bluuebunny/crossref_metadata_2025 huggingface dataset:
Small lattice relaxation at the D
X center as studied by extended x-ray absorption fine structure on Se-doped AlGaAs
Authors: Mizuta M. , Kitano T.
Published: July 2002

Lattice relaxation at the Se DX center in Al0.38Ga0.62As was determined by the extended x-ray absorption fine structure (EXAFS). During the EXAFS measurements the deep and shallow (metastable) states of the DX center were prepared and the resultant difference of the nearest-neighbor distance around Se between the two electronic states was found to be quite small (less than 0.04 Å). The result is indicative of small lattice relaxation at the DX center.
Self-consistent simulation of a parallel-plate rf discharge
Authors: Boswell R. W. , Morey I. J.
Published: July 2002

A particle-in-cell code with nonperiodic boundary conditions is used to stimulate a rf discharge. The simulations are started with a few ion-electron pairs which can initiate a breakdown after a few rf cycles with the plasma density increasing until a steady state is reached. The average potential of the plasma is greater than that on the boundaries so that ions drift towards the sheaths at the ambipolar rate. The ion loss at equilibrium is balanced mainly by ion creation due to jets of secondary electrons accelerated through the sheaths formed alternately on each electrode. The variation of the sheath width, the negative glow, and the average sheath potential are studied as a function of pressure and compared with Child–Langmuir theory.
Modulation of an intense beam by an external microwave source: Theory and simulation
Authors: Krall J. , Lau Y. Y.
Published: July 2002

A time-dependent, fully electromagnetic particle code is used to simulate the current modulation in an intense relativistic electron beam by an external rf source. It is shown that the intense beam may serve as a power amplifier with good phase stability, as suggested in earlier experiments. Increase in beam bunching by the dc space charge is demonstrated with a simple analytical model.
Electrical properties of InAs epilayers grown by molecular beam epitaxy on Si substrates
Authors: Kalem Ş. , Chyi J. , Litton C. W. , Morkoç H. , Kan S. C. , Yariv A.
Published: July 2002

InAs epitaxial films have been grown by molecular beam epitaxy on high-resistivity Si (20 Ω cm) substrates for the first time, and transport properties were investigated by Hall effect measurements down to 10 K. The electron mobilities peak at 75 K with a value of 4.5×104 cm2/(V s) in 6.7-μm-thick (n=2.6×1015 cm−3 ) unintentionally doped layers. It is shown that the temperature dependence of the Hall mobility can be explained by a combined ionized impurity-polar optical phonon scattering model in the thin InAs layers. Despite the existence of a large lattice mismatch, the results are indicative of a high quality material.
Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A
Authors: Lin Ridwan , Karpa Kelly , Kabbani Nadine , Goldman-Rakic Patricia , Levenson Robert
Published: July 2002

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor–FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton.
Transmembrane pores formed by synthetic
p
-octiphenyl β-barrels with internal carboxylate clusters: Regulation of ion transport by pH and Mg
2+
- complexed 8-aminonaphthalene-1,3,6-trisulfonate
Authors: Das Gopal , Matile Stefan
Published: July 2002

Design, synthesis, and study of a synthetic barrel-stave supramolecule with
p
-octiphenyl “staves,” β-sheet “hoops,” and hydrophobic exterior as well as internal carboxylate clusters are reported. Ion transport experiments indicate the formation of transmembrane pores at 5 < pH < 7 with nanomolar activity. Blockage of dye efflux from spherical bilayers by external Mg(OAc)
2
and internal 8-aminonaphthalene-1,3,6-trisulfonate is suggestive for weakly cooperative (
n
= 1.16) formation of aspartate-Mg
2+
-8-aminonaphthalene-1,3,6-trisulfonate complexes within the barrel-stave supramolecule (
K
D
= 2.9 mM). Corroborative evidence from structural studies by circular dichroism spectroscopy is provided and discussed with emphasis of the importance of internal charge repulsion for pore formation and future applications toward binding and catalysis within supramolecular synthetic pores.
Protective DNA vaccination against organ-specific autoimmunity is highly specific and discriminates between single amino acid substitutions in the peptide autoantigen
Authors: Weissert Robert , Lobell Anna , de Graaf Katrien L. , Eltayeb Sana Yousif , Andersson Roland , Olsson Tomas , Wigzell Hans
Published: July 2002

