bluuebunny/crossref_metadata_2025 huggingface dataset:
HIV-1 induces phenotypic and functional perturbations of B cells in chronically infected individuals
Authors: Moir Susan , Malaspina Angela , Ogwaro Kisani M. , Donoghue Eileen T. , Hallahan Claire W. , Ehler Linda A. , Liu Shuying , Adelsberger Joseph , Lapointe Réjean , Hwu Patrick , Baseler Michael , Orenstein Jan M. , Chun Tae-Wook , Mican Jo Ann M. , Fauci Anthony S.
Published: July 2002

A number of perturbations of B cells has been described in the setting of HIV infection; however, most remain poorly understood. To directly address the effect of HIV replication on B cell function, we investigated the capacity of B cells isolated from HIV-infected patients to respond to a variety of stimuli before and after reduction of viremia by effective antiretroviral therapy. B cells taken from patients with high levels of plasma viremia were defective in their proliferative responses to various stimuli. Viremia was also associated with the appearance of a subpopulation of B cells that expressed reduced levels of CD21. After fractionation into CD21high- and CD21low-expressing B cells, the CD21lowfraction showed dramatically reduced proliferation in response to B cell stimuli and enhanced secretion of immunoglobulins when compared with the CD21highfraction. Electron microscopic analysis of each fraction revealed cells with plasmacytoid features in the CD21lowB cell population but not in the CD21highfraction. These results indicate that HIV viremia induces the appearance of a subset of B cells whose function is impaired and which may be responsible for the hypergammaglobulinemia associated with HIV disease.
Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB
Authors: Mayr Bernhard M. , Canettieri Gianluca , Montminy Marc R.
Published: July 2002

Ser-133 phosphorylation of the cAMP-responsive element-binding protein (CREB) is sufficient to induce cellular gene expression in response to cAMP, but additional promoter-bound factors are required for target gene activation by CREB in response to mitogen/stress signals. To compare the relative effects of different signals on recruitment of the coactivator CREB-binding protein (CBP) to CREB in living cells, we developed a fluorescence resonance energy transfer (FRET) assay. cAMP promoted the interaction of CREB with CBP in a phosphorylation-dependent manner by FRET analysis, but mitogen/stress signals were far less effective in stimulating complex formation even though they induced comparable levels of Ser-133 phosphorylation. cAMP and non-cAMP stimuli were comparably active in promoting this interaction in the cytosol; the formation of CREB⋅CBP complexes in response to non-cAMP signals was specifically inhibited in the nucleus. Non-cAMP signals had no effect on intrinsic CREB- or CBP-binding activities by Far Western blot assay, thereby supporting the presence of a distinct CREB⋅CBP antagonist. Our studies indicate that the relative effects of cAMP and mitogen/stress signals on CREB⋅CBP complex formation impart selectivity to gene activation through CREB phosphorylated at Ser-133.
Social stress exacerbates stroke outcome by suppressing
Bcl-2
expression
Authors: DeVries A. Courtney , Joh Hung-Dong , Bernard Ora , Hattori Kimihiko , Hurn Patricia D. , Traystman Richard J. , Alkayed Nabil J.
Published: July 2002

The relationship between stressful life events and the onset of
disease is well documented. However, the role of psychological stress
as a risk factor for life-threatening cerebrovascular insults such as
stroke remains unspecified, but could explain individual variation in
stroke outcome. To discover the mechanisms through which psychological
stress may alter stroke outcome, we modeled the effects of chronic
social intimidation and stress on ischemia-induced
bcl-2
expression and early neuronal cell loss resulting from cerebral artery
occlusion in mice (C57BL/6). The
bcl-2
protooncogene
promotes cell survival and protects against apoptosis and
cellular necrosis in numerous neurodegenerative disorders, including
stroke. In our study, male mice were chronically exposed to aggressive
social stimuli before induction of a controlled, mild ischemic insult.
Stressed mice expressed ≈70% less
bcl-2
mRNA than
unstressed mice after ischemia. In addition, social stress greatly
exacerbated infarct in wild-type mice but not in transgenic mice that
constitutively express increased neuronal
bcl-2
. Despite
similar postischemic concentrations of corticosterone, the major stress
hormone in mice, high corticosterone concentrations were significantly
correlated with larger infarcts in wild-type mice but not
bcl-2
transgenic mice. Thus, enhanced
bcl-2
expression offsets the potentially deleterious
consequences of high postischemic plasma corticosterone concentrations.
Taken together, these data demonstrate that stressful prestroke social
milieu strongly compromises an endogenous molecular
mechanism of neuroprotection in injured brain and offer a new
behavioral target for stroke therapy.
Cytoplasmic dynein is associated with slow axonal transport.
Authors: Dillman J F , Dabney L P , Pfister K K
Published: July 2002

