bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell functions in granulocyte/macrophage colony-stimulating factor deficient mice
Authors: Wada Hisashi , Noguchi Yuji , Marino Michael W. , Dunn Ashley R. , Old Lloyd J.
Published: July 2002

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF −/− mice. To investigate the responses of CD8+and CD4+T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8+T cells with specificity for ovalbumin peptide could not be induced in GM-CSF −/− mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF −/− mice. Purified CD4+T cells from GM-CSF −/− mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-γ (IFN-γ) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4+T cells from GM-CSF −/− mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-γ and IL-4. To analyze the rescue effect of DC on CD4+T cells, supernatants from (i) CD4+T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4+T cells and KLH-pulsed spleen cells from GM-CSF −/− mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-γ production of CD4+T cells from GM-CSF −/− mice.
Stochastic mechanisms in gene expression
Authors: McAdams Harley H. , Arkin Adam
Published: July 2002

In cellular regulatory networks, genetic activity is controlled by molecular signals that determine when and how often a given gene is transcribed. In genetically controlled pathways, the protein product encoded by one gene often regulates expression of other genes. The time delay, after activation of the first promoter, to reach an effective level to control the next promoter depends on the rate of protein accumulation. We have analyzed the chemical reactions controlling transcript initiation and translation termination in a single such “genetically coupled” link as a precursor to modeling networks constructed from many such links. Simulation of the processes of gene expression shows that proteins are produced from an activated promoter in short bursts of variable numbers of proteins that occur at random time intervals. As a result, there can be large differences in the time between successive events in regulatory cascades across a cell population. In addition, the random pattern of expression of competitive effectors can produce probabilistic outcomes in switching mechanisms that select between alternative regulatory paths. The result can be a partitioning of the cell population into different phenotypes as the cells follow different paths. There are numerous unexplained examples of phenotypic variations in isogenic populations of both prokaryotic and eukaryotic cells that may be the result of these stochastic gene expression mechanisms.
Identification of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines
Authors: Siebert Paul D. , Huang Betty C. B.
Published: July 2002

Lactoferrin (LF), traditionally known as an iron-binding protein present in high concentrations in milk and various secretions, has emerged as a multifunctional protein involved in many aspects of the host defense against infection. Recently, LF has been shown to inhibit the growth of solid tumors and reduce experimental metastasis in mice, suggesting that LF also may play a role in the defense against tumorigenesis. Here we provide the sequence of the cDNA and promoter region, the chromosome assignment, and tissue expression pattern of a novel form of LF mRNA (ΔLF). The sequence of ΔLF mRNA is nearly identical to that of LF mRNA; however, at the 5′ end, we find a novel sequence that replaces the N-terminal signal peptide sequence of LF mRNA. We map the ΔLF mRNA to human chromosome 3 and find that both ΔLF and LF sequences colocalize to the same cloned 90- to 150-kb genomic DNA fragment. We further show that the ΔLF mRNA is the product of alternative splicing of the LF gene and likely is specified by use of an alternative promoter. Although we find ΔLF mRNA at various levels in 20 of 20 adult and fetal human tissues, we do not find ΔLF mRNA in any of 14 diverse tumor-derived cell lines.
Cell cycle independent interaction of CDC2 with the centrosome, which is associated with the nuclear matrix-intermediate filament scaffold
Authors: Pockwinse Shirwin M. , Krockmalnic Gabriela , Doxsey Stephen J. , Nickerson Jeffrey , Lian Jane B. , van Wijnen André J. , Stein Janet L. , Stein Gary S. , Penman Sheldon
Published: July 2002

The cell cycle regulating Cdc2 protein kinase helps orchestrate cell cycle dependent changes in cell structure and function. This report shows that Cdc2 is localized to the centrosome region and is tightly bound to the nuclear matrix-intermediate filament scaffold. Antibodies to Cdc2 and to the centrosome-specific protein, pericentrin, label the centrosome in an apparently cell cycle independent manner. Isolated centrosomes also label similarly with both antibodies. Essentially, all cells show Cdc2 labeling of the centrosomes, implying an independence of the stage in the cell cycle, a conclusion supported by studies of synchronized cells. In contrast to the labeling of every cell with the Cdc2 monoclonal antibody, fewer centrosomes were labeled with an antibody to the PSTAIRE domain of Cdc2. Embedment-free, immunogold electron micrographs of extracted cell whole mounts show the centrioles and a pericentriolar network of filaments. Both Cdc2 and pericentrin antibodies decorate the amorphous pericentriolar material, while the Cdc2 antibodies also decorate the centrioles themselves. The constitutive presence of Cdc2 at the centrosome suggests a continuing role in the dynamics of centrosome function throughout the cell cycle.
Differential expression of dystrophin isoforms and utrophin during dibutyryl-cAMP-induced morphological differentiation of rat brain astrocytes
Authors: Imamura Michihiro , Ozawa Eijiro
Published: July 2002

