bluuebunny/crossref_metadata_2025 huggingface dataset:
Supramolecular recognition: On the kinetic lability of thermodynamically stable host–guest association complexes
Authors: Goshe Andrew J. , Steele Ian M. , Ceccarelli Christopher , Rheingold Arnold L. , Bosnich B.
Published: July 2002

A molecular receptor consisting of a spacer bearing two cofacially disposed terpyridyl–palladium–ligand (terpy-Pd-L) units rigidly separated by about 7 Å has been investigated for molecular recognition of planar aromatic molecules. It is found that although the receptor forms stable 1:2 host–guest association complexes with 9-methylanthracene (9-MA), the guest undergoes very rapid site exchange within the receptor and with external free 9-MA. A crystal structure of the 2:1 adduct shows one 9-MA in the molecular cleft defined by the two terpy-Pd-L units and the other resides on an outside face of one terpy-Pd-L unit. To establish the site residency time of the guests, a number of tethered molecules were prepared. These involve an anthracene molecule tethered to a pyridine ligand bound to the palladium atoms to form intramolecular host–guest adducts. Rotating-frame Overhauser effects were used to infer the site residency of the anthracene guests in the receptor. Variable-temperature1H NMR spectroscopy of the intramolecular host–guest complexes has revealed that the site residency time of the anthracene guests is 1.6 × 10−5sec at 20°C and 1.3 sec at −90°C in acetone solution. Whereas the guests are thermodynamically stable, they are kinetically very labile. A crystal structure of one of the tethered host–guest adducts reveals the expected structure which is the same as that determined in solution by1H rotating-frame Overhauser enhancement spectroscopy experiments.
Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity
Authors: Tang Pingtao , Cheng Tammy P. , Agnello Davide , Wu Chang-You , Hissong Bruce D. , Watford Wendy T. , Ahn Hyun-Jong , Galon Jerome , Moss Joel , Vaughan Martha , O'Shea John J. , Gadina Massimo
Published: July 2002

Cytokines regulate lymphocyte development and differentiation, but precisely how they control these processes is still poorly understood. By using microarray technology to detect cytokine-induced genes, we identified a cDNA encoding Cybr, which was increased markedly in cells incubated with IL-2 and IL-12. The mRNA was most abundant in hematopoietic cells and tissues. The predicted amino acid sequence is similar to that of GRP-1-associated protein (GRASP), a recently identified retinoic acid-induced cytohesin-binding protein. Physical interaction, dependent on the coiled-coil domains of Cybr and cytohesin-1, was demonstrated by coimmunoprecipitation of the overexpressed proteins from 293T cells. Cytohesin-1, in addition to its role in cell adhesion, is a guanine nucleotide-exchange protein activator of ARF GTPases. Acceleration of guanosine 5′-O-(thiotriphosphate) binding to ARF by cytohesin-1in vitrowas enhanced by Cybr. Because the binding protein modified activation of ADP ribosylation factor by cytohesin-1, we designate this cytokine-inducible protein Cybr (cytohesin binder and regulator).
The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels
Authors: Koffa Maria D. , Graham Sheila V. , Takagaki Yoshio , Manley James L. , Clements J. Barklie
Published: July 2002

In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3′ untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.
Neuroprotective potential of a viral vector system induced by a neurological insult
Authors: Ozawa Clare R. , Ho Jill J. , Tsai David J. , Ho Dora Y. , Sapolsky Robert M.
Published: July 2002

Gene transfer into neurons via viral vectors for protection against acute necrotic insults has generated considerable interest. Most studies have used constitutive vector systems, limiting the ability to control transgene expression in a dose-dependent, time-dependent, or reversible manner. We have constructed defective herpes simplex virus vectors designed to be induced by necrotic neurological insults themselves. Such vectors contain a synthetic glucocorticoid-responsive promoter, taking advantage of the almost uniquely high levels of glucocorticoids—adrenal stress steroids—secreted in response to such insults. We observed dose-responsive and steroid-specific induction by endogenous and synthetic glucocorticoids in hippocampal cultures. Induction was likely to be rapid enough to allow transgenic manipulation of relatively early steps in the cascade of necrotic neuron death. The protective potential of such a vector was tested by inclusion of a neuroprotective transgene (the Glut-1 glucose transporter). Induction of this vector by glucocorticoids decreased glutamatergic excitotoxicity in culture. Finally, both exogenous glucocorticoids and excitotoxic seizures induced reporter gene expression driven from a glucocorticoid-responsive herpes simplex virus vector in the hippocampus
in vivo
.
Prostaglandin E2 receptors of the EP2 and EP4 subtypes regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells.
Authors: Fedyk E R , Phipps R P
Published: July 2002

