bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice
Authors: Marks-Konczalik Joanna , Dubois Sigrid , Losi Jacqueline M. , Sabzevari Helen , Yamada Nobuo , Feigenbaum Lionel , Waldmann Thomas A. , Tagaya Yutaka
Published: July 2002

A transgenic (Tg) mouse expressing human IL-15 was generated to define the role of IL-15 in the normal immune response. Overexpression of IL-15 resulted in an increase of NK, CD44hiCD8 memory T cells, and γδ T cells. Additionally, we observed the emergence of a novel type of NK-T cells with CD8αα′ expression. Due to the expansion and activation of NK cells, the IL-15Tg mouse showed enhanced innate immunity. In adaptive T cell immunity, the roles of IL-15 contrasted with those of IL-2. IL-15 inhibited IL-2-induced T cell death, which plays a role in the maintenance of peripheral self-tolerance. IL-15 thus seems to contribute to enhanced immune memory by selectively propagating memory T cells and by blocking T cell death mediated by IL-2.
Induction of ribosomal genes and hepatocyte hypertrophy by adenovirus-mediated expression of c-Myc
in vivo
Authors: Kim Sunkyu , Li Qing , Dang Chi V. , Lee Linda A.
Published: July 2002

Overexpression of c-Myc in immortalized cells increases cell
proliferation, inhibits cell differentiation, and promotes cell
transformation. Recent evidence suggests that these effects, however,
do not necessarily occur when c-Myc is overexpressed in primary
mammalian cells. We sought to determine the immediate effects of
transient overexpression of c-Myc in primary cells
in
vivo
by using recombinant adenovirus to overexpress human
MYC
in mouse liver. Mice were intravenously injected
with adenoviruses encoding
MYC
(Ad/Myc), E2F-1
(Ad/E2F-1), or β-galactosidase (Ad/LacZ). Transgene expression
was detectable 4 days after injection. Expression of ectopic c-Myc was
immediately accompanied by enlarged and dysmorphic hepatocytes in the
absence of significant cell proliferation or apoptosis. These
findings were not present in the livers of mice injected with
Ad/E2F-1 or Ad/LacZ. Prominent hepatocyte nuclei and nucleoli were
associated with the up-regulation of large- and small-subunit ribosomal
and nucleolar genes, suggesting that c-Myc may induce their expression
to increase cell mass. Our studies support a role for c-Myc in the
in vivo
control of vertebrate cell size and metabolism
independent of cell proliferation.
A functional imaging study of cooperation in two-person reciprocal exchange
Authors: McCabe Kevin , Houser Daniel , Ryan Lee , Smith Vernon , Trouard Theodore
Published: July 2002

Cooperation between individuals requires the ability to infer each
other's mental states to form shared expectations over mutual gains
and make cooperative choices that realize these gains. From evidence
that the ability for mental state attribution involves the use of
prefrontal cortex, we hypothesize that this area is involved in
integrating theory-of-mind processing with cooperative actions. We
report data from a functional MRI experiment designed to test this
hypothesis. Subjects in a scanner played standard two-person “trust
and reciprocity” games with both human and computer counterparts for
cash rewards. Behavioral data shows that seven subjects consistently
attempted cooperation with their human counterpart. Within this group
prefrontal regions are more active when subjects are playing a human
than when they are playing a computer following a fixed (and known)
probabilistic strategy. Within the group of five noncooperators, there
are no significant differences in prefrontal activation between
computer and human conditions.
Gambicin: A novel immune responsive antimicrobial peptide from the malaria vector
Anopheles gambiae
Authors: Vizioli Jacopo , Bulet Philippe , Hoffmann Jules A. , Kafatos Fotis C. , Müller Hans-Michael , Dimopoulos George
Published: July 2002