We have identified isoforms of dystrophin and utrophin, a dystrophin homologue, expressed in astrocytes and examined their expression patterns during dibutyryl-cAMP (dBcAMP)-induced morphological differentiation of astrocytes. Immunoblot and immunocytochemical analyses showed that full-length-type dystrophin (427 kDa), utrophin (395 kDa), and Dp71 (75 kDa), a small-type dystrophin isoform, were coexpressed in cultured nondifferentiated rat brain astrocytes and were found to be located in the cell membrane. During morphological differentiation of the astrocytes induced by 1 mM dBcAMP, the amount of Dp71 markedly increased, whereas that of dystrophin and utrophin decreased. Northern blot analyses revealed that dBcAMP regulates the mRNA levels of Dp71 and dystrophin but not that of utrophin. dBcAMP slightly increased the amount of the β-dystroglycan responsible for anchoring dystrophin isoforms and utrophin to the cell membrane. Immunocytochemical analyses showed that most utrophin was observed in the cytoplasmic area during astrocyte differentiation, whereas Dp71 was found along the cell membrane of the differentiated astrocytes. These findings suggest that most of the dystrophin/utrophin-dystroglycan complex on cell membrane in cultured astrocytes was replaced by the Dp71-dystroglycan complex during morphological differentiation. The cell biological roles of Dp71 are discussed.
Two distinct forms of long-term depression coexist at the mossy fiber-CA3 synapse in the hippocampus during development
Authors: Domenici Maria Rosaria , Berretta Nicola , Cherubini Enrico
Published: July 2002

During a critical period of postnatal development, between postnatal days 6 and 14, a high-frequency stimulation train (100 Hz for 1 s) to the mossy fibers induces a long-term depression (LTD) of synaptic efficacy of 29 ± 5.2%. This form of LTD is homosynaptic. It is independent of the activation of
N
-methyl-
d
-aspartate or metabotropic glutamate receptors but needs an increase in calcium into the postsynaptic cell for its induction. At the same synapse LTD also could be induced by low-frequency stimulation of the mossy fibers (1 Hz for 15 min). In this case the magnitude of the depression is 37 ± 4.2%. This form of LTD is
N
-methyl-
d
-aspartate independent but requires the activation of metabotropic glutamate receptors because it is prevented by (S)-α-methyl-4-carboxyphenylglycine (1 mM). Moreover its induction appears to be presynaptic, because, in contrast with the high-frequency one, it is not blocked by loading the postsynaptic cell with the calcium chelator EGTA or bis-(-
o
-aminophenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetic acid (BAPTA). Saturation of one form of LTD does not occlude the other, suggesting that high and low frequency-induced LTD depend on distinct mechanisms of induction and expression. Quantal (noise deconvolution) analysis of minimal excitatory postsynaptic potentials shows, similarly to high-frequency LTD, a decrease in quantal content without any change in quantal size after low-frequency LTD, suggesting that in both forms of LTD the site where maintenance mechanisms are located is presynaptic.
Targeted disruption of
Pax1
defines its null phenotype and proves haploinsufficiency
Authors: Wilm Bettina , Dahl Edgar , Peters Heiko , Balling Rudi , Imai Kenji
Published: July 2002

The murine paired box-containing gene
Pax1
is required for normal development of the vertebral column, the sternum, and the scapula. Previous studies have shown that three natural
Pax1
mouse mutants, the
undulated
alleles, exhibit phenotypes of different severity in these skeletal elements. Nevertheless, these analyses have not clarified whether the semidominant
Undulated short-tail
(
Un

s

) mutation, in which the complete
Pax1
locus is deleted, represents a null allele. Moreover, the analyses of the classical
undulated
mutants did not allow a conclusion with respect to haploinsufficiency of
Pax1
. To address both questions we have created a
Pax1
null allele in mice by gene targeting. Surprisingly, the phenotype of this defined mutation exhibits clear differences to that of
Un

s

. This result strongly indicates the contribution of additional gene(s) to the
Un

s

mutant phenotype. Furthermore, the phenotype of mice heterozygous for the null allele demonstrates that
Pax1
is haploinsufficient in some though not all skeletal elements which express
Pax1
during embryonic development.
Enhanced Gαq signaling: A common pathway mediates cardiac hypertrophy and apoptotic heart failure
Authors: Adams John W. , Sakata Yoshihito , Davis Michael G. , Sah Valerie P. , Wang Yibin , Liggett Stephen B. , Chien Kenneth R. , Brown Joan Heller , Dorn Gerald W.
Published: July 2002