DNA vaccines that encode encephalitogenic sequences in tandem can protect from subsequent experimental autoimmune encephalomyelitis induced with the corresponding peptide. The mechanism for this protection and, in particular, if it is specific for the amino acid sequence encoding the vaccine are not known. We show here that a single amino acid exchange in position 79 from serine (nonself) to threonine (self) in myelin basic protein peptide MBP68–85, which is a major encephalitogenic determinant for Lewis rats, dramatically alters the protection. Moreover, vaccines encoding the encephalitogenic sequence MBP68–85 do not protect against the second encephalitogenic sequence MBP89–101 in Lewis rats and vice versa. Thus, protective immunity conferred by DNA vaccination exquisitely discriminates between peptide target autoantigens. No bystander suppression was observed. The exact underlying mechanisms remain elusive because no simple correlation between impact onex vivoresponses and protection against disease were noted.
Combined expression of pTα and Notch3 in T cell leukemia identifies the requirement of preTCR for leukemogenesis
Authors: Bellavia Diana , Campese Antonio F. , Checquolo Saula , Balestri Anna , Biondi Andrea , Cazzaniga Giovanni , Lendahl Urban , Fehling Hans J. , Hayday Adrian C. , Frati Luigi , von Boehmer Harald , Gulino Alberto , Screpanti Isabella
Published: July 2002

Notch receptors are conserved regulators of cell fate and have been implicated in the regulation of T cell differentiation and lymphomagenesis. However, neither the generality of Notch involvement in leukemia, nor the molecules with which Notch may interact have been clarified. Recently, we showed that transgenic mice expressing the constitutively active intracellular domain of Notch3 in thymocytes and T cells developed early and aggressive T cell neoplasias. Although primarily splenic, the tumors sustained features of immature thymocytes, including expression of pTα, a defining component of the pre T cell receptor, known to be a potent signaling complex provoking thymocyte survival, proliferation, and activation. Thus, enforced expression of Notch3, which is ordinarily down-regulated as thymocytes mature, may sustain pre T cell receptor expression, causing dysregulated hyperplasia. This hypothesis has been successfully tested in this article by the observation that deletion of pTα in Notch3 transgenic mice abrogates tumor development, indicating a crucial role for pTα in T cell leukemogenesis. Parallel observations were made in humans, in that all T cell acute lymphoblastic leukemias examined showed expression of Notch3 and of the Notch target gene HES-1, as well as of pTα a and b transcripts, whereas the expression of all these genes was dramatically reduced or absent in remission. Together, these results suggest that the combined expression of Notch3 and pTα sustains T cell leukemogenesis and may represent pathognomonic molecular features of human T-ALL.
Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis
Authors: Narasimhan L. R. , Goodman William , Patel C. Kumar N.
Published: July 2002

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave anR2of 0.95 for breath ammonia and BUN correlation and anR2of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.
Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (
Ipomoea purpurea
)
Authors: Durbin Mary L. , Denton Amy L. , Clegg Michael T.
Published: July 2002

Mobile element dynamics in seven alleles of the chalcone synthase D
locus (CHS-D) of the common morning glory (
Ipomoea
purpurea
) are analyzed in the context of synonymous nucleotide
sequence distances for CHS-D exons. By using a nucleotide sequence of
CHS-D from the sister species
Ipomoea nil
(Japanese
morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. &
Iida, S. (1999)
Genes Genet. Syst.
74, 141–147], it is
also possible to determine the relative frequency of insertion and loss
of elements within the CHS-D locus between these two species. At least
four different types of transposable elements exist upstream of the
coding region, or within the single intron of the CHS-D locus in
I. purpurea
. There are three distinct families of
miniature inverted-repeat transposable elements (MITES), and some
recent transpositions of Activator/Dissociation (Ac/Ds)-like
elements (Tip100), of some short interspersed repetitive elements
(SINEs), and of an insertion sequence (InsIpCHSD) found in the
neighborhood of this locus. The data provide no compelling evidence of
the transposition of the mites since the separation of
I.
nil
and
I. purpurea
roughly 8 million years ago.
Finally, it is shown that the number and frequency of mobile elements
are highly heterogeneous among different duplicate CHS loci, suggesting
that the dynamics observed at CHS-D are locus-specific.
Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium
Authors: Uyttendaele H. , Ho J. , Rossant J. , Kitajewski J.
Published: July 2002

Notch proteins function as receptors for membrane-bound
ligands (Jagged and Delta-like) to regulate cell-fate determination. We
have investigated the role of Notch signaling in embryonic endothelium
of the mouse by expressing an activated form of the Notch4 protein in
vasculature under the regulation of the
Flk1
(VEGFR)
locus. Expression of activated Notch4 results in a growth and
developmental delay and embryonic lethality at about 10 days
postcoitum. The extent of the developing vasculature in mutant embryos
was restricted, fewer small vessels were seen, and vascular networks
were disorganized. The brain periphery of mutant embryos contained
large dilated vessels with evidence of compromised vessel-wall
integrity and large areas of necrosis; yolk-sac vasculature was
abnormal. Expression of an activated form of Notch4 in embryonic
vasculature leads to abnormal vessel structure and patterning,
implicating the Notch pathway in phases of vascular development
associated with vessel patterning and remodeling.
Detergent-phospholipid mixed micelles with a crystalline phospholipid core
Authors: Funari Sérgio S. , Nuscher Brigitte , Rapp Gert , Beyer Klaus
Published: July 2002