A novel mosquito antimicrobial peptide, gambicin, and the corresponding gene were isolated in parallel through differential display-PCR, an expressed sequence tag (EST) project, and characterization of an antimicrobial activity in a mosquito cell line by reverse-phase chromatography. The 616-bp gambicin ORF encodes an 81-residue protein that is processed and secreted as a 61-aa mature peptide containing eight cysteines engaged in four disulfide bridges. Gambicin lacks sequence homology with other known proteins. Like other
Anopheles gambiae
antimicrobial peptide genes, gambicin is induced by natural or experimental infection in the midgut, fatbody, and hemocyte-like cell lines. Within the midgut, gambicin is predominantly expressed in the anterior part. Both local and systemic gambicin expression is induced during early and late stages of natural malaria infection.
In vitro
experiments showed that the 6.8-kDa mature peptide can kill both Gram-positive and Gram-negative bacteria, has a morphogenic effect on a filamentous fungus, and is marginally lethal to
Plasmodium berghei
ookinetes. An oxidized form of gambicin isolated from the cell line medium was more active against bacteria than the nonoxidized form from the same medium.
Paired Ig-like receptor homologs in birds and mammals share a common ancestor with mammalian Fc receptors
Authors: Dennis Glynn , Kubagawa Hiromi , Cooper Max D.
Published: July 2002

Paired Ig-like receptors (PIR) that can reciprocally modulate cellular activation have been described in mammals. In the present study, we searched expressed sequence tag databases for PIR relatives to identify chicken expressed sequence tags predictive of ≈25% amino acid identity to mouse PIR. Rapid amplification of cDNA ends (RACE)-PCR extension of expressed sequence-tag sequences using chicken splenic cDNA as a template yielded two distinct cDNAs, the sequence analysis of which predicted protein products with related extracellular Ig-like domains. Chicken Ig-like receptor (CHIR)-A was characterized by its transmembrane segment with a positively charged histidine residue and short cytoplasmic tail, thereby identifying CHIR-A as a candidate-activating receptor. Conversely, CHIR-B was characterized by its nonpolar transmembrane segment and cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motifs, indicating that it may serve as an inhibitory receptor. The use of CHIR amino acid sequences in a search for other PIR relatives led to the recognition of mammalian Fc receptors as distantly related genes. Comparative analyses based on amino acid sequences and three-dimensional protein structures provided molecular evidence for common ancestry of the PIR and Fc receptor gene families.
Recombinant hepatitis E virus genomes infectious for primates: Importance of capping and discovery of a cis-reactive element
Authors: Emerson S. U. , Zhang M. , Meng X.-J. , Nguyen H. , St. Claire M. , Govindarajan S. , Huang Y. K. , Purcell R. H.
Published: July 2002

Hepatitis E virus recombinant genomes transcribed
in
vitro
from two cDNA clones differing by two nucleotides were
infectious for chimpanzees. However, one cDNA clone encoded a virus
that was attenuated for chimpanzees and unable to infect rhesus
monkeys. The second cDNA clone encoded a virus that infected both
chimpanzees and rhesus monkeys and caused acute hepatitis in both. One
mutation differentiating the two clones identified a cis-reactive
element that appeared to overlap the 3′ end of the capsid gene and part
of the 3′ noncoding region. Capping of the RNA transcripts was
essential for infectivity.
Thalamic bursting in rats during different awake behavioral states
Authors: Fanselow Erika E. , Sameshima Koichi , Baccala Luiz A. , Nicolelis Miguel A. L.
Published: July 2002

Thalamic neurons have two firing modes: tonic and bursting.
It was originally suggested that bursting occurs only during states
such as slow-wave sleep, when little or no information is relayed by
the thalamus. However, bursting occurs during wakefulness in the
visual and somatosensory thalamus, and could theoretically influence
sensory processing. Here we used chronically implanted electrodes to
record from the ventroposterior medial thalamic nucleus (VPM) and
primary somatosensory cortex (SI) of awake, freely moving rats during
different behaviors. These behaviors included quiet immobility,
exploratory whisking (large-amplitude whisker movements), and whisker
twitching (small-amplitude, 7- to 12-Hz whisker movements). We
demonstrated that thalamic bursting appeared during the oscillatory
activity occurring before whisker twitching movements, and continued
throughout the whisker twitching. Further, thalamic bursting occurred
during whisker twitching substantially more often than during the other
behaviors, and a neuron was most likely to respond to a stimulus if a
burst occurred ≈120 ms before the stimulation. In addition, the
amount of cortical area activated was similar to that during whisking.
However, when SI was inactivated by muscimol infusion, whisker
twitching was never observed. Finally, we used a statistical technique
called partial directed coherence to identify the direction of
influence of neural activity between VPM and SI, and observed that
there was more directional coherence from SI to VPM during whisker
twitching than during the other behaviors. Based on these findings, we
propose that during whisker twitching, a descending signal from SI
triggers thalamic bursting that primes the thalamocortical loop for
enhanced signal detection during the whisker twitching behavior.
Cryptococcus neoformansinteractions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages
Authors: Steenbergen J. N. , Shuman H. A. , Casadevall A.
Published: July 2002