Receptor-mediated Gq signaling promotes hypertrophic growth of cultured neonatal rat cardiac myocytes and is postulated to transduce
in vivo
cardiac pressure overload hypertrophy. Although initially compensatory, hypertrophy can proceed by unknown mechanisms to cardiac failure. We used adenoviral infection and transgenic overexpression of the alpha subunit of Gq to autonomously activate Gq signaling in cardiomyocytes. In cultured cardiac myocytes, overexpression of wild-type Gαq resulted in hypertrophic growth. Strikingly, expression of a constitutively activated mutant of Gαq, which further increased Gq signaling, produced initial hypertrophy, which rapidly progressed to apoptotic cardiomyocyte death. This paradigm was recapitulated during pregnancy in Gαq overexpressing mice and in transgenic mice expressing high levels of wild-type Gαq. The consequence of cardiomyocyte apoptosis was a transition from compensated hypertrophy to a rapidly progressive and lethal cardiomyopathy. Progression from hypertrophy to apoptosis
in vitro
and
in vivo
was coincident with activation of p38 and Jun kinases. These data suggest a mechanism in which moderate levels of Gq signaling stimulate cardiac hypertrophy whereas high level Gq activation results in cardiomyocyte apoptosis. The identification of a single biochemical stimulus regulating cardiomyocyte growth and death suggests a plausible mechanism for the progression of compensated hypertrophy to decompensated heart failure.
v-SNARE-dependent secretion is required for phagocytosis
Authors: Hackam David J. , Rotstein Ori D. , Sjolin Carola , Schreiber Alan D. , Trimble William S. , Grinstein Sergio
Published: July 2002

Phagosomes are generally believed to form by gradual apposition of the plasma membrane of leukocytes onto the surface of invading microorganisms. The internalization of the encapsulated particle is therefore predicted to reduce the surface area of the phagocyte. Contrary to this prediction, we observed that phagocytosis is associated with a net increase in cell surface area, suggesting the concomitant occurrence of exocytosis. Selective cleavage of components of the secretory machinery by microinjection or transfection of bacterial neurotoxins induced a pronounced inhibition of phagocytosis. These observations indicate that vesicle-solubleN-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis of endomembranes is essential for optimal completion of particle internalization during phagocytosis.
Chemotaxis in a gliding bacterium
Authors: Kearns Daniel B. , Shimkets Lawrence J.
Published: July 2002

Myxococcus xanthus
cells exhibit directed motility up phosphatidylethanolamine (PE) gradients, and we suggest that PE behaves as a chemoattractant. Computer-assisted stop-motion digital microscopy was used to record cell movements in slide culture. PE decreased cellular reversal frequency with molecular specificity that was correlated with the fatty acid composition. Synthetic dilauroyl (di C12:0) PE and dioleoyl (di C18:1 ω9c) PE suppressed direction reversals and stimulated movement up the gradient. Sensory adaptation occurred about 1 hr after the onset of stimulation. Null mutants in a methylated chemotaxis protein homolog (FrzCD) and a CheA/CheY homolog (FrzE) moved up a PE gradient at a reduced rate. The mutants displayed normal excitation but were defective in adaptation. A dominant, hyper-reversal mutant in the
M. xanthus
methyl accepting chemotaxis protein homolog,
frzCD224
, failed to respond to PE stimulation, which argued that PE was a transduced stimulus. Neither dilauroyl PE nor dioleoyl PE is present at high enough concentrations in vegetative or developmental PE to account for all of the chemotactic activity. It appears then that there are additional, as yet unknown, PE species that serve as autoattractants. We report on a discrete phospholipid chemoattractant in a gliding bacterium
Nitration of γ-tocopherol and oxidation of α-tocopherol by copper-zinc superoxide dismutase/H
2
O
2
/NO
2
−
: Role of nitrogen dioxide free radical
Authors: Singh Ravinder J. , Goss Steven P. A. , Joseph Joy , Kalyanaraman B.
Published: July 2002