Prostaglandin E2 (PGE2) is a potent lipid molecule with complex proinflammatory and immunoregulatory properties. PGE2 can shape the immune response by stimulating the production of IgE antibody by B lymphocytes and the synthesis of T-helper type 2 cytokines [e.g., interleukin (IL)-4, IL-10], while inhibiting production of Th1 cytokines (e.g., interferon-gamma, IL-12). It is unknown what type of receptor binds PGE2 and modulates these responses. Recent analyses in nonhematopoietic cells have identified six PGE2 receptors (EP1, EP2, EP3 alpha, EP3 beta, EP3 gamma, and EP4). This investigation examines quiescent B lymphocytes and reports that these cells express mRNA encoding EP1, EP2, EP3 beta, and EP4 receptors. The immunoregulatory functions of each receptor were investigated using small molecule agonists that preferentially bind EP receptor subtypes. Unlike agonists for EP1 and EP3, agonists that bound EP2 or EP2 and EP4 receptors strongly inhibited expression of class II major histocompatibility complex and CD23 and blocked enlargement of mouse B lymphocytes stimulated with IL-4 and/or lipopolysaccharide. PGE2 promotes differentiation and synergistically enhances IL-4 and lipopolysaccharide-driven B-cell immunoglobulin class switching to IgE. Agonists that bound EP2 or EP2 and EP4 receptors also strongly stimulated class switching to IgE. Experiments employing inhibitors of cAMP metabolism demonstrate that the mechanism by which EP2 and EP4 receptors regulate B lymphocyte activity requires elevation of cAMP. In conclusion, these data suggest that antagonists to EP2 and EP4 receptors will be important for diminishing allergic and IgE-mediated asthmatic responses.
Chaperone activity and structure of monomeric polypeptide binding domains of GroEL
Authors: Zahn Ralph , Buckle Ashley M. , Perrett Sarah , Johnson Christopher M. , Corrales Fernando J. , Golbik Ralph , Fersht Alan R.
Published: July 2002

The chaperonin GroEL is a large complex composed of 14 identical
57-kDa subunits that requires ATP and GroES for some of its activities.
We find that a monomeric polypeptide corresponding to residues 191 to
345 has the activity of the tetradecamer both in facilitating the
refolding of rhodanese and cyclophilin A in the absence of ATP and in
catalyzing the unfolding of native barnase. Its crystal structure,
solved at 2.5 Å resolution, shows a well-ordered domain with the same
fold as in intact GroEL. We have thus isolated the active site of the
complex allosteric molecular chaperone, which functions as a
“minichaperone.” This has mechanistic implications: the presence of
a central cavity in the GroEL complex is not essential for those
representative activities
in vitro
, and neither are the
allosteric properties. The function of the allosteric behavior on the
binding of GroES and ATP must be to regulate the affinity of the
protein for its various substrates
in vivo
, where the
cavity may also be required for special functions.
Ligand occupancy of the alpha-V-beta3 integrin is necessary for smooth muscle cells to migrate in response to insulin-like growth factor.
Authors: Jones J I , Prevette T , Gockerman A , Clemmons D R
Published: July 2002

Smooth muscle cells (SMCs) have been shown to migrate in response to insulin-like growth factor I (IGF-I). However, the mechanism mediating this response has not been determined. The migration rates of porcine and human vascular SMCs were assessed in a monolayer wounding assay. IGF-I and IGF-II induced increases of 141% and 97%, respectively, in the number of cells that migrated in 4 days. The presence of 0.2% fetal bovine serum in the culture medium was necessary for the IGFs to stimulate migration over uncoated plastic surfaces. However, if vitronectin was used as the substratum, IGF-I stimulated migration by 162% even in the absence of serum. To determine the role of integrins in mediating this migration, SMC surface proteins were labeled with 125I and immunoprecipitated with specific anti-integrin antibodies. Integrins containing alpha-V (vitronectin receptor), alpha5 (fibronectin receptor), and alpha3 (collagen/laminin receptor) subunits were the most abundant. IGF-I treatment caused a 73% reduction in alpha5-integrin subunit protein and a 25% increase in alpha-V subunit. More importantly, ligand binding of alpha-V-beta3 was increased by 2.4-fold. We therefore examined whether the function of the alpha-V-beta3 integrin was important for IGF-I-mediated migration. The disintegrin kistrin was shown by affinity crosslinking to specifically bind with high affinity to alpha-V-beta3 and not to alpha5-beta1 or other abundant integrins. The related disintegrin echistatin specifically inhibited 125I-labeled kistrin binding to alpha-V-beta3, while a structurally distinct disintegrin, decorsin, had 1000-fold lower affinity. The addition of increasing concentrations of either kistrin or echistatin inhibited IGF-I-induced migration, whereas decorsin had a minimal effect. The potency of these disintegrins in inhibiting IGF-I-induced migration paralleled their apparent affinity for the alpha-V integrin. Furthermore, an alpha-V-beta3 blocking antibody inhibited SMC migration by 80%. In summary, vitronectin receptor activation is a necessary component of IGF-I-mediated stimulation of smooth muscle migration, and alpha-V-beta3 integrin antagonists appear to be important reagents for modulating this process.
Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor
Authors: Meacci Elisabetta , Tsai Su-Chen , Adamik Ronald , Moss Joel , Vaughan Martha
Published: July 2002

Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the
Saccharomyces cerevisiae
Sec7 gene product, which is crucial in protein transport. More recently, the same protein has been reported to be a regulatory factor for the αLβ2 integrin in lymphocytes. Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function. ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP). Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP. Both soluble and particulate GEPs have been described. To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in
Escherichia coli
, on ARF activity were evaluated. Cytohesin-1 enhanced binding of
35
S-labeled guanosine 5′-[γ-thio]triphosphate [
35
S]GTP[γS] or [
3
H]GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP). Addition of cytohesin-1 to ARF3 with [
35
S]GTP[γS] bound, accelerated [
35
S]GTP[γS] release to a similar degree in the presence of unlabeled GDP or GTP[γS] and to a lesser degree with GDP[βS]; release was negligible without added nucleotide. Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function. In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain.
Src regulated by C-terminal phosphorylation is monomeric
Authors: Weijland Albert , Williams John C. , Neubauer Gitte , Courtneidge Sara A. , Wierenga Rik K. , Superti-Furga Giulio
Published: July 2002

The activity of the c-Src protein tyrosine kinase is regulated by phosphorylation of a tyrosine residue (Tyr-527) in the C-terminal tail of the molecule. Phosphorylation of Tyr-527 promotes association of the tail with the SH2 domain and a concomitant reduction of the enzymatic activity of Src. We asked the question whether regulation by C-terminal phosphorylation was accompanied by a change in the quaternary structure of the enzyme or if it occurred within a monomeric form of Src. For this purpose we purified to homogeneity a chicken Src form lacking the unique domain from
Schizosaccharomyces pombe
cells. The cells were engineered to express Src along with Csk, a protein kinase able to phosphorylate Tyr-527 efficiently. Mass spectrometric analysis showed that purified Src was homogeneously phosphorylated at Tyr-527. The enzyme was in the regulated form, because it could be activated by a phosphorylated peptide able to bind the SH2 domain with high affinity. Using gel filtration chromatography, dynamic light scattering, and ultracentrifugation, we found that the regulated form of Src was a monomer. We have obtained crystals diffracting to 2.4 Å with space group
P
2
1
2
1
2
1
and one molecule per asymmetric unit, in agreement with the monomeric state. These results indicate that the structural rearrangements of regulated Src are of an intramolecular nature.
Muscarinic cholinergic regulation of cardiac myocyte I
Ca-L
is absent in mice with targeted disruption of endothelial nitric oxide synthase
Authors: Han Xinqiang , Kubota Isao , Feron Olivier , Opel Douglas J. , Arstall Margaret A. , Zhao You-Yang , Huang Paul , Fishman Mark C. , Michel Thomas , Kelly Ralph A.
Published: July 2002

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (I
Ca-L
) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on I
Ca-L
and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOS
null
mice, with targeted disruption of the eNOS gene. Although both eNOS
null
and wild-type (WT) ventricular myocytes exhibited similar increases in I
Ca-L
in response to ISO, there was no measurable suppression of I
Ca-L
by CCh in cells from eNOS
null
mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOS
null
myocytes. Also, unlike myocytes from WT animals, eNOS
null
myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented I
Ca-L
in eNOS
null
cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K
+
channel by CCh was unaffected in atrial and ventricular eNOS
null
myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of I
Ca-L
in cardiac myocytes.
Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (
E
)-α-bisabolene synthase from grand fir (
Abies grandis
)
Authors: Bohlmann Jörg , Crock John , Jetter Reinhard , Croteau Rodney
Published: July 2002