An unusual micelle was discovered in mixtures of the nonionic detergent octaethyleneglycol-mono-
n
-dodecylether with disaturated phospholipids such as 1,2-dimyristoyl-
sn
-glycero-3-phosphocholine or 1,2-dipalmitoyl-
sn
-glycero-3-phosphocholine in water. These mixtures undergo a structural transition upon cooling through the chain-melting temperatures of the respective phospholipids, resulting in the formation of mixed micelles. Structural features of the micellar particles were studied here by synchrotron x-ray scattering. The translucent micellar solutions showed characteristic wide-angle reflections that were attributed to ordered hydrocarbon chains, whereas the absence of small-angle x-ray reflections indicated that there is no long-range order in these mixtures. The presence of ordered phospholipid acyl chains was confirmed by differential scanning calorimetry and isothermal titration calorimetry. The endothermic differential scanning calorimetry signals observed in the up-scan mode were tentatively ascribed to chain melting and mixing of the components. Isothermal titration of the mixed-micellar solutions into an excess of the detergent octaethyleneglycol-mono-
n
-dodecylether resulted in sudden uptake of the latent heat by the gel-state phospholipids. The heat uptake per mol of phospholipid decreased with increasing detergent/phospholipid molar ratio. A simple geometric model is presented assuming that the dominating particle species in the mixtures is a discoidal phospholipid aggregate with ordered acyl chains, surrounded by a toroidal detergent hoop. The model implies that the fraction of ordered phospholipid chains decreases with increasing detergent/phospholipid molar ratio, in agreement with the calorimetric results and high-resolution NMR spectroscopy.
Cyclic changes in estradiol regulate synaptic plasticity through the MAP kinase pathway
Authors: Bi Ruifen , Foy Michael R. , Vouimba Rose-Marie , Thompson Richard F. , Baudry Michel
Published: July 2002

Hippocampal synaptic structure and function exhibit marked variations during the estrus cycle of female rats. Estradiol activates the mitogen-activated protein (MAP) kinase pathway in numerous cell types, and MAP kinase has been shown to play a critical role in the mechanisms underlying synaptic plasticity. Here, we report that endogenous estrogen produces a tonic phosphorylation/activation of extracellular signal-regulated kinase 2 (ERK2)/MAP kinase throughout the female rat brain and an increase in tyrosine phosphorylation of NR2 subunits ofN-methyl-d-aspartate (NMDA) receptors. Moreover, cyclic changes in estrogen levels during the estrus cycle of female rats are associated with corresponding changes in the levels of activation of ERK2, the state of tyrosine phosphorylation of NR2 subunits of NMDA receptors, and the magnitude of long-term potentiation in hippocampus. Thus, cyclic changes in female sexual hormones result in marked variations in the state of activation of a major cellular signaling pathway critical for learning and memory and in a cellular model of learning and memory.
Function of GB1 and GB2 subunits in G protein coupling of GABA
B
receptors
Authors: Margeta-Mitrovic Marta , Jan Yuh Nung , Jan Lily Yeh
Published: July 2002

Many G protein-coupled receptors (GPCRs) have recently been shown
to dimerize, and it was suggested that dimerization may be a
prerequisite for G protein coupling. γ-aminobutyric acid type B
(GABA
B
) receptors (GPCRs for GABA, a major inhibitory
neurotransmitter in the brain) are obligate heterodimers of homologous
GB1 and GB2 subunits, neither of which is functional on its own. This
feature of GABA
B
receptors allowed us to examine which of
the eight intracellular segments of the heterodimeric receptor were
important for G protein activation. Replacing any of the three
intracellular loops of GB2 with their GB1 counterparts resulted in
nonfunctional receptors. The deletion of the complete GB2 C terminus
significantly attenuated the receptor function; however, the proximal
36 residues were sufficient for reconstitution of wild type-like
receptor activity. In contrast, the GB1 C terminus could be deleted and
GB1 intracellular loops replaced with their GB2 or mGluR1 equivalents
without affecting the receptor function. In addition, a large portion
of the GB1 i2 loop could be replaced with a random coil peptide without
any functional consequences. Thus, GB2 intracellular segments are
solely responsible for specific coupling of GABA
B
receptors
to their physiologic effectors, G
i
and G protein-activated
K
+
channels. These findings strongly support a model in
which a single GPCR monomer is sufficient for all of the specific G
protein contacts.
A bacterial artificial chromosome-based framework contig map of human chromosome 22q.
Authors: Kim U J , Shizuya H , Kang H L , Choi S S , Garrett C L , Smink L J , Birren B W , Korenberg J R , Dunham I , Simon M I
Published: July 2002

We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.
Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingested bacteria.
Authors: Evans T J , Buttery L D , Carpenter A , Springall D R , Polak J M , Cohen J
Published: July 2002