Cryptococcus neoformans(Cn) is a soil fungus that causes life-threatening meningitis in immunocompromised patients and is a facultative intracellular pathogen capable of replication inside macrophages. The mechanism by which environmental fungi acquire and maintain virulence for mammalian hosts is unknown. We hypothesized that the survival strategies forCnafter ingestion by macrophages and amoebae were similar. Microscopy, fungal and amoebae killing assays, and phagocytosis assays revealed thatCnis phagocytosed by and replicates inAcanthamoeba castellanii, which leads to death of amoebae. An acapsular strain ofCndid not survive when incubated with amoebae, but melanization protected these cells against killing by amoebae. A phospholipase mutant had a decreased replication rate in amoebae compared with isogenic strains. These observations suggest that cryptococcal characteristics that contribute to mammalian virulence also promote fungal survival in amoebae. Intracellular replication was accompanied by the accumulation of polysaccharide containing vesicles similar to those described inCn-infected macrophages. The results suggest that the virulence ofCnfor mammalian cells is a consequence of adaptations that have evolved for protection against environmental predators such as amoebae and provide an explanation for the broad host range of this pathogenic fungus.
Specific spatial learning deficits become severe with age in β-amyloid precursor protein transgenic mice that harbor diffuse β-amyloid deposits but do not form plaques
Authors: Koistinaho Milla , Ort Michael , Cimadevilla Jose M. , Vondrous Roman , Cordell Barbara , Koistinaho Jari , Bures Jan , Higgins Linda S.
Published: July 2002

Memory impairment progressing to dementia is the main
clinical symptom of Alzheimer's disease (AD). AD is characterized
histologically by the presence of β-amyloid (Aβ) plaques and
neurofibrillary tangles in specific brain regions. Although Aβ
derived from the Aβ precursor protein (β-APP) is believed to play a
central etiological role in AD, it is not clear whether soluble
and/or fibrillar forms are responsible for the memory deficit. We
have generated and previously described mice expressing human wild-type
β-APP
751
isoform in neurons. These transgenic mice
recapitulate early histopathological features of AD and form Aβ
deposits but no plaques. Here we describe a specific and progressive
learning and memory impairment in these animals. In the Morris water
maze, a spatial memory task sensitive to hippocampal damage, one
pedigree already showed significant differences in acquisition in
3-month-old mice that increased in severity with age and were expressed
clearly in 6-month- and 2-year-old animals. The second transgenic
pedigree displayed a milder impairment with a later age of onset.
Performance deficits significantly decreased during the 6 days of
training in young but not in aged transgenic animals. Both pedigrees of
the transgenic mice differed from wild-type mice by less expressed
increase of escape latencies after the platform position had been
changed in the reversal experiment and by failure to prefer the goal
quadrant in probe trials. Both pedigrees performed at wild-type level
in a number of other tests (open field exploration and passive and
active place avoidance). The results suggest that plaque formation is
not a necessary condition for the neuronal β-APP
751
transgene-induced memory impairment, which may be caused by β-APP
overexpression, isoform misexpression, or elevated soluble Aβ.
Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis.
Authors: Takiff H E , Cimino M , Musso M C , Weisbrod T , Martinez R , Delgado M B , Salazar L , Bloom B R , Jacobs W R
Published: July 2002

Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, of Streptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.
Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.
Authors: Lu Q , Inouye M
Published: July 2002

Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase.
Binding of thalidomide to alpha1-acid glycoprotein may be involved in its inhibition of tumor necrosis factor alpha production.
Authors: Turk B E , Jiang H , Liu J O
Published: July 2002

In addition to its well known sedative and teratogenic effects, thalidomide also possesses potent immunomodulatory and antiinflammatory activities, being most effective against leprosy and chronic graft-versus-host disease. The immunomodulatory activity of thalidomide has been ascribed to the selective inhibition of tumor necrosis factor alpha from monocytes. The molecular mechanism for the immunomodulatory effect of thalidomide remains unknown. To elucidate this mechanism, we synthesized an active photoaffinity label of thalidomide as a probe to identify the molecular target of the drug. Using the probe, we specifically labeled a pair of proteins of 43-45 kDa with high acidity from bovine thymus extract. Purification of these proteins and partial peptide sequence determination revealed them to be alpha1-acid glycoprotein (AGP). We show that the binding of thalidomide photoaffinity label to authentic human AGP is competed with both thalidomide and the nonradioactive photoaffinity label at concentrations comparable to those required for inhibition of production of tumor necrosis factor alpha from human monocytes, suggesting that AGP may be involved in the immunomodulatory activity of thalidomide.
Induction of intestinal epithelial proliferation by glucagon-like peptide 2.
Authors: Drucker D J , Erlich P , Asa S L , Brubaker P L
Published: July 2002