(
E
)-α-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (
Abies grandis
), and the olefin product of this cyclization reaction is considered to be the precursor in
Abies
species of todomatuic acid, juvabione, and related insect juvenile hormone mimics. A cDNA encoding (
E
)-α-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in
Escherichia coli
and shown to produce (
E
)-α-bisabolene as the sole product from farnesyl diphosphate. The expressed synthase has a deduced size of 93.8 kDa and a pI of 5.03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro
81
–Val
296
. Biosynthetically prepared (
E
)-α-[
3
H]bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes. These results suggest that induced (
E
)-α-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.
Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I
Authors: Prieto-Alamo Maria José , Laval Françoise
Published: July 2002

Hereditary tyrosinemia type I (HT1) is an autosomal recessive inborn error of metabolism caused by the deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolism pathway. This defect results in accumulation of succinylacetone (SA) that reacts with amino acids and proteins to form stable adducts via Schiff base formation, lysine being the most reactive amino acid. HT1 patients surviving beyond infancy are at considerable risk for the development of hepatocellular carcinoma, and a high level of chromosomal breakage is observed in HT1 cells, suggesting a defect in the processing of DNA. In this paper we show that the overall DNA-ligase activity is low in HT1 cells (about 20% of the normal value) and that Okazaki fragments are rejoined at a reduced rate compared with normal fibroblasts. No mutation was found by sequencing the ligase I cDNA from HT1 cells, and the level of expression of the ligase I mRNA was similar in normal and HT1 fibroblasts, suggesting the presence of a ligase inhibitor. SA was shown to inhibit
in vitro
the overall DNA-ligase activity present in normal cell extracts. The activity of purified T4 DNA-ligase, whose active site is also a lysine residue, was inhibited by SA in a dose-dependent manner. These results suggest that accumulation of SA reduces the overall ligase activity in HT1 cells and indicate that metabolism errors may play a role in regulating enzymatic activities involved in DNA replication and repair.
Ex vivo
nerve growth factor gene transfer to the basal forebrain in presymptomatic middle-aged rats prevents the development of cholinergic neuron atrophy and cognitive impairment during aging
Authors: Martínez-Serrano Alberto , Björklund Anders
Published: July 2002

Nerve growth factor (NGF) is able to restore spatial learning and reverse forebrain cholinergic neuron atrophy when administered intracerebrally to behaviorally impaired aged rats. In the present study, behaviorally unimpaired, middle-aged rats (14–16 months old) received transplants of
ex vivo
transduced, clonal NGF-secreting immortalized neural progenitor cells, bilaterally in the nucleus basalis and septum. During the subsequent 9 months the aged control animals developed the expected impairment in spatial learning in the water maze task, whereas the animals with NGF-secreting grafts maintained a performance level not different from the 12-month-old control rats. The marked age-induced atrophy (−25%) of the cholinergic neurons in medial septum and nucleus basalis, seen in the aged control rats, was not present in the NGF-treated aged animals.
3
H-labeled thymidine autoradiography showed that the transduced cells survived well and had become integrated into the host tissue surrounding the injection sites, and reverse transcription–PCR analysis revealed expression of the NGF transgene, at both 4 and 9 months postgrafting, in the grafted tissue. The results show that long-term supply of NGF from
ex vivo
transduced immortalized neural progenitor cells locally within the nucleus basalis and septum can prevent the subsequent development of age-dependent neuronal atrophy and behavioral impairments when the animals reach advanced age.
Direct observation of the anchoring process during the adsorption of fibrinogen on a solid surface by force-spectroscopy mode atomic force microscopy
Authors: Hemmerlé J. , Altmann S. M. , Maaloum M. , Hörber J. K. H. , Heinrich L. , Voegel J.-C. , Schaaf P.
Published: July 2002