Although the production of NO within rodent phagocytes is well-characterized, its production and function within human phagocytes are less clear. We show here that neutrophils within human buffy coat preparations stimulated with a mixture of interleukin 1, tumor necrosis factor alpha, and interferon gamma contain inducible NO synthase mRNA and protein, one of the enzymes responsible for NO production. The protein colocalizes with myeloperoxidase within neutrophil primary granules. Using an inhibitor of NO synthase, L-N-monomethyl arginine, we show that activity of this enzyme is required for the formation of nitrotyrosine around phagocytosed bacteria, most likely through the intermediate production of peroxynitrite, a reaction product of NO and superoxide anions.
Game theory and reciprocity in some extensive form experimental games
Authors: McCabe Kevin A. , Rassenti Stephen J. , Smith Vernon L.
Published: July 2002

We examine decision making in two-person extensive form game trees
using nine treatments that vary matching protocol, payoffs, and payoff
information. Our objective is to establish replicable principles of
cooperative versus noncooperative behavior that involve the use of
signaling, reciprocity, and backward induction strategies, depending on
the availability of dominated direct punishing strategies and the
probability of repeated interaction with the same partner. Contrary to
the predictions of game theory, we find substantial support for
cooperation under complete information even in various single-play
treatments.
Protein-RNA interactions in the active center of transcription elongation complex.
Authors: Markovtsov V , Mustaev A , Goldfarb A
Published: July 2002

By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex. Two of these sites are in the beta subunit and one is in the beta' subunit. During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript. It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent. Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment.
Distinct roles of C2A and C2B domains of synaptotagmin in the regulation of exocytosis in adrenal chromaffin cells
Authors: Ohara-Imaizumi Mica , Fukuda Mitsunori , Niinobe Michio , Misonou Hiroaki , Ikeda Ken , Murakami Takeshi , Kawasaki Masakazu , Mikoshiba Katsuhiko , Kumakura Konosuke
Published: July 2002

Synaptotagmin that contains two repeats of C2 regulatory domains is considered to be involved in neurotransmitter release. To reveal the roles of synaptotagmin in the regulation of exocytosis, we examined the effects of antibodies against C2A and C2B domains on Ca
2+
-evoked catecholamine (CA) release from digitonin-permeabilized adrenal chromaffin cells, resolving the Ca
2+
-evoked release into ATP-dependent priming and ATP-independent Ca
2+
-triggered steps. Anti-C2A antibody clearly reduced the ATP-independent release, suggesting that the C2A domain directly facilitate or promote Ca
2+
-triggered step, vesicular fusion. In contrast, anti-C2B antibody did not affect Ca
2+
-evoked release by itself, but significantly increased the spontaneous Ca
2+
-independent release. In addition, inositol high-polyphosphate series (IHPS) that bind the C2B domain inhibited both the ATP-independent Ca
2+
-evoked release and the spontaneous release in a dose-dependent manner. The inhibition by IHPS was totally reversed by anti-C2B antibody and significantly reversed by high concentration of Ca
2+
. These results suggest that IHPS binding to C2B domain arrests membrane fusion by presumably preventing interaction of synaptotagmin with phospholipids or with proteins of plasma membrane. Thus, IHPS binding to the C2B domain might keep the docked or primed vesicles away from spontaneous fusion at resting level of intracellular Ca
2+
. Binding of the increased intracellular Ca
2+
to the C2A domain may facilitate or trigger the vesicular fusion by releasing this suppression by IHPS.
Fluorescence
in situ
hybridization analysis of keratinocyte growth factor gene amplification and dispersion in evolution of great apes and humans
Authors: Zimonjic D. B. , Kelley M. J. , Rubin J. S. , Aaronson S. A. , Popescu N. C.
Published: July 2002

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence
in situ
hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome 15q15–21.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were localized to dispersed sites on chromosome 2q21, 9p11, 9q12–13, 18p11, 18q11, 21q11, and 21q21.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes 15 and 21. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.
APMLRARα transgene initiates murine acute promyelocytic leukemia
Authors: Brown Diane , Kogan Scott , Lagasse Eric , Weissman Irving , Alcalay Myriam , Pelicci Pier Giuseppe , Atwater Susan , Bishop J. Michael
Published: July 2002

The malignant cells of acute promyelocytic leukemia (APL) contain a reciprocal chromosomal translocation that fuses the promyelocytic leukemia gene (PML) with the retinoic acid receptor α gene (RARα). To test the hypothesis that the chimeraPMLRARα plays a role in leukemogenesis, we expressed aPMLRARα cDNA in myeloid cells of transgenic mice.PMLRARα transgenic mice exhibited impaired neutrophil maturation early in life, which progressed at a low frequency over the course of several months to overt APL. Both the preleukemic state and the leukemia could be transplanted to nontransgenic mice, and the transplanted preleukemia could progress to APL. The APL recapitulated features of the human disease, including a response to retinoic acid. Retinoic acid caused the leukemic cells to differentiatein vitroandin vivo, eliciting remissions of both the preleukemic state and APL in mice. Our results demonstrate thatPMLRARα impairs neutrophil differentiation and initiates the development of APL. The transgenic mice described here provide an apparently accurate model for human APL that includes clear evidence of tumor progression. The model should be useful for exploring the molecular pathogenesis of APL and the mechanisms of the therapeutic response to retinoic acid, as well as for preclinical studies of therapeutic regimens.
A neuromodulatory role of interleukin-1β in the hippocampus
Authors: Schneider H. , Pitossi F. , Balschun D. , Wagner A. , del Rey A. , Besedovsky H. O.
Published: July 2002

