.
Published: July 2002

The ability of actin to both polymerize into filaments and to depolymerize permits the rapid rearrangements of actin structures that are essential for actin’s function in most cellular processes. Filament polarity and dynamic properties are conferred by the hydrolysis of ATP on actin filaments. Release of inorganic phosphate (P
i
) from filaments after ATP hydrolysis promotes depolymerization. We identify a yeast actin mutation, Val-159 to Asn, which uncouples P
i
release from the conformational change that results in filament destabilization. Three-dimensional reconstructions of electron micrographs reveal a conformational difference between ADP-P
i
filaments and ADP filaments and show that ADP V159N filaments resemble ADP-P
i
wild-type filaments. Crystal structures of mammalian β-actin in which the nucleotide binding cleft is in the “open” and “closed” states can be used to model actin filaments in the ADP and ADP-P
i
conformations, respectively. We propose that these two conformations of G-actin may be related to two functional states of F-actin.
Long RNA hairpins that contain inosine are present in
Caenorhabditis elegans
poly(A)
+
RNA
Authors: Morse Daniel P. , Bass Brenda L.
Published: July 2002

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in
Caenorhabditis elegans
. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.
A brain sexual dimorphism controlled by adult circulating androgens
Authors: Cooke Bradley M. , Tabibnia Golnaz , Breedlove S. Marc
Published: July 2002

Reports of structural differences between the brains of men and women, heterosexual and homosexual men, and male-to-female transsexuals and other men have been offered as evidence that the behavioral differences between these groups are likely caused by differences in the early development of the brain. However, a possible confounding variable is the concentration of circulating hormones seen in these groups in adulthood. Evaluation of this possibility hinges on the extent to which circulating hormones can alter the size of mammalian brain regions as revealed by Nissl stains. We now report a sexual dimorphism in the volume of a brain nucleus in rats that can be completely accounted for by adult sex differences in circulating androgen. The posterodorsal nucleus of the medial amygdala (MePD) has a greater volume in male rats than in females, but adult castration of males causes the volume to shrink to female values within four weeks, whereas androgen treatment of adult females for that period enlarges the MePD to levels equivalent to normal males. This report demonstrates that adult hormone manipulations can completely reverse a sexual dimorphism in brain regional volume in a mammalian species. The sex difference and androgen responsiveness of MePD volume is reflected in the soma size of neurons there.
CpG island methylator phenotype in colorectal cancer
Authors: Toyota Minoru , Ahuja Nita , Ohe-Toyota Mutsumi , Herman James G. , Baylin Stephen B. , Issa Jean-Pierre J.
Published: July 2002

Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of
p16
and
THBS1
methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to
hMLH1
methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.
Characterization and catalytic properties of the sterol 14α-demethylase from
Mycobacterium tuberculosis
Authors: Bellamine Aouatef , Mangla Anil T. , Nes W. David , Waterman Michael R.
Published: July 2002

Sterol 14α-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the
Mycobacterium tuberculosis
genome project revealed that a protein having homology to mammalian 14α-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H
37
Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14α-demethylase. Expression of the
M. tuberculosis
CYP51 gene in
Escherichia coli
yields a P450, which, when purified to homogeneity, has the predicted molecular mass,
ca.
50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14α-demethylases. It catalyzes 14α-demethylation of lanosterol, 24,25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and
1
H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14α-methyl group and Δ
8(9)
-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14α-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.
Human eosinophils regulate human lung- and skin-derived fibroblast propertiesin vitro: A role for transforming growth factor β (TGF-β)
Authors: Levi-Schaffer Francesca , Garbuzenko Ekaterina , Rubin Ann , Reich Reuven , Pickholz Dalia , Gillery Philippe , Emonard Herve , Nagler Arnon , Maquart Francois A. Xavier
Published: July 2002

Eosinophils have been associated with fibrosis. To investigate their direct role in fibrosis, human peripheral blood eosinophil sonicate was added to human lung or dermal fibroblasts, and proliferation ([3H]thymidine) and collagen synthesis ([3H]proline) were evaluated. Proliferation was enhanced significantly in the monolayers in a dose-dependent manner. The activity of the eosinophil fibrogenic factor(s) remained unaltered when heated (56°C, 30 min). Supernatants of cultured eosinophils (20 min or 18 hr) also enhanced lung fibroblast proliferation, indicating that the preformed mitogenic factor(s) can be released both promptly and with a long kinetic. Eosinophils significantly decreased collagen production in lung fibroblasts while increasing it in dermal fibroblasts. However, eosinophils containing matrix metalloproteinase 9 (zymography) in latent form and tissue inhibitors of metalloproteinases 1 and 2 (reverse zymography) did not influence either fibroblast matrix metalloproteinases or tissue inhibitors of metalloproteinases. Eosinophil sonicate added to skin and lung fibroblasts in tridimensional collagen lattices significantly enhanced lattice contraction. Transforming growth factor β (TGF-β) is a major fibrogenic cytokine produced by eosinophils. Therefore, to assess its role, eosinophil sonicate was preincubated with anti-TGF-β neutralizing antibodies. This treatment partially inhibited proliferation of lung and collagen synthesis of dermal fibroblasts and suppressed the stimulation of lattice contraction, indicating the fibrogenic role of eosinophil-associated TGF-β. In conclusion, we have shown that eosinophils act as direct modulatory cells in fibroblast proliferation, collagen synthesis, and lattice contraction, in part, through TGF-β. These data corroborate the importance of eosinophils in skin and lung fibrosis.
Chip-based genotyping by mass spectrometry
Authors: Tang Kai , Fu Dong-Jing , Julien Dominique , Braun Andreas , Cantor Charles R. , Köster Hubert
Published: July 2002