Atomic force microscopy in a force-spectroscopy mode has been used to investigate the kinetics of the adsorption process of fibrinogen molecules on a silica surface. An original “approach/retraction” cycle of the tip/surface was used for this purpose. Fibrinogen molecules were adsorbed on the atomic force microscopy tip and were brought into contact with the silica surface for different interaction times varying from 5 to 2,000 ms. Multiple consecutive ruptures were observed. The mean number of ruptures
n
r
per cycle increases steadily with the interaction time as well as the mean strength
f
r
which varies from 300 pN for 5 ms to 1,400 pN for 2,000 ms. The minimal interaction time for a fibrinogen molecule to bind strongly to a silica surface during an adsorption process appears to lie between 50 and 200 ms. The histograms of the distances between two consecutive ruptures in one cycle exhibit maxima around 20–25 nm. This length is comparable to the characteristic distance between D and E globules of one fibrinogen molecule and suggests that fibrinogen molecules mainly adsorb through their D and E globules.
Expression of oligodendrocyte progenitor cell antigens by gliomas: Implications for the histogenesis of brain tumors
Authors: Shoshan Yigal , Nishiyama Akiko , Chang Ansi , Mörk Sverre , Barnett Gene H. , Cowell John K. , Trapp Bruce D. , Staugaitis Susan M.
Published: July 2002

The early events in neoplastic transformation can be understood only by comparison of the neoplastic cell with its nontransformed counterpart. The most common central nervous system gliomas traditionally are thought to arise from mature astrocytes and oligodendrocytes. We examined the possibility that gliomas arise from a population of glia that has properties of oligodendrocyte progenitors. These glial cells express the NG2 chondroitin sulfate proteoglycan and the α receptor of platelet-derived growth factor
in vivo
. We identified NG2 and the α receptor of platelet-derived growth factor expression in tissue from seven of seven oligodendrogliomas, three of three pilocytic astrocytomas, and one of five glioblastoma multiforme. These data provide evidence that glial tumors arise from glial progenitor cells. Molecules expressed by these progenitor cells should be considered as targets for novel therapeutics.
Differential protease-mediated turnover of H-NS and StpA revealed by a mutation altering protein stability and stationary-phase survival of
Escherichia coli
Authors: Johansson Jörgen , Uhlin Bernt Eric
Published: July 2002

The
Escherichia coli
proteins H-NS is recognized as an important component among the major nucleoid-associated proteins. In studies of
E. coli
strains with defects in H-NS, we discovered a mutant that phenotypically
r
estored
s
tationary-phase
v
iability (Rsv) of such strains. The Rsv phenotype was the result of a mutation that led to severalfold higher levels of the functionally and structurally related StpA protein. This mutation was a base pair change in the
stpA
structural gene, and the amino acid substitution in the StpA protein altered its turnover properties, suggesting a role for this residue in a cleavage site for proteolysis. We determined the stability of the StpA and the H-NS proteins and found that the StpA protein was degraded relatively rapidly in strains lacking functional H-NS, whereas H-NS remained stable irrespective of the presence/absence of StpA. Using protease-deficient mutants, we obtained evidence that the Lon protease was responsible for the degradation of StpA. The differential turnover of the nucleoid-associated proteins is suggested to contribute to the regulation of their stoichiometry and ratio in terms of homo- and heteromer formation. We conclude that StpA, in contrast to H-NS, is present mainly in heteromeric form in
E. coli
.
Parvalbumin gene transfer corrects diastolic dysfunction in diseased cardiac myocytes
Authors: Wahr Philip A. , Michele Daniel E. , Metzger Joseph M.
Published: July 2002

Heart failure frequently involves diastolic dysfunction that is characterized by a prolonged relaxation. This prolonged relaxation is typically the result of a decreased rate of intracellular Ca
2+
sequestration. No effective treatment for this decreased Ca
2+
sequestration rate currently exists. As an approach to possibly correct diastolic dysfunction, we hypothesized that expression of the Ca
2+
binding protein parvalbumin in cardiac myocytes would lead to increased rates of Ca
2+
sequestration and mechanical relaxation. Parvalbumin, which is normally absent in cardiac tissue, is known to act as a soluble relaxing factor in fast skeletal muscle fibers by acting as a delayed Ca
2+
sink. As a test of the hypothesis, gene transfer was used to express parvalbumin in isolated adult cardiac myocytes. We report here that expression of parvalbumin dramatically increases the rate of Ca
2+
sequestration and the relaxation rate in normal cardiac myocytes. Importantly, parvalbumin fully restored the relaxation rate in diseased cardiac myocytes isolated from an animal model of human diastolic dysfunction. These findings indicate that parvalbumin gene transfer offers unique potential as a possible direct treatment for diastolic dysfunction in failing hearts.
The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32
Authors: Vaudry David , Gonzalez Bruno J. , Basille Magali , Pamantung Tommy F. , Fontaine Marc , Fournier Alain , Vaudry Hubert
Published: July 2002

Caspase-3 knockout mice exhibit thickening of the internal granule
cell layer of the cerebellum. Concurrently, it has been shown that
intracerebral injection of pituitary adenylate cyclase-activating
polypeptide (PACAP) induces a transient increase of the thickness of
the cerebellar cortex. In the present study, we have investigated the
possible effect of PACAP on caspase activity in cultured cerebellar
granule cells from 8-day-old rat. Incubation of granule neurons with
PACAP for 24 h promoted cell survival and prevented DNA
fragmentation. Exposure of cerebellar granule cells to the specific
caspase-3 inhibitor
N
-benzyloxycarbonyl-Asp-Glu-Val-Asp
fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell
survival and inhibited apoptotic cell death. Time-course
studies revealed that PACAP causes a prolonged inhibition of caspase-3
activity without affecting caspase-1. Administration of graded
concentrations of PACAP for 3 h induced a dose-dependent
inhibition of caspase-3 activity. Incubation of granule cells with both
dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA)
mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of
cultured neurons with the protein kinase A inhibitor H89 and the
protein kinase C inhibitor chelerythrine abrogated the effect of PACAP
on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did
not affect the action of PACAP on caspase-3 activity. These data
demonstrate that PACAP prevents cerebellar granule neurons from
apoptotic cell death through a protein kinase A- and protein
kinase C-dependent inhibition of caspase-3 activity.
The Synaptic Protein Syntaxin1 Is Required for Cellularization of Drosophila Embryos
Authors: Burgess Robert W. , Deitcher David L. , Schwarz Thomas L.
Published: July 2002

Syntaxins are membrane proteins involved in vesicle trafficking and are required for the release of neurotransmitter at nerve terminals. The presence of syntaxins on target membranes has been hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins, the synaptobrevins or VAMPS. We have mutagenized syntaxin1 in Drosophila and have found that it links the mechanism of synaptic transmission to a distinct cell biological process: the cellularization of early embryos. This specialized form of cell division separates the 6,000 nuclei of the syncytial blastoderm into separate cells through the invagination of the surface membrane of the embryo. During this process, syntaxin1 protein is present on the newly forming lateral cell surfaces and invaginating cleavage furrows. This protein is derived both from maternal deposition of mRNA and protein and from early zygotic transcription. To analyze syntaxin1's role in early development, female germ line mosaics mutant for syntaxin1 expression were generated by mitotic recombination to reduce the maternal contribution. Visualizing the actin cytoskeleton and glycosylated surface proteins reveals that embryos with insufficient syntaxin1 have large acellular patches. The patches do not appear until cellularization begins, and the process fails entirely within these regions. These results provide genetic evidence that membrane trafficking is required for the cellularization of the syncytial blastoderm. We propose that the invagination of the surface membrane proceeds by the fusion of intracellular membrane vesicles with the surface. This reaction uses the same syntaxin1 protein as is required for neurotransmitter secretion at synapses. Thus, a single syntaxin can participate in trafficking steps that are functionally as distinct as synaptic transmission and cell division.
Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3α from Lung Dendritic Cells
Authors: Power Christine A. , Church Dennis J. , Meyer Alexandra , Alouani Sami , Proudfoot Amanda E.I. , Clark-Lewis Ian , Sozzani Silvano , Mantovani Alberto , Wells Timothy N.C.
Published: July 2002

Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3α or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3α is expressed predominantly in inflamed and mucosal tissues. MIP-3α produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34+-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3α was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1–5 and CXCR1 and CXCR2, suggesting that MIP-3α acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3α receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3α, and that its activation leads to pertussis toxin–sensitive and phospholipase C–dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.
L-selectin–mediated Leukocyte Adhesion In Vivo: Microvillous Distribution Determines Tethering Efficiency, But Not Rolling Velocity
Authors: Stein Jens V. , Cheng Guiying , Stockton Britt M. , Fors Brian P. , Butcher Eugene C. , von Andrian Ulrich H.
Published: July 2002

Adhesion receptors that are known to initiate contact (tethering) between blood-borne leukocytes and their endothelial counterreceptors are frequently concentrated on the microvilli of leukocytes. Other adhesion molecules are displayed either randomly or preferentially on the planar cell body. To determine whether ultrastructural distribution plays a role during tethering in vivo, we used pre-B cell transfectants expressing L- or E-selectin ectodomains linked to transmembrane/intracellular domains that mediated different surface distribution patterns. We analyzed the frequency and velocity of transfectant rolling in high endothelial venules of peripheral lymph nodes using an intravital microscopy model. Ectodomains on microvilli conferred a higher efficiency at initiating rolling than random distribution which, in turn, was more efficient than preferential expression on the cell body. The role of microvillous presentation was less accentuated in venules below 20 μm in diameter than in larger venules. In the narrow venules, tethering of cells with cell body expression may have been aided by forced margination through collision with erythrocytes. L-selectin transfected cells rolled 10-fold faster than E-selectin transfectants. Interestingly, rolling velocity histograms of cell lines expressing equivalent copy numbers of the same ectodomain were always similar, irrespective of the topographic distribution. Our data indicate that the distribution of adhesion receptors has a dramatic impact on contact initiation between leukocytes and endothelial cells, but does not play a role once rolling has been established.
Generation of Tumor-associated Cytotoxic T Lymphocytes Requires Interleukin 4 from CD8+ T Cells
Authors: Schüler Thomas , Kammertoens Thomas , Preiss Susanne , Debs Pierre , Noben-Trauth Nancy , Blankenstein Thomas
Published: July 2002

Activation of tumor-associated CD8+ cytotoxic T lymphocytes (CTLs) often requires antigen representation, e.g., by dendritic cells (DCs), and CD4+ T cell help. Previously, we showed that CTL-mediated tumor immunity required interleukin 4 (IL-4) during the immunization but not effector phase. To determine the source and target cells of IL-4, we performed adoptive T cell transfers using CD4+ and CD8+ T cells from IL-4−/− and IL-4R−/− mice and analyzed CTL generation. Even though necessary for CTL generation, CD4+ T cells did not need to express IL-4 or IL-4R. Surprisingly, CTL generation required IL-4 but not IL-4R expression by CD8+ T cells. As IL-4 (a) was expressed by naive CD8+ T cells within 24 h after antigen encounter, (b) IL-4 induced DC maturation, and (c) CTL development was impaired in T cell–reconstituted IL-4R−/− mice, CD8+ T cell–derived IL-4 appears to act on DCs. We conclude that CD4+ and CD8+ T cells provide different signals for DC activation during CTL generation.
Fes-Cre Targets Phosphatidylinositol Glycan Class a (Piga) Inactivation to Hematopoietic Stem Cells in the Bone Marrow
Authors: Keller Peter , Payne Jennifer L. , Tremml Gabi , Greer Peter A. , Gaboli Mirella , Pandolfi Pier P. , Bessler Monica
Published: July 2002

A somatic mutation in the X-linked phosphatidylinositol glycan class A (PIGA) gene causes the loss of glycosyl phosphatidylinositol (GPI)-linked proteins on blood cells from patients with paroxysmal nocturnal hemoglobinuria. Because all blood cell lineages may be affected it is thought that the mutation occurs in a hematopoietic stem cell. In transgenic mice, germline transmission of an inactive Piga gene is embryonic lethal. To inactivate the murine Piga gene in early hematopoiesis we therefore chose conditional gene inactivation using the Cre/loxP system. We expressed Cre recombinase under the transcription regulatory sequences of the human c-fes gene. FES-Cre inactivated PIGA in hematopoietic cells of mice carrying a floxed Piga allele (LF mice). PIGA− cells were found in all hematopoietic lineages of definitive but not primitive hematopoiesis. Their proportions were low in newborn mice but subsequently increased continuously to produce for the first time mice that have almost exclusively PIGA− blood cells. The loss of GPI-linked proteins occurred mainly in c-kit+CD34+Lin− progenitor cells before the CFU-GEMM stage. Using bone marrow reconstitution experiments with purified PIGA− cells we demonstrate that LF mice have long-term bone marrow repopulating cells that lack GPI-linked proteins, indicating that recombination of the floxed Piga allele occurs in the hematopoietic stem cell.
Surface Cytotoxic T Lymphocyte–associated Antigen 4 Partitions Within Lipid Rafts and Relocates to the Immunological Synapse under Conditions of Inhibition of T Cell Activation
Authors: Darlington Peter J. , Baroja Miren L. , Chau Thu A. , Siu Eric , Ling Vincent , Carreno Beatriz M. , Madrenas Joaquín
Published: July 2002

T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR–CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4–mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.
Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin
Authors: Freedman Jeffrey C. , Novak Terri S.
Published: July 2002