It is widely accepted that interleukin-1β (IL-1β), a cytokine produced not only by immune cells but also by glial cells and certain neurons influences brain functions during infectious and inflammatory processes. It is still unclear, however, whether IL-1 production is triggered under nonpathological conditions during activation of a discrete neuronal population and whether this production has functional implications. Here, we showin vivoandin vitrothat IL-1β gene expression is substantially increased during long-term potentiation of synaptic transmission, a process considered to underlie certain forms of learning and memory. The increase in gene expression was long lasting, specific to potentiation, and could be prevented by blockade of potentiation with theN-methyl-d-aspartate (NMDA) receptor antagonist, (±)-2-amino-5-phosphonopentanoic acid (AP-5). Furthermore, blockade of IL-1 receptors by the specific interleukin-1 receptor antagonist (IL-1ra) resulted in a reversible impairment of long-term potentiation maintenance without affecting its induction. These results show for the first time that the production of biologically significant amounts of IL-1β in the brain can be induced by a sustained increase in the activity of a discrete population of neurons and suggest a physiological involvement of this cytokine in synaptic plasticity.
A general mechanism for regulation of access to the translocon: Competition for a membrane attachment site on ribosomes
Authors: Möller Ines , Jung Martin , Beatrix Birgitta , Levy Robert , Kreibich Gert , Zimmermann Richard , Wiedmann Martin , Lauring Brett
Published: July 2002

For proteins to enter the secretory pathway, the membrane attachment site (M-site) on ribosomes must bind cotranslationally to the Sec61 complex present in the endoplasmic reticulum membrane. The signal recognition particle (SRP) and its receptor (SR) are required for targeting, and the nascent polypeptide associated complex (NAC) prevents inappropriate targeting of nonsecretory nascent chains. In the absence of NAC, any ribosome, regardless of the polypeptide being synthesized, binds to the endoplasmic reticulum membrane, and even nonsecretory proteins are translocated across the endoplasmic reticulum membrane. By occupying the M-site, NAC prevents all ribosome binding unless a signal peptide and SRP are present. The mechanism by which SRP overcomes the NAC block is unknown. We show that signal peptide-bound SRP occupies the M-site and therefore keeps it free of NAC. To expose the M-site and permit ribosome binding, SR can pull SRP away from the M-site without prior release of SRP from the signal peptide.
Graded transcriptional response to different concentrations of a single transactivator
Authors: Kringstein Andrew M. , Rossi Fabio M. V. , Hofmann Andreas , Blau Helen M.
Published: July 2002

Threshold mechanisms of transcriptional activation are thought to be critical for translating continuous gradients of extracellular signals into discrete all-or-none cellular responses, such as mitogenesis and differentiation. Indeed, unequivocal evidence for a graded transcriptional response in which the concentration of inducer directly correlates with the level of gene expression in individual eukaryotic cells is lacking. By using a novel binary tetracycline regulatable retroviral vector system, we observed a graded rather than a threshold mechanism of transcriptional activation in two different model systems. When polyclonal populations of cells were analyzed at the single cell level, a dose-dependent, stepwise increase in expression of the reporter gene, green fluorescent protein (GFP), was observed by fluorescence-activated cell sorting. These data provide evidence that, in addition to the generally observed all-or-none switch, the basal transcription machinery also can respond proportionally to changes in concentration of extracellular inducers and trancriptional activators.
Optimal modulation of a Brownian ratchet and enhanced sensitivity to a weak external force
Authors: Tarlie Martin B. , Astumian R. Dean
Published: July 2002

We studied the dynamics of a Brownian particle moving in a spatially anisotropic potential acted on by multiplicative temporal modulations so that
V
(
x
,
t
) =
g
(
t
)
U
(
x
). Using the concept of the “thermodynamic action,” we show that the class of modulation that maximizes the flow is a square-wave in time. We also show that adding a weak, homogenous force
F
in synergy with the square-wave modulation can cause particles of slightly different size to move in opposite directions. The synergetic change in velocity caused by
F
can be much greater than the drift velocity that would be caused by
F
alone.
Adhesion to fibronectin stimulates proliferation of wild-type and
bcr/abl
-transfected murine hematopoietic cells
Authors: Krämer Alwin , Hörner Susanne , Willer Andreas , Fruehauf Stefan , Hochhaus Andreas , Hallek Michael , Hehlmann Rüdiger
Published: July 2002