Injury, inflammation, or resection of the small intestine results in severe compromise of intestinal function. Nevertheless, therapeutic strategies for enhancing growth and repair of the intestinal mucosal epithelium are currently not available. We demonstrate that nude mice bearing subcutaneous proglucagon-producing tumors exhibit marked proliferation of the small intestinal epithelium. The factor responsible for inducing intestinal proliferation was identified as glucagon-like peptide 2 (GLP-2), a 33-aa peptide with no previously ascribed biological function. GLP-2 stimulated crypt cell proliferation and consistently induced a marked increase in bowel weight and villus growth of the jejunum and ileum that was evident within 4 days after initiation of GLP-2 administration. These observations define a novel biological role for GLP-2 as an intestinal-derived peptide stimulator of small bowel epithelial proliferation.
Ultrasensitivity in the mitogen-activated protein kinase cascade.
Authors: Huang C Y , Ferrell J E
Published: July 2002

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved series of three protein kinases implicated in diverse biological processes. Here we demonstrate that the cascade arrangement has unexpected consequences for the dynamics of MAPK signaling. We solved the rate equations for the cascade numerically and found that MAPK is predicted to behave like a highly cooperative enzyme, even though it was not assumed that any of the enzymes in the cascade were regulated cooperatively. Measurements of MAPK activation in Xenopus oocyte extracts confirmed this prediction. The stimulus/response curve of the MAPK was found to be as steep as that of a cooperative enzyme with a Hill coefficient of 4-5, well in excess of that of the classical allosteric protein hemoglobin. The shape of the MAPK stimulus/ response curve may make the cascade particularly appropriate for mediating processes like mitogenesis, cell fate induction, and oocyte maturation, where a cell switches from one discrete state to another.
Packing at the protein-water interface.
Authors: Gerstein M , Chothia C
Published: July 2002

We have determined the packing efficiency at the protein-water interface by calculating the volumes of atoms on the protein surface and nearby water molecules in 22 crystal structures. We find that an atom on the protein surface occupies, on average, a volume approximately 7% larger than an atom of equivalent chemical type in the protein core. In these calculations, larger volumes result from voids between atoms and thus imply a looser or less efficient packing. We further find that the volumes of individual atoms are not related to their chemical type but rather to their structural location. More exposed atoms have larger volumes. Moreover, the packing around atoms in locally concave, grooved regions of protein surfaces is looser than that around atoms in locally convex, ridge regions. This as a direct manifestation of surface curvature-dependent hydration. The net volume increase for atoms on the protein surface is compensated by volume decreases in water molecules near the surface. These waters occupy volumes smaller than those in the bulk solvent by up to 20%; the precise amount of this decrease is directly related to the extent of contact with the protein.
Primate photopigments and primate color vision.
Authors: Jacobs G H
Published: July 2002

The past 15 years have brought much progress in our understanding of several basic features of primate color vision. There has been particular success in cataloging the spectral properties of the cone photopigments found in retinas of a number of primate species and in elucidating the relationship between cone opsin genes and their photopigment products. Direct studies of color vision show that there are several modal patterns of color vision among groupings of primates: (i) Old World monkeys, apes, and humans all enjoy trichromatic color vision, although the former two groups do not seem prone to the polymorphic variations in color vision that are characteristic of people; (ii) most species of New World monkeys are highly polymorphic, with individual animals having any of several types of dichromatic or trichromatic color vision; (iii) less is known about color vision in prosimians, but evidence suggests that at least some diurnal species have dichromatic color vision; and (iv) some nocturnal primates may lack color vision completely. In many cases the photopigments and photopigment gene arrangements underlying these patterns have been revealed and, as a result, hints are emerging about the evolution of color vision among the primates.
Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress.
Authors: Gidrol X , Sabelli P A , Fern Y S , Kush A K
Published: July 2002

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.
Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppressionin vitro
Authors: Schlender Jörg , Schnorr Jens-Jörg , Spielhofer Pius , Cathomen Toni , Cattaneo Roberto , Billeter Martin A. , ter Meulen Volker , Schneider-Schaulies Sibylle
Published: July 2002

A marked suppression of immune function has long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. As a hallmark of measles virus (MV)-induced immunosuppression, peripheral blood lymphocytes (PBLs) isolated from patients exhibit a significantly reduced capacity to proliferate in response to mitogens, allogens, or recall antigens. In anin vitrosystem we show that proliferation of naive PBLs [responder cells (RCs)] in response to a variety of stimuli was significantly impaired after cocultivation with MV-infected, UV-irradiated autologous PBLs [presenter cells (PCs)]. We further observed that a 50% reduction in proliferation of RCs could still be observed when the ratio of PC to RC was 1:100. The effect was completely abolished after physical separation of the two populations, which suggests that soluble factors were not involved. Proliferative inhibition of the RCs was observed after short cocultivation with MV-infected cells, which indicates that surface contact between one or more viral proteins and the RC population was required. We identified that the complex of both MV glycoproteins, F and H, is critically involved in triggering MV-induced suppression of mitogen-dependent proliferation, since the effect was not observed (i) using a recombinant MV in which F and H were replaced with vesicular stomatitis virus G or (ii) when either of these proteins was expressed alone. Coexpression of F and H, however, lead to a significant proliferative inhibition in the RC population. Our data indicate that a small number of MV-infected PBLs can induce a general nonresponsiveness in uninfected PBLs by surface contact, which may, in turn, account for the general suppression of immune responses observed in patients with acute measles.
Convergent pathways for lipochitooligosaccharide and auxin signaling in tobacco cells
Authors: Röhrig Horst , Schmidt Jürgen , Walden Richard , Czaja Inge , Lubenow Helge , Wieneke Ursula , Schell Jeff , John Michael
Published: July 2002

Lipochitooligosaccharides (LCOs) are a novel class of plant growth
regulators that activate in tobacco protoplasts the expression of
AXI1
, a gene implicated in auxin signaling. Transient
assays with a chimeric P
AXI
-
GUS
expression
plasmid revealed that the
N
-octadecenoylated
monosaccharide Glc
N
has all structural requirements for
a biological active glycolipid, whereas the inactive
N
-acylated Gal
N
epimer inhibits LCO
action. Specific inhibition of LCO and auxin action shows that both
signals are transduced within the tobacco cell via separate pathways
that converge at or before
AXI1
transcription. Cytokinin
is suggested to be a common effector of LCO and auxin signaling. We
also show that activation of
AXI1
correlates with growth
factor-induced cell division.
A small engineered protein lacks structural uniqueness by increasing the side-chain conformational entropy
Authors: Furukawa Koji , Oda Masayuki , Nakamura Haruki
Published: July 2002

A small globular protein, the third repeat of the c-Myb DNA-binding
domain, which is composed of 54 amino acid residues, was engineered so
as to understand the structural uniqueness of native proteins. This
small protein has three α-helices that form a helix-turn-helix
structure, which is maintained by the hydrophobic core with three Ile
residues. One of the mutant proteins, with two of the buried Ile
(Ile-155 and Ile-181) substituted with Leu residues, showed multiple
conformations, as monitored by heteronuclear magnetic resonance
spectroscopy for
13
C- and
15
N-labeled proteins.
The increase in the side-chain conformational entropy, caused by
changing the Ile to a Leu residue on an α-helix, could engender the
lack of structural uniqueness. In native proteins, the conformations of
not only the β-branched side chains, but also those of the
neighboring bulky side chains, can be greatly restricted, depending
upon the local backbone structure.
Toward genetic dissection of high and low antibody responsiveness in Biozzi mice
Authors: Puel Anne , Mevel Jean-Claude , Bouthillier Yolande , Feingold Nicole , Fridman Wolf H. , Mouton Denise
Published: July 2002

Several distinct chromosomal segments were recently identified by cosegregation analysis of polymorphic markers with antibody responsiveness in an F2cross between high (H) and low (L) antibody responder lines of Biozzi mice. The effect associated with the relevant markers has now been investigated in backcross populations (toward the L line) bred from H and L mice made coisogenic at theH-2locus. The antibody titers, measured on days 5 and 14 of the primary response to sheep red blood cells, were considered to be two distinct quantitative phenotypes. The results of single or multilocus analyses demonstrated the significant involvement, at one or the two titration times, ofImgene(s) on four distinct chromosomes: 4, 8, 12, and 18. The regions on chromosomes 6 and 10 have a lesser but still suggestive effect. The contribution of each locus ranged from 3% to 13%, and together these loci accounted for about 40% of the phenotypic variance at each titration time. The data are compatible with an additive effect of the relevant loci and suggestive of some interaction effects. In a second backcross toward L line, the H line alleles of the putativeImgenes on chromosomes 6, 8, and 12 were isolated from each other and their effects were still detected.
The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase.
Authors: Damen J E , Liu L , Rosten P , Humphries R K , Jefferson A B , Majerus P W , Krystal G
Published: July 2002

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.
FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.
Authors: Henkel A W , Lübke J , Betz W J
Published: July 2002

Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion. This dye appears to label the membranes of recycled synaptic vesicles by being trapped during endocytosis. Stained terminals can subsequently be destained by repeating nerve stimulation in the absence of dye; the destaining evidently reflects escape of dye into the bathing medium from membranes of exocytosing synaptic vesicles. In the present study we tested two key aspects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually released into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobenzidine. This created an electron-dense reaction product that could be visualized in the electron microscope. Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated into the medium from tubocurarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle-forming detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude.
The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.
Authors: Bobkov A A , Bobkova E A , Lin S H , Reisler E
Published: July 2002

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.
The C-terminal region of human angiogenin has a dual role in enzymatic activity.
Authors: Russo N , Nobile V , Di Donato A , Riordan J F , Vallee B L
Published: July 2002

The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123 which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function.
Fos and Jun do not bend the AP-1 recognition site.
Authors: Sitlani A , Crothers D M
Published: July 2002

We have used a solution-based DNA cyclization assay and a gel-phasing method to show that contrary to previous reports [Kerppola, T. K. & Curran, T. (1991) Cell 66, 317-326], basic region leucine zipper proteins Fos and Jun do not significantly bend their AP-1 recognition site. We have constructed two sets of DNA constructs that contain the 7-bp 5'-TGACTCA-3' AP-1 binding site, from either the yeast or the human collagenase gene, which is well separated from and phased by 3-4 helical turns against an A tract-directed bend. The cyclization probabilities of DNAs with altered phasings are not significantly affected by Fos-Jun binding. Similarly, Fos-Jun and Jun-Jun bound to differently phased DNA constructs show insignificant variations in gel mobilities. Both these methods independently indicate that Fos and Jun bend their AP-1 target site by <5 degrees, an observation that has important implications in understanding their mechanism of transcriptional regulation.
Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion.
Authors: Spannagel A W , Green G M , Guan D , Liddle R A , Faull K , Reeve J R
Published: July 2002

Cholecystokinin (CCK) secretion in rats and humans is inhibited by pancreatic proteases and bile acids in the intestine. It has been hypothesized that the inhibition of CCK release caused by pancreatic proteases is due to proteolytic inactivation of a CCK-releasing peptide present in intestinal secretion. To purify the putative luminal CCK-releasing factor (LCRF), intestinal secretions were collected by perfusing a modified Thiry-Vella fistula of jejunum in conscious rats. From these secretions, the peptide was concentrated by ultrafiltration followed by low-pressure reverse-phase chromatography and purified by reverse-phase high-pressure liquid chromatography. Purity was confirmed by high-performance capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stimulate pancreatic protein secretion when infused into the proximal small intestine of conscious rats. Partially purified fractions strongly stimulated both pancreatic secretion and CCK release while CCK receptor blockade abolished the pancreatic response. Amino acid analysis and mass spectral analysis showed that the purified peptide is composed of 70-75 amino acid residues and has a mass of 8136 Da. Microsequence analysis of LCRF yielded an amino acid sequence for 41 residues as follows: STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV. When infused intraduodenally, the purified peptide stimulated pancreatic protein and fluid secretion in a dose-related manner in conscious rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography using antisera raised to synthetic LCRF-(1-6) abolished the CCK releasing activity of intestinal secretions. These studies demonstrate, to our knowledge, the first chemical characterization of a luminally secreted enteric peptide functioning as an intraluminal regulator of intestinal hormone release.
The role of London forces in defining noncentrosymmetric order of high dipole moment–high hyperpolarizability chromophores in electrically poled polymeric thin films
Authors: Dalton Larry R. , Harper Aaron W. , Robinson Bruce H.
Published: July 2002

Graphs of second harmonic generation coefficients and electro-optic coefficients (measured by ellipsometry, attenuated total reflection, and two-slit interference modulation) as a function of chromophore number density (chromophore loading) are experimentally observed to exhibit maxima for polymers containing chromophores characterized by large dipole moments and polarizabilities. Modified London theory is used to demonstrated that this behavior can be attributed to the competition of chromophore-applied electric field and chromophore–chromophore electrostatic interactions. The comparison of theoretical and experimental data explains why the promise of exceptional macroscopic second-order optical nonlinearity predicted for organic materials has not been realized and suggests routes for circumventing current limitations to large optical nonlinearity. The results also suggest extensions of measurement and theoretical methods to achieve an improved understanding of intermolecular interactions in condensed phase materials including materials prepared by sequential synthesis and block copolymer methods.
A fast, high resolution, second-order central scheme for incompressible flows
Authors: Kupferman Raz , Tadmor Eitan
Published: July 2002

A high resolution, second-order central difference method for incompressible flows is presented. The method is based on a recent second-order extension of the classic Lax–Friedrichs scheme introduced for hyperbolic conservation laws (Nessyahu H. & Tadmor E. (1990)
J. Comp. Physics.
87, 408-463; Jiang G.-S. & Tadmor E. (1996) UCLA CAM Report 96-36,
SIAM J. Sci. Comput
., in press) and augmented by a new discrete Hodge projection. The projection is exact, yet the discrete Laplacian operator retains a compact stencil. The scheme is fast, easy to implement, and readily generalizable. Its performance was tested on the standard periodic double shear-layer problem; no spurious vorticity patterns appear when the flow is underresolved. A short discussion of numerical boundary conditions is also given, along with a numerical example.
Characterization of
VPS41
, a gene required for vacuolar trafficking and high-affinity iron transport in yeast
Authors: Radisky Derek C. , Snyder William B. , Emr Scott D. , Kaplan Jerry
Published: July 2002

Mutations in the yeast gene
VPS41
give rise to poor growth on low iron medium, severe alterations in vacuolar morphology, and cause the missorting of membranous and soluble vacuolar proteins. Our studies predict that
VPS41
encodes a hydrophilic protein of 992 amino acids that contains no obvious signal sequence or hydrophobic domains. The deduced Vps41p sequence contains a domain rich in glutamic and aspartic residues, as well as a domain with resemblance to a region of clathrin heavy chain. We have also identified and sequenced putative
VPS41
homologues from
Caenorhabditis elegans
, plants, and humans. The
VPS41
homologues (but not the yeast
VPS41
itself) contain a conserved cysteine-rich RING-H2 zinc finger at their COOH termini. Biochemical experiments suggest that
VPS41
functions in post-Golgi protein processing: the deletion mutant exhibits defective high affinity transport due to impaired Fet3p activity and also exhibits defects in the processing and sorting of multiple vacuolar hydrolases.
Ly-6C regulates endothelial adhesion and homing of CD8+T cells by activating integrin-dependent adhesion pathways
Authors: Hänninen Arno , Jaakkola Ilkka , Salmi Marko , Simell Olli , Jalkanen Sirpa
Published: July 2002

Ly-6C belongs to the Ly-6 family of glycosyl phosphatidylinositol-anchored surface glycoproteins and is expressed on a subset of mature CD8+T cells. Ly-6C ligation can mediate T cell activation and causes interleukin 2 secretion in cytolytic T cell clones. We characterize herein a new mAb 1G7.G10 against Ly-6C that recognizes an epitope involved in lymphocyte adhesion and in lymphocyte homing. Pretreatment of lymph node lymphocytes and of purified CD8+T cells (but not of lymphocytes depleted of CD8+T cells) with 1G7.G10 reduced theirin vitrobinding to lymph node high endothelial venules by 28% and 34%, respectively. This effect was bypassed by cross-linking Ly-6C molecules with 1G7.G10 and a second-step antibody. Thein vivohoming of (donor) CD8+T lymphocytes to lymph nodes was reduced by Ly-6C blocking with 1G7.G10 (whole antibody) or with its fragments [F(ab) or F(ab)2] by 20% or by 32% and 48%, respectively. Cross-linking of Ly-6Cin vitroinduced very late antigen-4 and lymphocyte function-associated antigen 1-mediated aggregation of CD8+T cells, suggesting that ligand binding to Ly-6C leads to activation of integrins. This activation may facilitate homing of Ly-6C+CD8+T cellsin vivo.
Slow dimer dissociation of the TATA binding protein dictates the kinetics of DNA binding
Authors: Coleman Robert A. , Pugh B. Franklin
Published: July 2002

The association of the TATA binding protein (TBP) to eukaryotic promoters is a possible rate-limiting step in gene expression. Slow promoter binding might be related to TBP’s ability to occlude its DNA binding domain through dimerization. Using a “pull-down” based assay, we find that TBP dimers dissociate slowly (
t
½ = 6–10 min), and thus present a formidable kinetic barrier to TATA binding. At 10 nM, TBP appears to exist as a mixed population of monomers and dimers. In this state, TATA binding displays burst kinetics that appears to reflect rapid binding of monomers and slow dissociation of dimers. The kinetics of the slow phase is in excellent agreement with direct measurements of the kinetics of dimer dissociation.
Cloning of a novel T-cell protein FYB that binds FYN and SH2-domain-containing leukocyte protein 76 and modulates interleukin 2 production
Authors: da Silva Antonio J. , Li Zhuwen , de Vera Cristina , Canto Eduardo , Findell Paul , Rudd Christopher E.
Published: July 2002

T cell receptor ζ (TcRζ)/CD3 ligation initiates a signaling cascade that involves src kinases p56lckand ζ-associated protein 70, leading to the phosphorylation of substrates such as TcRζ, Vav, SH2-domain-containing leukocyte protein 76 (SLP-76), cbl, and p120/130. FYN binding protein (FYB or p120/130) associates with p59fyn, the TcRζ/CD3 complex, and becomes tyrosine-phosphorylated in response to receptor ligation. In this study, we report the cDNA cloning of human and murine FYB and show that it is restricted in expression to T cells and myeloid cells and possesses an overall unique hydrophilic sequence with several tyrosine-based motifs, proline-based type I and type II SH3 domain binding motifs, several putative lysine/glutamic acid-rich nuclear localization motifs, and a SH3-like domain. In addition to binding the src kinase p59fyn, FYB binds specifically to the hematopoietic signaling protein SLP-76, an interaction mediated by the SLP-76 SH2 domain. In keeping with this, expression of FYB augmented interleukin 2 secretion from a T cell hybridoma, DC27.10, in response to TcRζ/CD3 ligation. FYB is therefore a novel hematopoietic protein that acts as a component of the FYN and SLP-76 signaling cascades in T cells.
Nuclear localization of basonuclin in human keratinocytes and the role of phosphorylation
Authors: Iuchi Shiro , Green Howard
Published: July 2002

Basonuclin is a zinc-finger protein found in basal cells of the epidermis. In human keratinocyte cultures, basonuclin is susceptible to serine-phosphorylation and the addition of the phosphatase inhibitor, okadaic acid, promotes accumulation of basonuclin in the cytoplasm. The region of basonuclin containing the nuclear localization signal of basonuclin is necessary for nuclear localization of the protein and Ser-541, located immediately C-terminal to the nuclear localization signal, is the principal phosphorylation site
in vitro
. A nearly complete basonuclin transiently expressed in cultured keratinocytes localizes predominantly in the nucleus, but substitution of aspartic acid for Ser-541 promotes cytoplasmic localization. The same substitution of Ser-537 has a similar but weaker effect. Substitution of both serine residues by alanine leads to nuclear localization. These results show that nuclear localization of basonuclin depends on serine dephosphorylation, primarily of Ser-541. Different subcellular locations of basonuclin in different keratinocyte subtypes are therefore most likely to be controlled by the state of phosphorylation of Ser-541.
Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-κB control
Authors: Chu Zhi-Liang , McKinsey Timothy A. , Liu Lily , Gentry Jennifer J. , Malim Michael H. , Ballard Dean W.
Published: July 2002

Members of the NF-κB/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor α (TNF). Although NF-κB appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-κB is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-κB and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blockedin vivoby coexpressing a dominant form of IκB that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-κB induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-κB via an IκB targeting mechanism. Functional coupling of NF-κB and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.