Current-voltage curves for DIDS-insensitive Cl− conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl− conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K+ and Cl− were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl− effluxes, induced by valinomycin or gramicidin at varied [K+]o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K+]o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio PK/PCl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of PCl. Fitting the constant field current-voltage equation to the measured Cl− currents yields PCl = 0.13 h−1 with DIDS, compared to 0.49 h−1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl− current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.
Tyrosine Decaging Leads to Substantial Membrane Trafficking during Modulation of an Inward Rectifier Potassium Channel
Authors: Tong Yanhe , Brandt Gabriel S. , Li Ming , Shapovalov George , Slimko Eric , Karschin Andreas , Dougherty Dennis A. , Lester Henry A.
Published: July 2002

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K+ currents and also to substantial (15–26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K+ channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor 32P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.
Sets of minimal Hausdorff dimension for quasiconformal maps
Authors: Tyson Jeremy
Published: July 2002

For any

1
≤
α
≤
n

1\le \alpha \le n

, there is a compact set

E
⊂

R

n

E\subset \mathbb {R}^n

of (Hausdorff) dimension

α
\alpha

whose dimension cannot be lowered by any quasiconformal map

f
:

R

n

→

R

n

f:\mathbb {R}^n\to \mathbb {R}^n

. We conjecture that no such set exists in the case

α
>
1

\alpha >1

. More generally, we identify a broad class of metric spaces whose Hausdorff dimension is minimal among quasisymmetric images.
Local derivations of reflexive algebras
Authors: Wu Jing
Published: July 2002

Let

A

\mathcal A

be a reflexive algebra in Banach space

X
X

such that both

O
+

≠
O

O_+\not =O

and

X
−

≠
X

X_-\not =X

in

Lat

A

\operatorname {Lat}\,\mathcal A

, the invariant subspace lattice of

A

\mathcal A

, then every derivation of

A

\mathcal A

into itself is spatial. Furthermore, if

X
X

is additionally reflexive, then the set of all inner derivations of

A

\mathcal A

into itself is topologically algebraically reflexive.
Combinatorial 𝐵_{𝑛}-analogues of Schubert polynomials
Authors: Fomin Sergey , Kirillov Anatol
Published: July 2002

Combinatorial

B

n

B_{n}

-analogues of Schubert polynomials and corresponding symmetric functions are constructed and studied. The development is based on an exponential solution of the type

B
B

Yang-Baxter equation that involves the nilCoxeter algebra of the hyperoctahedral group.
Dragon Promoter Finder: recognition of vertebrate RNA
polymerase II promoters
Authors: Bajic Vladimir B. , Seah Seng Hong , Chong Allen , Zhang Guanglan , Koh Judice L. Y. , Brusic Vladimir
Published: July 2002

Summary: Dragon Promoter Finder (DPF) locates RNA polymerase II promoters in DNA sequences of vertebrates by predicting Transcription Start Site (TSS) positions. DPF’s algorithm uses sensors for three functional regions (promoters, exons and introns) and an Artificial Neural Network (ANN). Results on a large and diverse evaluation set indicate that DPF exhibits a superior predicting ability for TSS location compared to three other promoter-finding programs.

Availability: http://sdmc.krdl.org.sg:8080/promoter/

Contact: bajicv@krdl.org.sg

* To whom correspondence should be addressed.
Development of an improved CAD scheme for automated detection of lung nodules in digital chest images
Authors: Xu Xin‐Wei , Doi Kunio , Kobayashi Takeshi , MacMahon Heber , Giger Maryellen L.
Published: July 2002

Lung cancer is the leading cause of cancer deaths in men and women in the United States, with a 5‐year survival rate of only about 13%. However, this survival rate can be improved to 47% if the disease is diagnosed and treated at an early stage. In this study, we developed an improved computer‐aided diagnosis (CAD) scheme for the automated detection of lung nodules in digital chest images to assist radiologists, who could miss up to 30% of the actually positive cases in their daily practice. Two hundred PA chest radiographs, 100 normals and 100 abnormals, were used as the database for our study. The presence of nodules in the 100 abnormal cases was confirmed by two experienced radiologists on the basis of CT scans or radiographic follow‐up. In our CAD scheme, nodule candidates were selected initially by multiple gray‐level thresholding of the difference image (which corresponds to the subtraction of a signal‐enhanced image and a signal‐suppressed image) and then classified into six groups. A large number of false positives were eliminated by adaptive rule‐based tests and an artificial neural network (ANN). The CAD scheme achieved, on average, a sensitivity of 70% with 1.7 false positives per chest image, a performance which was substantially better as compared with other studies. The CPU time for the processing of one chest image was about 20 seconds on an IBM RISC/6000 Powerstation 590. We believe that the CAD scheme with the current performance is ready for initial clinical evaluation.