Neuronal function is dependent on the transport of materials from the cell body to the synapse via anterograde axonal transport. Anterograde axonal transport consists of several components that differ in both rate and protein composition. In fast transport, membranous organelles are moved along microtubules by the motor protein kinesin. The cytoskeleton and the cytomatrix proteins move in the two components of slow transport. While the mechanisms underlying slow transport are unknown, it has been hypothesized that the movement of microtubules in slow transport is generated by sliding. To determine whether dynein, a motor protein that causes microtubule sliding in flagella, may play a role in slow axonal transport, we identified the transport rate components with which cytoplasmic dynein is associated in rat optic nerve. Nearly 80% of the anterogradely moving dynein was associated with slow transport, whereas only approximately 15% of the dynein was associated with the membranous organelles of anterograde fast axonal transport. A segmental analysis of the transport of dynein through contiguous regions of the optic nerve and tract showed that dynein is associated with the microfilaments and other proteins of slow component b. Dynein from this transport component has the capacity to bind microtubules in vitro. These results are consistent with the hypothesis that cytoplasmic dynein generates the movement of microtubules in slow axonal transport. A model is presented to illustrate how dynein attached to the slow component b complex of proteins is appropriately positioned to generate force of the correct polarity to slide microtubules down the axon.
Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.
Authors: Berg R J , van Kranen H J , Rebel H G , de Vries A , van Vloten W A , Van Kreijl C F , van der Leun J C , de Gruijl F R
Published: July 2002

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.
Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain.
Authors: Forster M J , Dubey A , Dawson K M , Stutts W A , Lal H , Sohal R S
Published: July 2002

The hypothesis that age-associated impairment of cognitive and motor functions is due to oxidative molecular damage was tested in the mouse. In a blind study, senescent mice (aged 22 months) were subjected to a battery of behavioral tests for motor and cognitive functions and subsequently assayed for oxidative molecular damage as assessed by protein carbonyl concentration in different regions of the brain. The degree of age-related impairment in each mouse was determined by comparison to a reference group of young mice (aged 4 months) tested concurrently on the behavioral battery. The age-related loss of ability to perform a spatial swim maze task was found to be positively correlated with oxidative molecular damage in the cerebral cortex, whereas age-related loss of motor coordination was correlated with oxidative molecular damage within the cerebellum. These results support the view that oxidative stress is a causal factor in brain senescence. Furthermore, the findings suggest that age-related declines of cognitive and motor performance progress independently, and involve oxidative molecular damage within different regions of the brain.
p53 as a target for cancer vaccines: recombinant canarypox virus vectors expressing p53 protect mice against lethal tumor cell challenge.
Authors: Roth J , Dittmer D , Rea D , Tartaglia J , Paoletti E , Levine A J
Published: July 2002

The p53 protein is an attractive target for immunotherapy, because mutations in the p53 gene are the most common genetic alterations found in human tumors. These mutations result in high levels of p53 protein in the tumor cell, whereas the expression level of wild-type p53 in nonmalignant tissue is usually much lower. Several canarypox virus recombinants expressing human or murine p53 in wild-type or mutant form were constructed. Immunization with these viruses protected BALB/c mice from a challenge with an isogenic and highly tumorigenic mouse fibroblast tumor cell line expressing high levels of mutant p53. The tumor protection was equally effective regardless of whether wild-type or mutant p53 was used for the immunization, indicating that the immunologic response was not dependent on any particular p53 mutation and that immunization with this live virus vaccine works effectively against mutant p53 protein expressed in a tumor cell. In tumors escaping immunologic rejection, the expression of the p53 protein was commonly down-regulated.
Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells.
Authors: Reichel C , Mathur J , Eckes P , Langenkemper K , Koncz C , Schell J , Reiss B , Maas C
Published: July 2002

The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.
Kinetics of self-assembling microtubules: an "inverse problem" in biochemistry.
Authors: Flyvbjerg H , Jobs E , Leibler S
Published: July 2002

Experimental time series for a nonequilibrium reaction may in some cases contain sufficient data to determine a unique kinetic model for the reaction by a systematic mathematical analysis. As an example, a kinetic model for the self-assembly of microtubules is derived here from turbidity time series for solutions in which microtubules assemble. The model may be seen as a generalization of Oosawa's classical nucleation-polymerization model. It reproduces the experimental data with a four-stage nucleation process and a critical nucleus of 15 monomers.
The glucose sensor protein glucokinase is expressed in glucagon-producing alpha-cells.
Authors: Heimberg H , De Vos A , Moens K , Quartier E , Bouwens L , Pipeleers D , Van Schaftingen E , Madsen O , Schuit F
Published: July 2002

Expression of glucokinase in hepatocytes and pancreatic 6-cells is of major physiologic importance to mammalian glucose homeostasis. Liver glucokinase catalyzes the first committed step in the disposal of glucose, and beta-cell glucokinase catalyzes a rate-limiting step required for glucose-regulated insulin release. The present study reports the expression of glucokinase in rat glucagon-producing alpha-cells, which are negatively regulated by glucose. Purified rat alpha-cells express glucokinase mRNA and protein with the same transcript length, nucleotide sequence, and immunoreactivity as the beta-cell isoform. Glucokinase activity accounts for more than 50% of glucose phosphorylation in extracts of alpha-cells and for more than 90% of glucose utilization in intact cells. The glucagon-producing tumor MSL-G-AN also contained glucokinase mRNA, protein, and enzymatic activity. These data indicate that glucokinase may serve as a metabolic glucose sensor in pancreatic alpha-cells and, hence, mediate a mechanism for direct regulation of glucagon release by extracellular glucose. Since these cells do not express Glut2, we suggest that glucose sensing does not necessarily require the coexpression of Glut2 and glucokinase.
Increased tricarboxylic acid cycle flux in rat brain during forepaw stimulation detected with 1H[13C]NMR.
Authors: Hyder F , Chase J R , Behar K L , Mason G F , Siddeek M , Rothman D L , Shulman R G
Published: July 2002

NMR spectroscopy was used to test recent proposals that the additional energy required for brain activation is provided through nonoxidative glycolysis. Using localized NMR spectroscopic methods, the rate of C4-glutamate isotopic turnover from infused [1-(13)C]glucose was measured in the somatosensory cortex of rat brain both at rest and during forepaw stimulation. Analysis of the glutamate turnover data using a mathematical model of cerebral glucose metabolism showed that the tricarboxylic acid cycle flux [(V(TCA)] increased from 0.49 +/- 0.03 at rest to 1.48 +/- 0.82 micromol/g/min during stimulation (P < 0.01). The minimum fraction of C4-glutamate derived from C1-glucose was approximately 75%, and this fraction was found in both the resting and stimulated rats. Hence, the percentage increase in oxidative cerebral metabolic rate of glucose use (CMRglc) equals the percentage increases in V(TCA) and cerebral metabolic rate of oxygen consumption (CMRO2). Comparison with previous work for the same rat model, which measured total CMRglc [Ueki, M., Linn, F. & Hossman, K. A. (1988) J. Cereb. Blood Flow Metab. 8, 486-4941, indicates that oxidative CMRglc supplies the majority of energy during sustained brain activation.
16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the Green Non-Sulfur bacteria.
Authors: Giovannoni S J , Rappé M S , Vergin K L , Adair N L
Published: July 2002

Microorganisms play an important role in the biogeochemistry of the ocean surface layer, but spatial and temporal structures in the distributions of specific bacterioplankton species are largely unexplored, with the exceptions of those organisms that can be detected by either autofluorescence or culture methods. The use of rRNA genes as genetic markers provides a tool by which patterns in the growth, distribution, and activity of abundant bacterioplankton species can be studied regardless of the ease with which they can be cultured. Here we report an unusual cluster of related 16S rRNA genes (SAR202, SAR263, SAR279, SAR287, SAR293, SAR307) cloned from seawater collected at 250 m in the Sargasso Sea in August 1991, when the water column was highly stratified and the deep chlorophyll maximum was located at a depth of 120 m. Phylogenetic analysis and an unusual 15-bp deletion confirmed that the genes were related to the Green Non-Sulfur phylum of the domain Bacteria. This is the first evidence that representatives of this phylum occur in the open ocean. Oligonucleotide probes were used to examine the distribution of the SAR202 gene cluster in vertical profiles (0-250 m) from the Atlantic and Pacific Oceans, and in discrete (monthly) time series (O and 200 m) (over 30 consecutive months in the Western Sargasso Sea. The data provide robust statistical support for the conclusion that the SAR202 gene cluster is proportionately most abundant at the lower boundary of the deep chlorophyll maximum (P = 2.33 x 10(-5)). These results suggest that previously unsuspected stratification of microbial populations may be a significant factor in the ecology of the ocean surface layer.
Phosphorylated Ser/Arg-rich proteins: limiting factors in the assembly of 200S large nuclear ribonucleoprotein particles.
Authors: Yitzhaki S , Miriami E , Sperling R , Sperling J
Published: July 2002