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide (O
2
•−
) to O
2
and H
2
O
2
. In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of H
2
O
2
, leading to self-inactivation and formation of a potent enzyme-bound oxidant. We report in this study that lipid peroxidation of
l
-α-lecithin liposomes was enhanced greatly during the SOD/H
2
O
2
reaction in the presence of nitrite anion (NO
2
−
) with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of NO
2
−
also greatly enhanced α-tocopherol (α-TH) oxidation by SOD/H
2
O
2
in saturated 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UV-studies was α-tocopheryl quinone. When 1,2-diauroyl-sn-glycero-3-phosphatidylcholine liposomes containing γ-tocopherol (γ-TH) were incubated with SOD/H
2
O
2
/NO
2
−
, the major product identified was 5-NO
2
-γ-TH. Nitrone spin traps significantly inhibited the formation of α-tocopheryl quinone and 5-NO
2
-γ-TH. NO
2
−
inhibited H
2
O
2
-dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of NO
2
−
by an SOD-bound oxidant to the nitrogen dioxide radical (
•
NO
2
). In this study, we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that NO
2
−
is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis-linked SOD mutants.
Nerve growth factor acutely reduces chemical transmission by means of postsynaptic TrkA-like receptors in squid giant synapse
Authors: Moreno Herman , Nadal Marcela , Leznik Elena , Sugimori Mutsuyuki , Lax Irit , Schlessinger Joseph , Llinás Rodolfo
Published: July 2002

Tyrosine phosphorylation has been shown to be an important modulator of synaptic transmission in both vertebrates and invertebrates. Such findings hint toward the existence of extracellular ligands capable of activating this widely represented signaling mechanism at or close to the synapse. Examples of such ligands are the peptide growth factors which, on binding, activate receptor tyrosine kinases. To gain insight into the physiological consequences of receptor tyrosine kinase activation in squid giant synapse, a series of growth factors was tested in this preparation. Electrophysiological, pharmacological, and biochemical analysis demonstrated that nerve growth factor (NGF) triggers an acute and specific reduction of the postsynaptic potential amplitude, without affecting the presynaptic spike generation or presynaptic calcium current. The NGF target is localized at a postsynaptic site and involves a new TrkA-like receptor. The squid receptor crossreacts with antibodies generated against mammalian TrkA, is tyrosine phosphorylated in response to NGF stimulation, and is blocked by specific pharmacological inhibitors. The modulation described emphasizes the important role of growth factors on invertebrate synaptic transmission.
Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by α-toxin
Authors: Otto-Bruc Annie E. , Fariss Robert N. , Van Hooser J. Preston , Palczewski Krzysztof
Published: July 2002

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho → Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca
2+
ions, acting through Ca
2+
-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an
in vitro
assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca
2+
effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal α-toxin, including an α-toxin mutant that forms pores whose permeability is modulated by Zn
2+
. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer
N
-(2-aminoethyl) biotinamide hydrochloride. Application of [γ-
32
P]ATP to α-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca
2+
]
free
, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca
2+
]
free
conditions, even when rods were loaded with ATP and the pores were closed by Zn
2+
. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca
2+
and Ca
2+
-binding proteins, including recoverin.
rexB
of bacteriophage λ is an anti-cell death gene
Authors: Engelberg–Kulka Hanna , Reches Myriam , Narasimhan Sudarsan , Schoulaker-Schwarz Rachel , Klemes Yoel , Aizenman Einat , Glaser Gad
Published: July 2002

In
Escherichia coli
, programmed cell death is mediated through “addiction modules” consisting of two genes; the product of one gene is long-lived and toxic, whereas the product of the other is short-lived and antagonizes the toxic effect. Here we show that the product of λ
rexB
, one of the few genes expressed in the lysogenic state of bacteriophage λ, prevents cell death directed by each of two addiction modules,
phd-doc
of plasmid prophage P1 and the
rel mazEF
of
E. coli
, which is induced by the signal molecule guanosine 3′,5′-bispyrophosphate (ppGpp) and thus by amino acid starvation. λRexB inhibits the degradation of the antitoxic labile components Phd and MazE of these systems, which are substrates of ClpP proteases. We present a model for this anti-cell death effect of λRexB through its action on the ClpP proteolytic subunit. We also propose that the λ
rex
operon has an additional function to the well known phenomenon of exclusion of other phages; it can prevent the death of lysogenized cells under conditions of nutrient starvation. Thus, the
rex
operon may be considered as the “survival operon” of phage λ.
CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice
Authors: Millan Cynthia L. Brazolot , Weeratna Risini , Krieg Arthur M. , Siegrist Claire-Anne , Davis Heather L.
Published: July 2002