Cells of most tissues require adhesion to a surface to grow. However, for hematopoietic cells, both stimulation and inhibition of proliferation by adhesion to extracellular matrix components have been described. Furthermore, it has been suggested that progenitor cells from chronic myelogenous leukemia show decreased β
1
integrin-mediated adhesion to fibronectin, resulting in increased proliferation and abnormal trafficking. However, we show here that the chronic myelogenous leukemia-specific fusion protein p210bcr/abl stimulates the expression of α
5
β
1
integrins and induces adhesion to fibronectin when expressed in the myeloid cell line 32D. Moreover, proliferation of both p210bcr/abl-transfected 32D (32Dp210) cells and untransfected 32D cells is stimulated by immobilized fibronectin. Cell cycle analysis revealed that nonadherent 32D and 32Dp210 cells are arrested in late G
1
or early S phase, whereas the adherent fractions continue cycling. Although both adherent and nonadherent p210bcr/abl-transfected and parental 32D cells express equal amounts of cyclin A, a protein necessary for cell cycle progression at the G
1
/S boundary, cyclin A complexes immunoprecipitated from 32D cells cultured on immobilized fibronectin were found to be catalytically inactive in nonadherent but not in adherent cells. In addition, as compared with untransfected 32D cells, cyclin A immunoprecipitates from 32Dp210 cells exhibited a greatly elevated kinase activity and remained partially active irrespective of the adhesion status. The lack of cyclin A/cyclin-dependent kinase (CDK) 2 activity in nonadherent 32D cells appeared to result from increased expression and cyclin A complex formation of the CDK inhibitor p27
Kip1
. Taken together, our results indicate that adhesion stimulates cell cycle progression of hematopoietic cells by down-regulation of p27
Kip1
, resulting in activation of cyclin A/CDK2 complexes and subsequent transition through the G
1
/S adhesion checkpoint.
Signal recognition particle components in the nucleolus
Authors: Politz Joan C. , Yarovoi Serge , Kilroy Susan M. , Gowda Krishne , Zwieb Christian , Pederson Thoru
Published: July 2002

The signal recognition particle (SRP) is a ribonucleoprotein
composed of an
Alu
domain and an S domain. The S domain
contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54,
SRP68, and SRP72. SRP interacts with ribosomes to bring translating
membrane and secreted proteins to the endoplasmic reticulum (ER) for
proper processing. Additionally, SRP RNA is a member of a family of
small nonribosomal RNAs found recently in the nucleolus, suggesting
that the nucleolus is more plurifunctional than previously realized. It
was therefore of interest to determine whether other SRP components
localize to this intranuclear site. In transfected rat fibroblasts,
green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized
to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also
accumulated in the ER, consistent with its affinity for the ER-bound
SRP receptor. SRP54 was detected in the cytoplasm as a green
fluorescent protein fusion and in immunofluorescence studies, but was
not detected in the nucleolus.
In situ
hybridization
experiments also revealed endogenous SRP RNA in the
nucleolus. These results demonstrate that SRP RNA and three SRP
proteins visit the nucleolus, suggesting that partial SRP assembly, or
another unidentified activity of the SRP components, occurs at the
nucleolus. SRP54 apparently interacts with nascent SRP beyond the
nucleolus, consistent with
in vitro
reconstitution
experiments showing that SRP19 must bind to SRP RNA before SRP54 binds.
Our findings support the notion that the nucleolus is the site of
assembly and/or interaction between the family of ribonucleoproteins
involved in protein synthesis, in addition to ribosomes themselves.
Redox regulation of chemokine receptor expression
Authors: Saccani Alessandra , Saccani Simona , Orlando Simone , Sironi Marina , Bernasconi Sergio , Ghezzi Pietro , Mantovani Alberto , Sica Antonio
Published: July 2002

Cytokines and reactive oxygen intermediates (ROI) are frequent companions at sites of acute inflammation. We have shown previously that in human monocytes, bacterial lipopolysaccharide, IL-1, and tumor necrosis factor-α induce a rapid down-regulation of the monocyte chemotactic protein-1 receptor CCR2 (CC chemokine receptor-2). These stimuli also induce production of ROI. In this paper, we investigate the influence of antioxidants and/or ROI on chemokine-receptor expression. In human monocytes, the antioxidant pyrrolidine dithiocarbamate (PDTC) rapidly inhibited CCR2 (95–100% of inhibition) and CCR5 (77–100% of inhibition) mRNA expression by strongly decreasing transcript stability. CCR2 half-life was decreased from 1.5 h to 45 min; CCR5 half-life was decreased from 2 h to 70 min. This inhibitory activity also included CXCR4 (CXC chemokine receptor-4) but not CXCR2 receptor and, although to a lesser extent, was shared by the antioxidantsN-acetyl-l-cysteine and 2-mercaptoethanol. In contrast, the ROI-generating system xanthine/xanthine oxidase increased CCR5 and CXCR4 mRNA expression and counteracted the inhibitory effect of PDTC. Accordingly, H2O2and the glutathione-depleting drug buthionine sulfoximine increased to different extents CCR2, CCR5, and CXCR4 mRNA expression. The PDTC-mediated inhibition of CCR5 and CXCR4 mRNA expression was associated with decreased chemotactic responsiveness (>90% inhibition) and with a marked inhibition of surface-receptor expression. In contrast, xanthine/xanthine oxidase opposed the bacterial lipopolysaccharide- and tumor necrosis factor-α-mediated inhibition of CCR5 and CXCR4 mRNA expression and increased both the CCR5 surface expression and the cell migration (3-fold) in response to macrophage inflammatory protein-1β. These results suggest that the redox status of cells is a crucial determinant in the regulation of the chemokine system.
A mutant herpes simplex virus type 1 thymidine kinase reporter gene shows improved sensitivity for imaging reporter gene expression with positron emission tomography
Authors: Gambhir Sanjiv S. , Bauer Eileen , Black Margaret E. , Liang Qianwa , Kokoris Mark S. , Barrio Jorge R. , Iyer Meera , Namavari Mohammad , Phelps Michael E. , Herschman Harvey R.
Published: July 2002

We are developing assays for noninvasive, quantitative imaging of reporter genes with positron emission tomography (PET), for application both in animal models and in human gene therapy. We report here a method to improve the detection of lower levels of PET reporter gene expression by utilizing a
mutant
herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) as a PET reporter gene. The HSV1-sr39tk mutant was identified from a library of site-directed mutants. Accumulation (net uptake) of the radioactively labeled substrates [8-
3
H]penciclovir ([8-
3
H]PCV), and 8-[
18
F]fluoropenciclovir (FPCV) in C6 rat glioma cells expressing HSV1-sr39tk is increased by a factor of ≈2.0 when compared with C6 cells expressing wild-type HSV1-tk. The increased imaging sensitivity of HSV1-sr39tk when FPCV is used is also demonstrated
in vivo
both with tumor cells stably transfected with either HSV1-tk or HSV1-sr39tk, and after hepatic delivery of HSV1-tk or HSV1-sr39tk by using adenoviral vectors. The use of HSV1-sr39tk as a PET reporter gene and FPCV as a PET reporter probe results in significantly enhanced sensitivity for imaging reporter gene expression
in vivo
.
Secretory leukocyte protease inhibitor in punch biopsies from human colonic mucosa
Authors: Nyström Max , Westin Ulla Peterson , Linder Carina , Ohlsson Kjell
Published: July 2002

Secretory leukocyte protease inhibitor (SLPI) is a wellknown protease inhibitor. Its function is thought to be protease/protease‐inhibitor balance. Free proteolytic activity, mainly pancreatic elastase, anionic trypsin and granulocytic elastase, has been demonstrated in faecal extracts from patients with ulcerative colitis. We wanted to verify that SLPI is actually secreted from normal human colonic mucosa. Also, we wanted to ascertain whether studies of SLPI secretion based on punch biopsies were dependent on biopsy area or on biopsy circumference. Normal colonic mucosa was sampled during surgery for colonic cancer. A total of 36 samples from four patients were used. Mucosa preparation was carried out using a punch biopsy technique, and samples of 3, 4 and 6 mm diameter were used. All media contained SLPI at varying concentrations. When expressed in terms of the sample area, the secretion per millimetre‐squared seemed to decrease with increasing area. When calculated as secretion per circumference, secretion seemed to be constant. In conclusion, SLPI was secreted from normal human colonic mucosa. The SLPI secretion seemed dependent on the circumference of the biopsy rather
than on the area of the biopsy.
The Mechanisms of Action of Interleukin‐1 on Rabbit Intestinal Epithelial Cells
Authors: Homaidan Fadia R. , Zhao Liming , Chakroun Iman , Martin Carla A. , Burakoff Robert
Published: July 2002

Interleukin‐1 (IL‐1) is an inflammatory mediator that increases Cl- secretion in intestinal epithelial cells. To identify the signal transduction pathway(s) involved in IL‐1′s action, cells were treated with IL‐1 and the levels of cyclooxygenase (COX) enzymes, prostaglandin E2 (PGE2) and phospholipase A2‐activating protein (PLAP), and the activity of phospholipase A2 (PLA2) were measured. IL‐1 caused concentrationand time‐dependent increases in the levels of PLA2 activity, and/or in the levels of PLAP, COX‐2 and PGE2. The IL‐induced increase in PGE2 levels was biphasic, with the first peak due to the increase in PLAP levels, and the second peak due to the increase in COX‐2 levels. This increase in PGE2 levels may provide a mechanism for acute and chronic inflammation in the intestine.
Incorporation and Effect of Arachidonic Acid on the Growth of Human Myeloma Cell Lines
Authors: Desplat V. , Dulery C. , Praloran V. , Denizot Y.
Published: July 2002

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2‐treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling‐chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12‐ and 15‐hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.
Twenty-Eight-Day Toxicity Study of Melatonin Infused Subcutaneously in Rats Via an Osmotic Pump
Authors: Prevo M. , Johnson P. , Chester A.
Published: July 2002

This 28-day study in rats evaluated the safety of melatonin delivered continuously subcutaneously by an ALZET Osmotic Pump. ALZET Osmotic Pumps 2ML4 continuously delivered 60 μ1/day of vehicle (polyethylene glycol 400), 0.03%, 0.3%, or 3% melatonin subcutaneously for 28 days to Sprague-Dawley rats (19/sex/group). The dose of melatonin delivered based on weekly group mean body weights (n = 10) was approximately 0.050,0.50, and 4.8 mg/kg day for the male groups and 0.074, 0.75, and 7.3 mg/kg day for the female groups. An additional group (19/sex) underwent surgery, but no osmotic pumps were implanted (sham control). No deaths or changes in clinical observations occurred that were attributed to melatonin. No drug effect occurred in body weights, hematology, clinical chemistry, urinalyses, or gross pathology. A dose-related trend of increasing serum melatonin concentrations occurred in males and females. In males, there was a trend toward decreasing serum prolactin concentrations with time at all levels of melatonin treatment. No difference in serum follicle-stimulating hormone (FSH) concentrations occurred between treated groups. Most of the samples were at the limit of detection for the serum luteinizing hormone (LH) assay (0.157 ng/ml). A dose-related increase occurred in urine 6-sulphatoxymelatonin (the primary metabolite) concentrations in melatonin-treated male and female groups. No treatment-related organ weight or histopathology changes were present in rats infused with 0.03% or 0.3% melatonin. Two of 10 males administered 3.0% melatonin had decreased testes weights and testicular degenerative changes composed of reduced or absent spermatogenesis, spermatidic giant cells, and edema. The cause of the observed testicular degenerative changes in the high-dose group and possible reversibility should be investigated in follow-up studies of longer duration.
Overview of the Fourth International Conference on Harmonization
Authors: Christian Mildred S.
Published: July 2002

This overview of the Fourth International Conference on Harmonization (ICH 4), held in Brussels, Belgium, 16-18 July 1997, summarizes results and provides information regarding the nonclinical workshop and issued nonclinical testing guidelines [Draft Final (Step 4) Guidelines recommended for adoption to the regulatory bodies of the EU, Japan, and the USA]. ICH 4 completed the initial harmonization targets for the three ICH Regions [European Commission-European Union (EU) and European Federation of Pharmaceutical Industries' Association (EFPIA); Ministry of Health and Welfare (MHW) Japan and Japan Pharmaceutical Manufacturers Association (JPMA); and the U.S. Food and Drug Administration (FDA) and Pharmaceutical Research and Manufacturers of America (PhRMA)] and the International Federation of Pharmaceutical Manufacturers Associations (IFPMA). Proceedings will be available in book form. Earlier conferences, guidelines, and proceedings are available on CD-ROM and in print and diskette form from the ICH Secretariat, c/o IFPMA, 3 rue de St.-Jean, P.O. Box 9, 1211 Geneva 18, Switzerland; Tel +41 (22) 340 12 00; Fax +41 (22) 345 82 75. Similar information is obtainable from the ICH Home Page (http://www.ifpma.org/ich1.html) and the Pharm Web home page (http://www.pharmweb.net).
Regulatory Perspective on in Vitro Assays as Predictors of Phototoxicity and Photo Co-Carcinogenicity
Authors: Ellis Amy L.
Published: July 2002

Drugs from a variety of chemical classes used for a wide range of therapeutic indications can be photosensitizers in humans. Several drugs are phototoxic in animal models as well; there are no nonclinical data for many. In vitro tests have been developed as predictors of phototoxicity and although they have been used as screens, none have replaced the in vivo tests done in rodents (usually mice or guinea pigs) since these have been good predictors of clinical phototoxicity. Some phototoxic drug classes are co-carcinogens with ultraviolet radiation (UVA and/or UVB) in hairless mice, specifically psoralens, retinoids, and fluo-roquinolones. Treatment with 8-methoxypsoralen and ultraviolet A radiation for psoriasis is also carcinogenic in humans. It has been suggested that in vitro photogenotoxicity assays using microorganisms or mammalian cells may be predictive of photo co-carcinogenicity. Some attractions of these in vitro assays, compared to the hairless mouse photo co-carcinogenicity assay, are their generally shorter duration and lower cost as well as reducing the number of animals used in research. Currently, personnel at the Food and Drug Administration (FDA) are examining the available data on phototoxicity, photogenotoxicity, and photo co-carcinogenicity to determine how this information can best be used toregulate and label drug products, and considering which assays should be recommended under specific circumstances.