We have previously shown that specific nuclear pre-mRNA transcripts and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are packaged in large nuclear ribonucleoprotein (InRNP) particles that sediment at the 200S region in sucrose gradients. The InRNP particles contain all uridine-rich small nuclear ribonucleoprotein complexes required for pre-mRNA splicing, as well as protein splicing factors. In this paper we show that all of the phosphorylated, mAb 104 detectable, Ser/Arg-rich essential splicing factors (SR proteins) in the nucleoplasm are integral components of the InRNP particles, whereas only part of the essential splicing factor U2AF65 (U2 snRNP auxiliary factor) and the polypyrimidine tract binding protein (PTB) are associated with these particles. This finding suggests a limiting role for SR proteins in the assembly of the InRNP particles. We further show that the structural integrity of InRNP particles is sensitive to variations in the phosphorylation levels of the SR proteins.
Expression and analysis of presenilin 1 in a human neuronal system: localization in cell bodies and dendrites.
Authors: Cook D G , Sung J C , Golde T E , Felsenstein K M , Wojczyk B S , Tanzi R E , Trojanowski J Q , Lee V M , Doms R W
Published: July 2002

Mutations in the recently identified presenilin 1 gene on chromosome 14 cause early onset familial Alzheimer disease (FAD). Herein we describe the expression and analysis of the protein coded by presenilin 1 (PS1) in NT2N neurons, a human neuronal model system. PS1 was expressed using recombinant Semliki Forest virions and detected by introduced antigenic tags or antisera to PS1-derived peptides. Immunoprecipitation revealed two major PS1 bands of approximately 43 and 50 kDa, neither of which were N-glycosylated or O-glycosylated. Immunoreactive PS1 was detected in cell bodies and dendrites of NT2N neurons but not in axons or on the cell surface. PS1 was also detected in BHK cells, where it was also intracellular and colocalized with calnexin, a marker for the rough endoplasmic reticulum. A mutant form of PS1 linked to FAD did not differ from the wild-type protein at the light microscopic level. The model system described here will enable studies of the function of PS1 in human neurons and the role of mutant PS1 in FAD.
Salmonella typhimurium invasion induces apoptosis in infected macrophages.
Authors: Monack D M , Raupach B , Hromockyj A E , Falkow S
Published: July 2002

Invasive Salmonella typhimurium induces dramatic cytoskeletal changes on the membrane surface of mammalian epithelial cells and RAW264.7 macrophages as part of its entry mechanism. Noninvasive S. typhimurium strains are unable to induce this membrane ruffling. Invasive S. typhimurium strains invade RAW264.7 macrophages in 2 h with 7- to 10-fold higher levels than noninvasive strains. Invasive S. typhimurium and Salmonella typhi, independent of their ability to replicate intracellularly, are cytotoxic to RAW264.7 macrophages and, to a greater degree, to murine bone marrow-derived macrophages. Here, we show that the macrophage cytotoxicity mediated by invasive Salmonella is apoptosis, as shown by nuclear morphology, cytoplasmic vacuolization, and host cell DNA fragmentation. S. typhimurium that enter cells causing ruffles but are mutant for subsequent intracellular replication also initiate host cell apoptosis. Mutant S. typhimurium that are incapable of inducing host cell membrane ruffling fail to induce apoptosis. The activation state of the macrophage plays a significant role in the response of macrophages to Salmonella invasion, perhaps indicating that the signal or receptor for initiating programmed cell death is upregulated in activated macrophages. The ability of Salmonella to promote apoptosis may be important for the initiation of infection, bacterial survival, and escape of the host immune response.
The N-end rule: functions, mysteries, uses.
Authors: Varshavsky A
Published: July 2002

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Similar but distinct versions of the N-end rule operate in all organisms examined, from mammals to fungi and bacteria. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system. I discuss the mechanisms and functions of this pathway, and consider its applications.
Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase
1
gene
Authors: Venugopal Radjendirane , Jaiswal Anil K.
Published: July 2002

Twenty-four base pairs of the human antioxidant response element
(hARE) are required for high basal transcription of the NAD(P)H:quinone
oxidoreductase
1
(NQO
1
) gene and its induction
in response to xenobiotics and antioxidants. hARE is a unique
cis-element that contains one perfect and one imperfect AP1 element
arranged as inverse repeats separated by 3 bp, followed by a “GC”
box. We report here that Jun, Fos, Fra, and Nrf nuclear transcription
factors bind to the hARE. Overexpression of cDNA derived combinations
of the nuclear proteins Jun and Fos or Jun and Fra1 repressed
hARE-mediated chloramphenicol acetyltransferase (CAT) gene expression
in transfected human hepatoblastoma (Hep-G2) cells. Further experiments
suggested that this repression was due to overexpression of c-Fos and
Fra1, but not due to Jun proteins. The Jun (c-Jun, Jun-B, and Jun-D)
proteins in all the possible combinations were more or less ineffective
in repression or upregulation of hARE-mediated gene expression.
Interestingly, overexpression of Nrf1 and Nrf2 individually in Hep-G2
and monkey kidney (COS1) cells significantly increased CAT gene
expression from reporter plasmid hARE-thymidine kinase-CAT in
transfected cells that were inducible by β-naphthoflavone and
tert
-butyl hydroquinone. These results indicated that
hARE-mediated expression of the NQO
1
gene and its induction
by xenobiotics and antioxidants are mediated by Nrf1 and Nrf2. The
hARE-mediated basal expression, however, is repressed by overexpression
of c-Fos and Fra1.
Learning and recall of form discriminations during reversible cooling deactivation of ventral-posterior suprasylvian cortex in the cat.
Authors: Lomber S G , Payne B R , Cornwell P
Published: July 2002

Extrastriate visual cortex of the ventral-posterior suprasylvian gyrus (vPS cortex) of freely behaving cats was reversibly deactivated with cooling to determine its role in performance on a battery of simple or masked two-dimensional pattern discriminations, and three-dimensional object discriminations. Deactivation of vPS cortex by cooling profoundly impaired the ability of the cats to recall the difference between all previously learned pattern and object discriminations. However, the cats' ability to learn or relearn pattern and object discriminations while vPS was deactivated depended upon the nature of the pattern or object and the cats' prior level of exposure to them. During cooling of vPS cortex, the cats could neither learn the novel object discriminations nor relearn a highly familiar masked or partially occluded pattern discrimination, although they could relearn both the highly familiar object and simple pattern discriminations. These cooling-induced deficits resemble those induced by cooling of the topologically equivalent inferotemporal cortex of monkeys and provides evidence that the equivalent regions contribute to visual processing in similar ways.
A functional Rev-erb alpha responsive element located in the human Rev-erb alpha promoter mediates a repressing activity.
Authors: Adelmant G , Bègue A , Stéhelin D , Laudet V
Published: July 2002

Rev-erb alpha belongs to the nuclear receptor superfamily, which contains receptors for steroids, thyroid hormones, retinoic acid, and vitamin D, as well as "orphan" receptors. No ligand has been found for Rev-erb alpha to date, making it one of these orphan receptors. Similar to some other orphan receptors, Rev-erb alpha has been shown to bind DNA as a monomer on a specific sequence called a Rev-erb alpah responsive element (RevRE), but its transcriptional activity remains unclear. In this paper, we characterize a functional RevRE located in the human Rev-erb alpha promoter itself. We also present evidence that (i) Rev-erb alpha mediates transcriptional repression of its own promoter in vitro, (ii) this repressing effect strictly depends on the binding of Rev-erb alpha to its responsive element and is transferable to a heterologous promoter; and (iii) Rev-erb alpha binds to this responsive sequence as a homodimer.
Prolactin increases CD4/CD8 cell ratio in thymus-grafted congenitally athymic nude mice.
Authors: Gaufo G O , Diamond M C
Published: July 2002

One distinctive effect on T-cell development was analyzed by selectively increasing serum prolactin (PRL) concentration in thymus-grafted congenitally athymic nude mice and by neutralizing PRL in suspension cultures of thymus from 1-day-old neonatal mice. Flow cytometric analysis of single-positive CD4+ and CD8+ cells derived from inguinal lymph nodes revealed a CD4/CD8 cell ratio of 2.2 +/- 0.18 (mean +/- SEM) in thymus-grafted nude mice that is similar to the ratio for immune-competent BALB/c mice (2.0 +/- 0.06). Addition of the pituitary to thymus-grafted nude mice significantly elevated serum PRL (P < 0.005) and increased the CD4/CD8 cell ratio (2.8 +/- 0.12; P < 0.005), demonstrating preferential stimulation of CD4+ cell development. T cells in nude mice receiving sham (submandibular salivary gland) or pituitary grafts alone were below detectable levels. Suspension cultures of neonatal thymus treated with anti-mouse PRL antiserum resulted in 20% and 30% decreases in double-positive CD4+8+ thymocytes and thymocyte viability, respectively. A 10-fold increase in double-negative CD4-8- thymocytes expressing the interleukin 2 receptor alpha chain, CD25, was also observed concurrently. Our findings illustrate an important way in which PRL may participate in two interrelated mechanisms: the regulation of peripheral single-positive cells and the maintenance of thymocyte viability during the double-positive stage of intrathymic differentiation.
Chromogranin B (secretogranin I) promotes sorting to the regulated secretory pathway of processing intermediates derived from a peptide hormone precursor.
Authors: Natori S , Huttner W B
Published: July 2002

Chromogranin B (CgB, secretogranin I) is a widespread constituent of neuroendocrine secretory granules whose function is unknown. To determine whether CgB affects the sorting of peptide hormone and neuropeptide precursors to secretory granules, we overexpressed CgB in AtT-20 cells, which exhibit an only moderate capacity to sort proopiomelanocortin and proteolytic fragments derived therefrom. In mock-transfected AtT-20 cells, a substantial proportion of newly synthesized proopiomelanocortin and its two primary proteolytic products generated in the trans-Golgi network, the N-terminal 23-kDa fragment containing adrenocorticotropin and the C-terminal beta-lipotropin fragment, was secreted via the constitutive pathway. Two- to three-fold overexpression of CgB markedly reduced the constitutive secretion of the 23-kDa fragment, but not beta-lipotropin and tripled the amount of adrenocorticotropin generated and stored in secretory granules. Our results indicate the existence of neuroendocrine-specific helper proteins which promote the sorting from the trans-Golgi network to secretory granules of certain processing intermediates derived from peptide hormone and neuropeptide precursors and demonstrate that CgB functions as such.
Scrapie susceptibility-linked polymorphisms modulate the
in vitro
conversion of sheep prion protein to protease-resistant forms
Authors: Bossers Alex , Belt Peter B. G. M. , Raymond Gregory J. , Caughey Byron , de Vries Ruth , Smits Mari A.
Published: July 2002

Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrP
Sc
) of the host-encoded cellular prion protein (PrP
C
) mainly in the central nervous system. Polymorphisms in the PrP gene are linked to differences in susceptibility for prion diseases. The mechanisms underlying these effects are still unknown. Here we describe studies of the influence of sheep PrP polymorphisms on the conversion of PrP
C
into protease-resistant forms. In a cell-free system, sheep PrP
Sc
induced the conversion of sheep PrP
C
into protease-resistant PrP (PrP-res) similar or identical to PrP
Sc
. Polymorphisms present in either PrP
C
or PrP
Sc
had dramatic effects on the cell-free conversion efficiencies. The PrP variant associated with a high susceptibility to scrapie and short survival times of scrapie-affected sheep was efficiently converted into PrP-res. The wild-type PrP variant associated with a neutral effect on susceptibility and intermediate survival times was converted with intermediate efficiency. The PrP variant associated with scrapie resistance and long survival times was poorly converted. Thus the
in vitro
conversion characteristics of the sheep PrP variants reflect their linkage with scrapie susceptibility and survival times of scrapie-affected sheep. The modulating effect of the polymorphisms in PrP
C
and PrP
Sc
on the cell-free conversion characteristics suggests that, besides the species barrier, polymorphism barriers play a significant role in the transmissibility of prion diseases.
Positionally cloned human disease genes: Patterns of evolutionary conservation and functional motifs
Authors: Mushegian Arcady R. , Bassett Douglas E. , Boguski Mark S. , Bork Peer , Koonin Eugene V.
Published: July 2002

Positional cloning has already produced the sequences of more than 70 human genes associated with specific diseases. In addition to their medical importance, these genes are of interest as a set of human genes isolated solely on the basis of the phenotypic effect of the respective mutations. We analyzed the protein sequences encoded by the positionally cloned disease genes using an iterative strategy combining several sensitive computer methods. Comparisons to complete sequence databases and to separate databases of nematode, yeast, and bacterial proteins showed that for most of the disease gene products, statistically significant sequence similarities are detectable in each of the model organisms. Only the nematode genome encodes apparent orthologs with conserved domain architecture for the majority of the disease genes. In yeast and bacterial homologs, domain organization is typically not conserved, and sequence similarity is limited to individual domains. Generally, human genes complement mutations only in orthologous yeast genes. Most of the positionally cloned genes encode large proteins with several globular and nonglobular domains, the functions of some or all of which are not known. We detected conserved domains and motifs not described previously in a number of proteins encoded by disease genes and predicted functions for some of them. These predictions include an ATP-binding domain in the product of hereditary nonpolyposis colon cancer gene (a MutL homolog), which is conserved in the HS90 family of chaperone proteins, type II DNA topoisomerases, and histidine kinases, and a nuclease domain homologous to bacterial RNase D and the 3′-5′ exonuclease domain of DNA polymerase I in the Werner syndrome gene product.
At
GRP7, a nuclear RNA-binding protein as a component of a circadian-regulated negative feedback loop in
Arabidopsis thaliana
Authors: Heintzen Christian , Nater Mena , Apel Klaus , Staiger Dorothee
Published: July 2002

The endogenous clock that drives circadian rhythms is thought to communicate temporal information within the cell via cycling downstream transcripts. A transcript encoding a glycine-rich RNA-binding protein,
Atgrp7
, in
Arabidopsis thaliana
undergoes circadian oscillations with peak levels in the evening. The
At
GRP7 protein also cycles with a time delay so that
Atgrp7
transcript levels decline when the
At
GRP7 protein accumulates to high levels. After
At
GRP7 protein concentration has fallen to trough levels,
Atgrp7
transcript starts to reaccumulate. Overexpression of
At
GRP7 in transgenic
Arabidopsis
plants severely depresses cycling of the endogenous
Atgrp7
transcript. These data establish both transcript and protein as components of a negative feedback circuit capable of generating a stable oscillation.
At
GRP7 overexpression also depresses the oscillation of the circadian-regulated transcript encoding the related RNA-binding protein
At
GRP8 but does not affect the oscillation of transcripts such as
cab
or catalase mRNAs. We propose that the
At
GRP7 autoregulatory loop represents a “slave” oscillator in
Arabidopsis
that receives temporal information from a central “master” oscillator, conserves the rhythmicity by negative feedback, and transduces it to the output pathway by regulating a subset of clock-controlled transcripts.
RanGTP-mediated nuclear export of karyopherin α involves its interaction with the nucleoporin Nup153
Authors: Moroianu Junona , Blobel Günter , Radu Aurelian
Published: July 2002

Using binding assays, we discovered an interaction between karyopherin α2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin β1, termed β1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an
in vitro
assay in which export of endogenous nuclear karyopherin α from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin α antibodies, we found that karyopherin α export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of karyopherin α was inhibited by peptides representing the interacting domains of Nup153 and karyopherin α2, indicating that the binding reactions detected
in vitro
are physiologically relevant and verifying our mapping data. Moreover, β1*, although it inhibited import, did not inhibit export of karyopherin α. Hence, karyopherin α import into and export from nuclei are asymmetric processes.
Epidermal growth factor receptor activation induces nuclear targeting of cyclooxygenase-2, basolateral release of prostaglandins, and mitogenesis in polarizing colon cancer cells
Authors: Coffey Robert J. , Hawkey Chris J. , Damstrup Lars , Graves-Deal Ramona , Daniel Vincent C. , Dempsey Peter J. , Chinery Rebecca , Kirkland Susan C. , DuBois Raymond N. , Jetton Thomas L. , Morrow Jason D.
Published: July 2002

Nonsteroidal antiinflammatory drugs reduce the risk of colon cancer, possibly via cyclooxygenase (COX) inhibition. The growth factor-inducible COX-2, which is overexpressed in neoplastic colonic tissue, is an attractive target to mediate this effect. Herein we have exploited the ability of a human colon cancer cell line, HCA-7 Colony 29, to polarize when cultured on Transwell (Costar) filters to study COX-2 production and the vectorial release of prostaglandins (PGs). Administration of type α transforming growth factor to the basolateral compartment, in which the epidermal growth factor receptor (EGFR) resides, results in a marked induction of COX-2 immunoreactivity at the base of the cells and the unexpected appearance of COX-2 in the nucleus. The increase in COX-2 protein is associated with a dose- and time-dependent increase in PG levels in the basolateral, but not apical, medium. Amphiregulin is the most abundantly expressed EGFR ligand in these cells, and the protein is present at the basolateral surface. EGFR blockade reduces baseline COX-2 immunoreactivity, PG levels, and mitogenesis in a concentration-dependent manner. Two specific COX-2 inhibitors, SC-58125 and NS 398, also, in a dose-dependent manner, attenuate baseline and type α transforming growth factor-stimulated mitogenesis, although PG levels are decreased >90% at all concentrations of inhibitor tested. These findings show that activation of the EGFR stimulates COX-2 production and its translocation to the nucleus, vectorial release of PGs, and mitogenesis in polarized HCA-7 Colony 29 cells.
Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2
Authors: Kuziel William A. , Morgan Sharon J. , Dawson Tracey C. , Griffin Stephanie , Smithies Oliver , Ley Klaus , Maeda Nobuyo
Published: July 2002

CC chemokine receptor 2 (CCR2) is a prominent receptor for the monocyte chemoattractant protein (MCP) group of CC chemokines. Mice generated by gene targeting to lack CCR2 exhibit normal leukocyte rolling but have a pronounced defect in MCP-1-induced leukocyte firm adhesion to microvascular endothelium and reduced leukocyte extravasation. Constitutive macrophage trafficking into the peritoneal cavity was not significantly different between CCR2-deficient and wild-type mice. However, after intraperitoneal thioglycollate injection, the number of peritoneal macrophages in CCR2-deficient mice did not rise above basal levels, whereas in wild-type mice the number of macrophages at 36 h was ≈3.5 times the basal level. The CCR2-deficient mice showed enhanced early accumulation and delayed clearance of neutrophils and eosinophils. However, by 5 days neutrophils and eosinophils in both CCR2-deficient and wild-type mice had returned to near basal levels, indicating that resolution of this inflammatory response can occur in the absence of macrophage influx and CCR2-mediated activation of the resident peritoneal macrophages. After intravenous injection with yeast β-glucan, wild-type mice formed numerous large, well-defined granulomas throughout the liver parenchyma, whereas CCR2-deficient mice had much fewer and smaller granulomas. These results demonstrate that CCR2 is a major regulator of induced macrophage traffickingin vivo.
Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF
121
and VEGF
165
but not VEGF
189
Authors: Cheng Shi-Yuan , Nagane Motoo , Huang H.-J. Su , Cavenee Webster K.
Published: July 2002