Silicon chips with immobilized target DNAs were used for accurate genotyping by mass spectrometry. Genomic DNAs were amplified with PCR, and the amplified products were covalently attached to chip wells via
N
-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) chemistry. Primer annealing, extension, and termination were performed on a 1-μl scale directly in the chip wells in parallel. Diagnostic products thus generated were detected
in situ
by using matrix-assisted laser desorption ionization mass spectrometry. This miniaturized method has the potential for accurate, high-throughput, low-cost identification of genetic variations.
Truncated BRCA2 is cytoplasmic: Implications for cancer-linked mutations
Authors: Spain Brian H. , Larson Christopher J. , Shihabuddin Lamya S. , Gage Fred H. , Verma Inder M.
Published: July 2002

BRCA2
mutations predispose carriers mainly to breast cancer. The vast majority of
BRCA2
mutations are predicted to result in a truncated protein product. The smallest known cancer-associated deletion removes from the C terminus only 224 of the 3,418 residues constituting BRCA2, suggesting that these terminal amino acids are crucial for BRCA2 function. A series of green fluorescent protein (GFP)-tagged BRCA2 deletion mutants revealed that nuclear localization depends on two nuclear localization signals that reside within the final 156 residues of BRCA2. Consistent with this observation, an endogenous truncated BRCA2 mutant (6174delT) was found to be cytoplasmic. Together, these studies provide a simple explanation for why the vast majority of BRCA2 mutants are nonfunctional: they do not translocate into the nucleus.
Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid
Authors: Calhoun Michael E. , Burgermeister Patrick , Phinney Amie L. , Stalder Martina , Tolnay Markus , Wiederhold Karl-Heinz , Abramowski Dorothee , Sturchler-Pierrat Christine , Sommer Bernd , Staufenbiel Matthias , Jucker Mathias
Published: July 2002

Transgenic mice that overexpress mutant human amyloid precursor protein (APP) exhibit one hallmark of Alzheimer’s disease pathology, namely the extracellular deposition of amyloid plaques. Here, we describe significant deposition of amyloid β (Aβ) in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in aging APP23 mice that had striking similarities to that observed in human aging and Alzheimer’s disease. Amyloid deposition occurred preferentially in arterioles and capillaries and within individual vessels showed a wide heterogeneity (ranging from a thin ring of amyloid in the vessel wall to large plaque-like extrusions into the neuropil). CAA was associated with local neuron loss, synaptic abnormalities, microglial activation, and microhemorrhage. Although several factors may contribute to CAA in humans, the neuronal origin of transgenic APP, high levels of Aβ in cerebrospinal fluid, and regional localization of CAA in APP23 mice suggest transport and drainage pathways rather than local production or blood uptake of Aβ as a primary mechanism underlying cerebrovascular amyloid formation. APP23 mice on an
App
-null background developed a similar degree of both plaques and CAA, providing further evidence that a neuronal source of APP/Aβ is sufficient to induce cerebrovascular amyloid and associated neurodegeneration.
Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor
α
(RAR
α
) and oncogenic RAR
α
fusion proteins
Authors: Zhu Jun , Gianni Maurizio , Kopf Eliezer , Honoré Nicole , Chelbi-Alix Mounira , Koken Marcel , Quignon Frédérique , Rochette-Egly Cécile , de Thé Hugues
Published: July 2002

Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia/retinoic acid receptor α (PML/RAR
α
) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML/RAR
α
cleavage, RA triggers degradation of both PML/RAR
α
and RAR
α
. Similarly, in non-APL cells, RA directly targeted RAR
α
and RAR
α
fusions to the proteasome degradation pathway. Activation of either RAR
α
or RXR
α
by specific agonists induced degradation of both proteins. Conversely, a mutation in RAR
α
that abolishes heterodimer formation and DNA binding, blocked both RAR
α
and RXR
α
degradation. Mutations in the RAR
α
DNA-binding domain or AF-2 transcriptional activation region also impaired RAR
α
catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation.