bluuebunny/crossref_metadata_2025 huggingface dataset:
Dendritic cell modulation by 1α,25 dihydroxyvitamin D3and its analogs: A vitamin D receptor-dependent pathway that promotes a persistent state of immaturityin vitroandin vivo
Authors: Griffin Matthew D. , Lutz Ward , Phan Vy A. , Bachman Lori A. , McKean David J. , Kumar Rajiv
Published: July 2002

Dendritic cells (DCs) play a central role in regulating immune activation and responses to self. DC maturation is central to the outcome of antigen presentation to T cells. Maturation of DCs is inhibited by physiological levels of 1α,25 dihydroxyvitamin D3[1α,25(OH)2D3] and a related analog, 1α,25(OH)2-16-ene-23-yne-26,27-hexafluoro-19-nor-vitamin D3(D3analog). Conditioning of bone marrow cultures with 10−10M D3analog resulted in accumulation of immature DCs with reduced IL-12 secretion and without induction of transforming growth factor β1. These DCs retained an immature phenotype after withdrawal of D3analog and exhibited blunted responses to maturing stimuli (CD40 ligation, macrophage products, or lipopolysaccharide). Resistance to maturation depended on the presence of the 1α,25(OH)2D3receptor (VDR). In anin vivomodel of DC-mediated antigen-specific sensitization, D3analog-conditioned DCs failed to sensitize and, instead, promoted prolonged survival of subsequent skin grafts expressing the same antigen. To investigate the physiologic significance of 1α,25(OH)2D3/VDR-mediated modulation of DC maturity we analyzed DC populations from mice lacking VDR. Compared with wild-type animals, VDR-deficient mice had hypertrophy of subcutaneous lymph nodes and an increase in mature DCs in lymph nodes but not spleen. We conclude that 1α,25(OH)2D3/VDR mediates physiologically relevant inhibition of DC maturity that is resistant to maturational stimuli and modulates antigen-specific immune responsesin vivo.
Parallel detection of violations of color constancy
Authors: Foster David H. , Nascimento Sérgio M. C. , Amano Kinjiro , Arend Larry , Linnell Karina J. , Nieves Juan Luis , Plet Sabrina , Foster Jeffrey S.
Published: July 2002

The perceived colors of reflecting surfaces generally remain stable despite changes in the spectrum of the illuminating light. This color constancy can be measured operationally by asking observers to distinguish illuminant changes on a scene from changes in the reflecting properties of the surfaces comprising it. It is shown here that during fast illuminant changes, simultaneous changes in spectral reflectance of one or more surfaces in an array of other surfaces can be readily detected almost independent of the numbers of surfaces, suggesting a preattentive, spatially parallel process. This process, which is perfect over a spatial window delimited by the anatomical fovea, may form an early input to a multistage analysis of surface color, providing the visual system with information about a rapidly changing world in advance of the generation of a more elaborate and stable perceptual representation.
Statistical significance of protein structure prediction by threading
Authors: Mirny Leonid A. , Finkelstein Alexey V. , Shakhnovich Eugene I.
Published: July 2002

In this study, we estimate the statistical significance of structure prediction by threading. We introduce a single parameter ɛ that serves as a universal measure determining the probability that the best alignment is indeed a native-like analog. Parameter ɛ takes into account both length and composition of the query sequence and the number of decoys in threading simulation. It can be computed directly from the query sequence and potential of interactions, eliminating the need for sequence reshuffling and realignment. Although our theoretical analysis is general, here we compare its predictions with the results of gapless threading. Finally we estimate the number of decoys from which the native structure can be found by existing potentials of interactions. We discuss how this analysis can be extended to determine the optimal gap penalties for any sequence-structure alignment (threading) method, thus optimizing it to maximum possible performance.
Presenilin-dependent γ-secretase activity modulates thymocyte development
Authors: Doerfler Petra , Shearman Mark S. , Perlmutter Roger M.
Published: July 2002

In neuronal cells, presenilin-dependent γ-secretase activity cleaves amyloid precursor proteins to release Aβ peptides, and also catalyzes the release of the intracellular domain of the transmembrane receptor Notch. Accumulation of aberrant Aβ peptides appears to be causally related to Alzheimer's disease. Inhibition of Aβ peptide production is therefore a potential target for therapeutic intervention. Notch proteins play an important role in cell fate determination in many different organisms and at different stages of development, for example in mammalian T cell development. We therefore addressed whether structurally diverse γ-secretase inhibitors impair Notch function by studying thymocyte development in murine fetal thymic organ cultures. Here we show that high concentrations of the most potent inhibitors blocked thymocyte development at the most immature stage. In contrast, lower concentrations or less potent inhibitors impaired differentiation at a later stage, most notably suppressing the development of CD8 single-positive T cells. These phenotypes are consistent with an impairment of Notch signaling by γ-secretase inhibitors and define a strict Notch dose dependence of consecutive stages during thymocyte development.
Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells
Authors: Ting Alice Y. , Kain Kristin H. , Klemke Richard L. , Tsien Roger Y.
Published: July 2002

The complexity and specificity of many forms of signal
transduction are widely believed to require spatial compartmentation of
protein kinase and phosphatase activities, yet existing methods for
measuring kinase activities in cells lack generality or spatial or
temporal resolution. We present three genetically encoded fluorescent
reporters for the tyrosine kinases Src, Abl, and epidermal growth
factor (EGF) receptor. The reporters consist of fusions of cyan
fluorescent protein (CFP), a phosphotyrosine binding domain, a
consensus substrate for the relevant kinase, and yellow fluorescent
protein (YFP). Stimulation of kinase activities in living cells with
addition of growth factors causes 20–35% changes in the ratios of
yellow to cyan emissions because of phosphorylation-induced changes in
fluorescence resonance energy transfer (FRET). Platelet-derived growth
factor (PDGF) stimulated Abl activity most strongly in actin-rich
membrane ruffles, supporting the importance of this tyrosine kinase in
the regulation of cell morphology. These results establish a
general strategy for nondestructively imaging dynamic protein tyrosine
kinase activities with high spatial and temporal resolution in single
living cells.
Multiclass cancer diagnosis using tumor gene expression signatures
Authors: Ramaswamy Sridhar , Tamayo Pablo , Rifkin Ryan , Mukherjee Sayan , Yeang Chen-Hsiang , Angelo Michael , Ladd Christine , Reich Michael , Latulippe Eva , Mesirov Jill P. , Poggio Tomaso , Gerald William , Loda Massimo , Lander Eric S. , Golub Todd R.
Published: July 2002

The optimal treatment of patients with cancer depends on
establishing accurate diagnoses by using a complex combination of
clinical and histopathological data. In some instances, this task is
difficult or impossible because of atypical clinical presentation or
histopathology. To determine whether the diagnosis of multiple common
adult malignancies could be achieved purely by molecular
classification, we subjected 218 tumor samples, spanning 14
common tumor types, and 90 normal tissue samples to oligonucleotide
microarray gene expression analysis. The expression levels of 16,063
genes and expressed sequence tags were used to evaluate the accuracy of
a multiclass classifier based on a support vector machine algorithm.
Overall classification accuracy was 78%, far exceeding the accuracy of
random classification (9%). Poorly differentiated cancers resulted in
low-confidence predictions and could not be accurately classified
according to their tissue of origin, indicating that they are
molecularly distinct entities with dramatically different gene
expression patterns compared with their well differentiated
counterparts. Taken together, these results demonstrate the feasibility
of accurate, multiclass molecular cancer classification and suggest a
strategy for future clinical implementation of molecular cancer
diagnostics.
Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection
Authors: Kanai Anthony J. , Pearce Linda L. , Clemens Paula R. , Birder Lori A. , VanBibber Michelle M. , Choi So-Young , de Groat William C. , Peterson Jim
Published: July 2002

Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca
2+
-dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophin-deficient (
mdx
) mice.
mdx
cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wild-type cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome
c
oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.
Cytochrome
c
release from mitochondria proceeds by a two-step process
Authors: Ott Martin , Robertson John D. , Gogvadze Vladimir , Zhivotovsky Boris , Orrenius Sten
Published: July 2002

Cytochrome
c
is often released from mitochondria during the early stages of apoptosis, although the precise mechanisms regulating this event remain unclear. In this study, with isolated liver mitochondria, we demonstrate that cytochrome
c
release requires a two-step process. Because cytochrome
c
is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin, this interaction must first be disrupted to generate a soluble pool of this protein. Specifically, solubilization of cytochrome
c
involves a breaching of the electrostatic and/or hydrophobic affiliations that this protein usually maintains with cardiolipin. Once cytochrome
c
is solubilized, permeabilization of the outer mitochondrial membrane by Bax is sufficient to allow the extrusion of this protein into the extramitochondrial environment. Neither disrupting the interaction of cytochrome
c
with cardiolipin, nor permeabilizing the outer membrane with Bax, alone, is sufficient to trigger this protein's release. This mechanism also extends to conditions of mitochondrial permeability transition insofar as cytochrome
c
release is significantly depressed when the electrostatic interaction between cytochrome
c
and cardiolipin remains intact. Our results indicate that the release of cytochrome
c
involves a distinct two-step process that is undermined when either step is compromised.
Abolition of male sexual behaviors in mice lacking estrogen receptors α and β (αβERKO)
Authors: Ogawa Sonoko , Chester April E. , Hewitt Sylvia Curtis , Walker Vickie R. , Gustafsson Jan-Åke , Smithies Oliver , Korach Kenneth S. , Pfaff Donald W.
Published: July 2002

Male mice with a knockout of the estrogen receptor (ER)-α gene, a
ligand-activated transcription factor, showed reduced levels of
intromissions and no ejaculations whereas simple mounting behavior was
not affected. In contrast, all components of sexual behaviors were
intact in male mice lacking the novel ER-β gene. Here we measure the
extent of phenotype in mice that lack both ER-α and ER-β genes
(αβERKO). αβERKO male mice did not show any components of
sexual behaviors, including simple mounting behavior. Nor did they show
ultrasonic vocalizations during behavioral tests with receptive female
mice. On the other hand, reduced aggressive behaviors of αβERKO
mice mimicked those of single knockout mice of ER-α gene (αERKO).
They showed reduced levels of lunge and bite aggression, but rarely
showed offensive attacks. Thus, either one of the ERs is sufficient for
the expression of simple mounting in male mice, indicating a redundancy
in function. Offensive attacks, on the other hand, depend specifically
on the ER-α gene. Different patterns of natural behaviors require
different patterns of functions by ER genes.
Cryoelectron microscopy of λ phage DNA condensates in vitreous ice: The fine structure of DNA toroids
Authors: Hud Nicholas V. , Downing Kenneth H.
Published: July 2002

DNA toroids produced by the condensation of λ phage DNA with
hexammine cobalt (III) have been investigated by cryoelectron
microscopy. Image resolution obtained by this technique has
allowed unprecedented views of DNA packing within toroidal condensates.
Toroids oriented coplanar with the microscope image plane exhibit
circular fringes with a repeat spacing of 2.4 nm. For some toroids
these fringes are observed around almost the entire circumference of
the toroid. However, for most toroids well-defined fringes are limited
to less than one-third of the total toroid circumference. Some toroids
oriented perpendicular to the image plane reveal DNA polymers organized
in a hexagonal close-packed lattice; however, for other toroids
alternative packing arrangements are observed. To aid interpretation of
electron micrographs, three-dimensional model toroids were
generated with perfect hexagonal DNA packing throughout, as well as
more physically realistic models that contain crossover points between
DNA loops. Simulated transmission electron microscopy images of
these model toroids in different orientations faithfully reproduce most
features observed in cryoelectron micrographs of actual toroids.
Neural plasticity in adults with amblyopia.
Authors: Levi D M , Polat U
Published: July 2002

Amblyopia is a neuronal abnormality of vision that is often considered irreversible in adults. We found strong and significant improvement of Vernier acuity in human adults with naturally occurring amblyopia following practice. Learning was strongest at the trained orientation and did not transfer to an untrained task (detection), but it did transfer partially to the untrained eye (primarily at the trained orientation). We conclude that this perceptual learning reflects alterations in early neural processes that are localized beyond the site of convergence of the two eyes. Our results suggest a significant degree of plasticity in the visual system of adults with amblyopia.
Genetic separation of tumor growth and hemorrhagic phenotypes in an estrogen-induced tumor.
Authors: Wendell D L , Herman A , Gorski J
Published: July 2002

Chronic administration of estrogen to the Fischer 344 (F344) rat induces growth of large, hemorrhagic pituitary tumors. Ten weeks of diethylstilbestrol (DES) treatment caused female F344 rat pituitaries to grow to an average of 109.2 +/- 6.3 mg (mean +/- SE) versus 11.3 +/- 1.4 mg for untreated rats, and to become highly hemorrhagic. The same DES treatment produced no significant growth (8.9 +/- 0.5 mg for treated females versus 8.7 +/- 1.1 for untreated females) or morphological changes in Brown Norway (BN) rat pituitaries. An F1 hybrid of F344 and BN exhibited significant pituitary growth after 10 weeks of DES treatment with an average mass of 26.3 +/- 0.7 mg compared with 8.6 +/- 0.9 mg for untreated rats. Surprisingly, the F1 hybrid tumors were not hemorrhagic and had hemoglobin content and outward appearance identical to that of BN. Expression of both growth and morphological changes is due to multiple genes. However, while DES-induced pituitary growth exhibited quantitative, additive inheritance, the hemorrhagic phenotype exhibited recessive, epistatic inheritance. Only 5 of the 160 F2 pituitaries exhibited the hemorrhagic phenotype; 36 of the 160 F2 pituitaries were in the F344 range of mass, but 31 of these were not hemorrhagic, indicating that the hemorrhagic phenotype is not merely a consequence of extensive growth. The hemorrhagic F2 pituitaries were all among the most massive, indicating that some of the genes regulate both phenotypes.
The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.
Authors: Mott H R , Carpenter J W , Zhong S , Ghosh S , Bell R M , Campbell S L
Published: July 2002

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.
Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks.
Authors: Byrne P F , McMullen M D , Snook M E , Musket T A , Theuri J M , Widstrom N W , Wiseman B R , Coe E H
Published: July 2002

Interpretation of quantitative trait locus (QTL) studies of agronomic traits is limited by lack of knowledge of biochemical pathways leading to trait expression. To more fully elucidate the biological significance of detected QTL, we chose a trait that is the product of a well-characterized pathway, namely the concentration of maysin, a C-glycosyl flavone, in silks of maize, Zea mays L. Maysin is a host-plant resistance factor against the corn earworm, Helicoverpa zea (Boddie). We determined silk maysin concentrations and restriction fragment length polymorphism genotypes at flavonoid pathway loci or linked markers for 285 F2 plants derived from the cross of lines GT114 and GT119. Single-factor analysis of variance indicated that the p1 region on chromosome 1 accounted for 58.0% of the phenotypic variance and showed additive gene action. The p1 locus is a transcription activator for portions of the flavonoid pathway. A second QTL, represented by marker umc 105a near the brown pericarp1 locus on chromosome 9, accounted for 10.8% of the variance. Gene action of this region was dominant for low maysin, but was only expressed in the presence of a functional p1 allele. The model explaining the greatest proportion of phenotypic variance (75.9%) included p1, umc105a, umc166b (chromosome 1), r1 (chromosome 10), and two epistatic interaction terms, p1 x umc105a and p1 x r1. Our results provide evidence that regulatory loci have a central role and that there is a complex interplay among different branches of the flavonoid pathway in the expression of this trait.
Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan.
Authors: Pluschke G , Vanek M , Evans A , Dittmar T , Schmid P , Itin P , Filardo E J , Reisfeld R A
Published: July 2002

A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.
Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours.
Authors: Mori T , Binder B , Johnson C H
Published: July 2002

To ascertain whether the circadian oscillator in the prokaryotic cyanobacterium Synechococcus PCC 7942 regulates the timing of cell division in rapidly growing cultures, we measured the rate of cell division, DNA content, cell size, and gene expression (monitored by luminescence of the PpsbAI::luxAB reporter) in cultures that were continuously diluted to maintain an approximately equal cell density. We found that populations dividing at rates as rapid as once per 10 h manifest circadian gating of cell division, since phases in which cell division slows or stops recur with a circadian periodicity. The data clearly show that Synechococcus cells growing with doubling times that are considerably faster than once per 24 h nonetheless express robust circadian rhythms of cell division and gene expression. Apparently Synechococcus cells are able to simultaneously sustain two timing circuits that express significantly different periods.
The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.
Authors: Stearns-Kurosawa D J , Kurosawa S , Mollica J S , Ferrell G L , Esmon C T
Published: July 2002

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.
Mnemonic neuronal activity in somatosensory cortex.
Authors: Zhou Y D , Fuster J M
Published: July 2002

Single-unit activity was recorded from the hand areas of the somatosensory cortex of monkeys trained to perform a haptic delayed matching to sample task with objects of identical dimensions but different surface features. During the memory retention period of the task (delay), many units showed sustained firing frequency change, either excitation or inhibition. In some cases, firing during that period was significantly higher after one sample object than after another. These observations indicate the participation of somatosensory neurons not only in the perception but in the short-term memory of tactile stimuli. Neurons most directly implicated in tactile memory are (i) those with object-selective delay activity, (ii) those with nondifferential delay activity but without activity related to preparation for movement, and (iii) those with delay activity in the haptic-haptic delayed matching task but no such activity in a control visuo-haptic delayed matching task. The results indicate that cells in early stages of cortical somatosensory processing participate in haptic short-term memory.
A few selected applications of surface nonlinear optical spectroscopy.
Authors: Shen Y R
Published: July 2002

As a second-order nonlinear optical process, sum-frequency generation is highly surface-specific and accordingly has been developed into a very powerful and versatile surface spectroscopic tool. It has found many unique applications in different disciplines and thus provided many exciting new research opportunities in surface and surface-related science. Selected examples are discussed here to illustrate the power of the technique.
Absence of fetal liver hematopoiesis in mice deficient in transcriptional coactivator core binding factor beta.
Authors: Sasaki K , Yagi H , Bronson R T , Tominaga K , Matsunashi T , Deguchi K , Tani Y , Kishimoto T , Komori T
Published: July 2002

Core binding factor beta (CBF beta) is considered to be a transcriptional coactivator that dimerizes with transcription factors core binding factor alpha 1 (CBFA1), -2, and -3, and enhances DNA binding capacity of these transcription factors. CBF beta and CBFA2, which is also called acute myeloid leukemia 1 gene, are frequently involved in chromosomal translocations in human leukemia. To elucidate the function of CBF beta, mice carrying a mutation in the Cbfb locus were generated. Homozygous mutant embryos died between embryonic days 11.5-13.5 due to hemorrhage in the central nervous system. Mutant embryos had primitive erythropoiesis in yolk sac but lacked definitive hematopoiesis in fetal liver. In the yolk sac of mutant embryos, no erythroid or myeloid progenitors of definitive hematopoietic origin were detected, and the expression of flk-2/flt-3, the marker gene for early precursor cells of definitive hematopoiesis, was absent. These data suggest that Cbfb is essential for definitive hematopoiesis in liver, especially for the commitment to early hematopoietic precursor cells.
Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells.
Authors: Mohler W A , Blau H M
Published: July 2002

Complementing reporter genes provide biological indicators of coincident expression of proteins in cells. We have adapted intracistronic complementation of the Escherichia coli lacZ gene for use in mammalian cells. Enzymatic activity detectable by quantitative biochemical assay, flow cytometry, or microscopy is produced upon convergent expression of two distinct mutant lacZ peptides within single cells, or upon fusion of cells expressing such mutants. A novel fluorescent substrate for beta-galactosidase (Fluor-X-Gal) increases detection and permits simultaneous microscopic visualization of other fluorescent markers. The enzymatic complementation described here should facilitate studies of cell fusion, cell lineage, and signal transduction, by producing activity only when two proteins are expressed at the same time and place in intact cells.
The effect of overexpression of the protein tyrosine phosphatase PTPMEG on cell growth and on colony formation in soft agar in COS-7 cells
Authors: Gu Minxiang , Meng Kun , Majerus Philip W.
Published: July 2002

We established stable COS-7 cell lines overexpressing recombinant
PTPMEG and an inactive mutant form in which the active site cysteine is
mutated to serine (PTPMEGCS). We found that both endogenous
and recombinant enzyme were primarily located in the membrane and
cytoskeletal fractions of COS-7 cells. Endogenous PTPMEG
accounts for only 1/3000th of the total tyrosine phosphatase activity
in COS-7 cells and transfected cells expressed 2- to 7-fold higher
levels of the enzyme. These levels of overexpression did not result in
detectable changes in either total tyrosine phosphatase activity or the
state of protein tyrosine phosphorylation as determined by
immunoblotting of cell homogenates with anti-phosphotyrosine
antibodies. Despite the low levels of activity for PTPMEG, we found
that overexpressing cells grew slower and reached confluence at a lower
density than vector transfected cells. Surprisingly,
PTPMEGCS-transfected cells also reach confluence at a lower density
than vector-transfected cells, although they grow to higher density
than PTPMEG-transfected cells. Both constructs inhibited the ability of
COS-7 cells to form colonies in soft agar, with the native PTPMEG
having a greater effect (30-fold) than PTPMEGCS (10-fold). These
results indicate that in COS-7 cells both PTPMEG and PTPMEGCS inhibit
cell proliferation, reduce the saturation density, and block the
ability of these cells to grow without adhering to a solid matrix.
Immune response to a differentiation antigen induced by altered antigen: A study of tumor rejection and autoimmunity
Authors: Naftzger Clarissa , Takechi Yoshizumi , Kohda Hironobu , Hara Isao , Vijayasaradhi Setaluri , Houghton Alan N.
Published: July 2002

Recognition of self is emerging as a theme for the immune
recognition of human cancer. One question is whether the immune system
can actively respond to normal tissue autoantigens expressed by cancer
cells. A second but related question is whether immune recognition of
tissue autoantigens can actually induce tumor rejection. To address
these issues, a mouse model was developed to investigate immune
responses to a melanocyte differentiation antigen, tyrosinase-related
protein 1 (or gp75), which is the product of the
brown
locus. In mice, immunization with purified syngeneic gp75 or syngeneic
cells expressing gp75 failed to elicit antibody or cytotoxic T-cell
responses to gp75, even when different immune adjuvants and cytokines
were included. However, immunization with altered sources of gp75
antigen, in the form of either syngeneic gp75 expressed in insect cells
or human gp75, elicited autoantibodies to gp75. Immunized mice rejected
metastatic melanomas and developed patchy depigmentation in their
coats. These studies support a model of tolerance maintained to a
melanocyte differentiation antigen where tolerance can be broken by
presenting sources of altered antigen (e.g., homologous xenogeneic
protein or protein expressed in insect cells). Immune responses induced
with these sources of altered antigen reacted with various processed
forms of native, syngeneic protein and could induce both tumor
rejection and autoimmunity.
Mapping nucleosome position at single base-pair resolution by using site-directed hydroxyl radicals.
Authors: Flaus A , Luger K , Tan S , Richmond T J
Published: July 2002

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.
Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences.
Authors: Herb A , Higuchi M , Sprengel R , Seeburg P H
Published: July 2002

RNA editing by adenosine deamination in brain-expressed pre-mRNAs for glutamate receptor (GluR) subunits alters gene-specified codons for functionally critical positions, such as the channel's Q/R site. We show by transcript analysis of minigenes transiently expressed in PC-12 cells that, in contrast to GluR-B pre-mRNA, where the two editing sites (Q/R and R/G) require base pairing with nearby intronic editing site complementary sequences (ECSs), editing in GluR5 and GluR6 pre-mRNAs recruits an ECS located as far as 1900 nucleotides distal to the Q/R site. The exon-intron duplex structure of the GluR5 and GluR6 pre-mRNAs appears to be a substrate of double-stranded RNA-specific adenosine deaminase. This enzyme when coexpressed in HEK 293 cells preferentially targets the adenosine of the Q/R site and of an unpaired position in the ECS which is highly edited in brain.
Different interleukin 2 receptor beta-chain tyrosines couple to at least two signaling pathways and synergistically mediate interleukin 2-induced proliferation.
Authors: Friedmann M C , Migone T S , Russell S M , Leonard W J
Published: July 2002

One of the earliest events induced by interleukin 2 (IL-2) is tyrosine phosphorylation of cellular proteins, including the IL-2 receptor beta chain (IL-2Rbeta). Simultaneous mutation of three tyrosines (Y338, Y392, and Y510) in the IL-2Rbeta cytoplasmic domain abrogated IL-2-induced proliferation, whereas mutation of only Y338 or of Y392 and Y510 inhibited proliferation only partially. While Y392 and Y510 were critical for IL-2-induced activation of signal transducers and activators of transcription (STAT proteins), Y338 was required for Shc-IL-2Rbeta association and for IL-2-induced tyrosine phosphorylation of Shc. Thus, activation of both Jak-STAT and Shc-coupled signaling pathways requires specific IL-2Rbeta tyrosines that together act in concert to mediate maximal proliferation. In COS-7 cells, overexpression of Jak1 augmented phosphorylation of Y338 as well as Y392 and Y510, suggesting that the role for this Jak kinase may extend beyond the Jak-STAT pathway.
Heterometallic hybrids of homometallic human hemoglobins.
Authors: Huang Y , Yonetani T , Tsuneshige A , Hoffman B M , Ackers G K
Published: July 2002

Hybridization experiments between normal Hb tetramers (Fe2+ Hb) and those with four metal-substituted hemes (i.e., replacement of Fe2+ by Co2+, Mg2+, Mn2+, Mn3+, Ni2+, or Zn2+) have revealed unexpected behavior. These homometallic Hbs have previously served as models that mimic the deoxy or oxy properties of normal Fe2+ Hb. In this study, hybrids were composed of one alpha 1 beta 1 dimer that is metal-substituted at both hemes, in association with a second dimer alpha 2 beta 2 that has normal Fe2+ hemes. Both metal-substituted subunits are unligated, whereas the two Fe2+ subunits either are both unligated or both ligated with O2, CO, or CN. It was found that four of the metal-substituted Hbs (Mg2+ Hb, Mn2+ Hb, Ni2+ Hb, and Zn2+ Hb) did not form detectable amounts of heterometallic hybrids with normal Fe2+ Hb even though (i) their homometallic parents formed tight tetrameric complexes with stabilities similar to that of Fe2+ Hb and (ii) hybrids with metal substitution at both alpha sites or both beta sites are known to form readily. This striking positional effect was independent of whether the normal Fe2+ hemes were ligated and of which ligand was used. These findings indicate that surprisingly large changes in tetramer behavior can arise from small and subtle perturbations at the heme sites. Possible origins of these effects are considered.
Chick pineal clock regulates serotonin
N
-acetyltransferase mRNA rhythm in culture
Authors: Bernard Marianne , Klein David C. , Zatz Martin
Published: July 2002

Melatonin production in the chick pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin
N
-acetyltransferase (arylalkylamine
N
-acetyltransferase, AA-NAT; EC
2.3.1.87
), the penultimate enzyme in melatonin synthesis. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not
de novo
protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.
Involvement of caspase-dependent activation of cytosolic phospholipase A2in tumor necrosis factor-induced apoptosis
Authors: Wissing Dorte , Mouritzen Helle , Egeblad Mikala , Poirier Guy G. , Jäättelä Marja
Published: July 2002

Tumor necrosis factor (TNF)-induced apoptosis is mediated by caspases, which are cysteine proteases related to interleukin 1β-converting enzyme. We report here that TNF-induced activation of caspases results in the cleavage and activation of cytosolic phospholipase A2(cPLA2) and that activated cPLA2contributes to apoptosis. Inhibition of caspases by expression of a cowpox virus-derived inhibitor, CrmA, or by a specific tetrapeptide inhibitor of CPP32/caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited TNF-induced activation of cPLA2and apoptosis. TNF-induced activation of cPLA2was accompanied by a cleavage of the 100-kDa cPLA2to a 70-kDa proteolytic fragment. This cleavage was inhibited by Ac-DEVD-CHO in a similar manner as that of poly(ADP)ribose polymerase, a known substrate of CPP32/caspase-3. Interestingly, specific inhibition of cPLA2enzyme activity by arachidonyl trifluoromethylketone (AACOCF3) partially inhibited TNF-induced apoptosis without inhibition of caspase activity. Thus, our results suggest a novel caspase-dependent activation pathway for cPLA2during apoptosis and identify cPLA2as a mediator of TNF-induced cell death acting downstream of caspases.
Evidence for phosphorylation and oligomeric assembly of presenilin 1
Authors: Seeger Mary , Nordstedt Christer , Petanceska Suzana , Kovacs Dora M. , Gouras Gunnar K. , Hahne Solveig , Fraser Paul , Levesque Lyne , Czernik Andrew J. , George-Hyslop Peter St , Sisodia Sangram S. , Thinakaran Gopal , Tanzi Rudolph E. , Greengard Paul , Gandy Sam
Published: July 2002

Pathogenic mutations in presenilin 1 (PS1) are associated with ≈50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12,13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20–23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage λ protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.
The second finger of Urbs1 is required for iron-mediated repression of
sid1
in
Ustilago maydis
Authors: An Zhiqiang , Zhao Qin , McEvoy James , Yuan Walter M. , Markley John L. , Leong Sally A.
Published: July 2002

The
urbs1
gene encodes a transcriptional regulator of siderophore biosynthesis in
Ustilago maydis
. Biological and DNA-binding activities of the two putative zinc-finger motifs of Urbs1 were studied by analyzing mutants containing altered finger domains. The mutated
urbs1
alleles from three previously described
N
′-methyl-
N
′-nitro-
N
-nitrosoguanidine (NTG) mutants were mapped and cloned by a gap-repair procedure. Sequence analyses revealed single amino acid substitutions in two of the NTG mutants. Both mutations (G-507 to D in
urbs1–1
and P-491 to L in
urbs1–3
), which are located in the Urbs1 C-terminal finger domain, reduced DNA-binding activity by 10-fold and were sufficient to confer a
urbs1
–minus phenotype. The third NTG
urbs1
mutant (
urbs1–2
) also contained a mutation in one of the conserved amino acids (P-518 to S) in the C-terminal finger domain, but this mutation alone was not sufficient to confer a
urbs1
–minus phenotype. A second frame shift mutation was identified in
urbs1–2
and is necessary for the
urbs1
–minus phenotype. In an analysis of the function of the N-terminal finger of Urbs1, the conserved amino acid Arg-350 was mutated to leucine. A Urbs1 protein with this mutation complemented a
urbs1
null mutant strain. By contrast, a similar mutation in the C-terminal domain abolished the ability of Urbs1 to regulate siderophore biosynthesis and greatly reduced its ability to bind target DNA.
A role for Sp and nuclear receptor transcription factors in a cardiac hypertrophic growth program
Authors: Sack Michael N. , Disch Dennis L. , Rockman Howard A. , Kelly Daniel P.
Published: July 2002

During cardiac hypertrophy, the chief myocardial energy source switches from fatty acid β-oxidation (FAO) to glycolysis—a reversion to fetal metabolism. The expression of genes encoding myocardial FAO enzymes was delineated in a murine ventricular pressure overload preparation to characterize the molecular regulatory events involved in the alteration of energy substrate utilization during cardiac hypertrophy. Expression of genes involved in the thioesterification, mitochondrial import, and β-oxidation of fatty acids was coordinately down-regulated after 7 days of right ventricular (RV) pressure overload. Results of RV pressure overload studies in mice transgenic for the promoter region of the gene encoding human medium-chain acyl-CoA dehydrogenase (MCAD, which catalyzes a rate-limiting step in the FAO cycle) fused to a chloramphenicol acetyltransferase reporter confirmed that repression of MCAD gene expression in the hypertrophied ventricle occurred at the transcriptional level. Electrophoretic mobility-shift assays performed with MCAD promoter fragments and nuclear protein extracts prepared from hypertrophied and control RV identified pressure overload-induced protein/DNA interactions at a regulatory unit shown previously to confer control of MCAD gene transcription during cardiac development. Antibody “supershift” studies demonstrated that members of the Sp (Sp1, Sp3) and nuclear hormone receptor [chicken ovalbumin upstream promoter transcription factor (COUP-TF)/erbA-related protein 3] families interact with the pressure overload-responsive unit. Cardiomyocyte transfection studies confirmed that COUP-TF repressed the transcriptional activity of the MCAD promoter. The DNA binding activities and nuclear expression of Sp1/3 and COUP-TF in normal fetal mouse heart were similar to those in the hypertrophied adult heart. These results identify a transcriptional regulatory mechanism involved in the reinduction of a fetal metabolic program during pressure overload-induced cardiac hypertrophy.
A nonrandom dynamic component in the synaptic noise of a central neuron
Authors: Faure Philippe , Korn Henri
Published: July 2002

Continuous segments of synaptic noise were recorded
in vivo
from teleost Mauthner cells and were studied with the methods of nonlinear analysis. As in many central neurons, this ongoing activity is dominated by consecutive inhibitory postsynaptic potentials. Recurrence plots and first or third order Poincaré maps combined with surrogate shuffling revealed nonrandom patterns consistent with the notion that synaptic noise is a continuously varying mixture of periodic and chaotic phases. Chaos was further demonstrated by the occurrence of unstable periodic orbits. The nonrandom component of the noise is reproducibly and persistently reduced when the level of background sound, a natural stimulus for networks afferent to the Mauthner cell, is briefly elevated. These data are consistent with a model involving a reciprocally connected inhibitory network, presynaptic to the Mauthner cell and its intrinsic properties. The presence of chaos in the inhibitory synaptic noise that regulates the excitability of the Mauthner cell and its sensitivity to external stimuli suggests that it modulates this neuron’s function, namely to trigger a fast escape motor reaction following unexpected sensory information.
Experience-dependent, asymmetric expansion of hippocampal place fields
Authors: Mehta Mayank R. , Barnes Carol A. , McNaughton Bruce L.
Published: July 2002

Theories of sequence learning based on temporally asymmetric, Hebbian long-term potentiation predict that during route learning the spatial firing distributions of hippocampal neurons should enlarge in a direction opposite to the animal’s movement. On a route AB, increased synaptic drive from cells representing A would cause cells representing B to fire earlier and more robustly. These effects appeared within a few laps in rats running on closed tracks. This provides indirect evidence for Hebbian synaptic plasticity and a functional explanation for why place cells become directionally selective during route following, namely, to preserve the synaptic asymmetry necessary to encode the sequence direction.