The vascular endothelial growth factor (VEGF) has been shown to be a significant mediator of angiogenesis during a variety of normal and pathological processes, including tumor development. Human U87MG glioblastoma cells express the three VEGF isoforms: VEGF
121
, VEGF
165
, and VEGF
189
. Here, we have investigated whether these three isoforms have distinct roles in glioblastoma angiogenesis. Clones that overexpressed each isoform were derived and inoculated into mouse brains. Mice that received VEGF
121
- and VEGF
165
-overexpressing cells developed intracerebral hemorrhages after 60–90 hr. In contrast, mice implanted with VEGF
189
-overexpressing cells had only slightly larger tumors than those caused by parental cells and little evidence of hemorrhage at these early times after implantation, whereas, after longer periods of growth, enhanced angiogenicity and tumorigenicity were apparent. There was rapid blood vessel growth and breakdown around the tumors caused by cells overexpressing VEGF
121
and VEGF
165
, whereas there was similar vascularization but no eruption in the vicinity of those tumors caused by cells overexpressing VEGF
189
, and none on the border of the tumors caused by the parental cells. Thus, by introducing VEGF-overexpressing glioblastoma cells into the brain, we have established a reproducible and predictable
in vivo
model of tumor-associated intracerebral hemorrhage caused by the enhanced expression of single molecular species. Such a model should be useful for uncovering the role of VEGF isoforms in the mechanisms of angiogenesis and for investigating intracerebral hemorrhage due to ischemic stroke or congenital malformations.
Periplasmic superoxide dismutase protectsSalmonellafrom products of phagocyte NADPH-oxidase and nitric oxide synthase
Authors: De Groote Mary Ann , Ochsner Urs A. , Shiloh Michael U. , Nathan Carl , McCord Joe M. , Dinauer Mary C. , Libby Stephen J. , Vazquez-Torres Andrés , Xu Yisheng , Fang Ferric C.
Published: July 2002

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking theSalmonella typhimurium sodCgene encoding Cu,Zn-SOD demonstrates significant homology to λ phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria.Salmonelladeficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, asodCmutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogensin vivo.
The seven-transmembrane spanning topography of the Alzheimer disease-related presenilin proteins in the plasma membranes of cultured cells
Authors: Dewji Nazneen N. , Singer S. J.
Published: July 2002

To ascertain the membrane topography of the multi-transmembrane spanning presenilin proteins PS-1 and PS-2, anti-peptide antibodies were raised to several specific amino acid sequences in the two proteins, and, after their specificity was ascertained, the anti-peptide antibodies were used in immunofluorescent labeling of live PS-transfected, cultured DAMI cells, which are impermeable to the antibodies, as well as of their fixed and permeabilized counterparts. In such experiments, antibodies that specifically stain the intact live cells must label epitopes of the PS proteins that are on the exterior face of the plasma membrane whereas those antibodies that do not stain the live cells but do stain the fixed and permeabilized cells must label epitopes that face the cytoplasmic side of the membrane. The results obtained were entirely in accord with the predictions of the seven-transmembrane spanning topography (like that of rhodopsin and the β-adrenergic receptor) and were totally inconsistent with the expectations for either the six- or eight-transmembrane topographies that have been proposed.
Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets
Authors: Barba G. , Harper F. , Harada T. , Kohara M. , Goulinet S. , Matsuura Y. , Eder G. , Schaff Zs. , Chapman M. J. , Miyamura T. , Bréchot C.
Published: July 2002

There is now abundant evidence to substantiate an important role of hepatitis C virus (HCV) core protein in cellular gene expression as well as in the viral cycle. Thus the subcellular localization of this protein has important implications. However, several studies have shown controversial results: the HCV core has been, indeed, described as cytoplasmic or nuclear depending on the size of the protein or on the genotype analyzed. We have studied the localization of the HCV core protein in two different cell lines, one nonhepatic (CHO) and the other hepatic (HepG2). Double immunofluorescence staining using a nuclear membrane marker and confocal analysis showed the core protein pattern to be cytoplasmic and globular. This pattern is not cell cycle-regulated. Electron microscopy analysis revealed the nature of the globular staining observed in immunofluorescence. The HCV core protein accumulated at the surface of lipid droplets that were also the unique morphological feature of nonhepatic core transfected cells. The lipid droplets were isolated by sequential ultracentrifugation on the basis of their density; biochemical analysis revealed a prevalence of triglycerides. In addition the core protein colocalized with apolipoprotein AII at the surface of the lipid droplets as revealed by confocal microscopy. Moreover analysis of liver biopsies from chronically HCV-infected chimpanzees revealed that HCV core is cytoplasmic and localized on the endoplasmic reticulum and on lipid droplets. These results clearly define the subcellular localization of the HCV core protein and suggest a relationship between the expression of the HCV core protein and cellular lipid metabolism.