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1–14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0–10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.
Improvement of neurological deficits in 6-hydroxydopamine-lesioned rats after transplantation with allogeneic simian virus 40 large tumor antigen gene-induced immortalized dopamine cells
Authors: Clarkson Edward D. , Rosa Francisco G. La , Edwards-Prasad Judith , Weiland David A. , Witta Samir E. , Freed Curt R. , Prasad Kedar N.
Published: July 2002

The replacement of dopamine (DA) by DA neuron transplants in the treatment of advanced Parkinson disease (PD) is a rational approach. Because of limitations associated with fetal tissue transplants, a clone (1RB
3
AN
27
) of simian virus 40 large tumor antigen (LTa) gene-induced immortalized DA neurons were used in this study. These allogeneic immortalized dopamine neurons, when grafted into striata of normal rats, did not divide, did not form tumors, did not produce LTa, did not extend neurites to host neurons, and were not rejected, for as long as 13 months after transplantation. Grafted cells when recultured
in vitro
resumed cell proliferation and LTa production, suggesting the presence of a LTa gene-inhibiting factor in the brain. The grafting of undifferentiated and differentiated 1RB
3
AN
27
cells or differentiated murine neuroblastoma (NBP
2
) cells into striata of 6-hydroxydopamine-lesioned rats (an animal model of PD) caused a time-dependent improvement in neurological deficits (reduction in the methamphetamine-induced turning rate). At 3 months after transplantation, 100% of the animals receiving differentiated 1RB
3
AN
27
cells, 63% of the animals receiving undifferentiated 1RB
3
AN
27
cells, 56% of the animals receiving differentiated NBP
2
cells, and 0% of the sham-transplanted animals showed improvements in neurological deficits. At 6 months after transplantation, there was a progressive increase in spontaneous recovery in sham-transplanted animals. These results suggest that immortalized DA neurons should be further studied for their potential use in transplant therapy in advanced PD patients.
Induction of transporter associated with antigen processing by interferon γ confers endothelial cell cytoprotection against natural killer-mediated lysis
Authors: Ayalon Oran , Hughes Eric A. , Cresswell Peter , Lee Jennifer , O’Donnell Lynn , Pardi Ruggero , Bender Jeffrey R.
Published: July 2002

T lymphocytes react minimally with nonactivated endothelial cells (ECs). However, natural killer (NK) lymphocyte interactions with resting ECs are rapid, avid, and result in endothelial activation and/or cytotoxicity. The molecular basis for these interactions and EC sensitivity to NK-mediated lysis is unclear. To address the EC-specific nature of NK sensitivity, we used syngeneic human umbilical vein ECs, dermal microvascular ECs, dermal fibroblasts, and B lymphoblastoid cell lines in calcein-AM retention NK assays with allogeneic NK effector cells and found the EC lines consistently more NK-sensitive. Because NK inhibitory receptors are engaged by membrane major histocompatibility complex (MHC) I molecules and MHC I-deficient targets are NK-sensitive, we investigated the quantitative levels of membrane MHC I on the panel of syngeneic lines. Highly sensitive ECs expressed similar (or higher) levels of membrane MHC I than their syngeneic NK-resistant counterparts. Pretreatment of ECs with γ interferon (IFN-γ) conferred protection against NK-mediated lysis, with much more rapid kinetics (2–6 hr) than those required for membrane MHC I hyperinduction (>8 hr). These kinetics are consistent with induction of transporter associated with antigen processing (TAP) expression and function. As opposed to NK-resistant cell lines, TAP-1 was undetectable in resting ECs. Recombinant expression of the TAP inactivator ICP47 by adenoviral-mediated transduction was used to selectively inhibit IFN-γ-mediated EC TAP function. ICP47 expression abrogated EC cytoprotection conferred by IFN-γ. We demonstrate a relationship between both basal and induced TAP-1 expression/function and EC sensitivity to NK-mediated cytotoxicity. We discuss the influence of an induced MHC I-associated peptide repertoire on vascular vulnerability to cytotoxic lymphocytes.
High affinity ligands from
in vitro
selection: Complex targets
Authors: Morris Kevin N. , Jensen Kirk B. , Julin Carol M. , Weil Michael , Gold Larry
Published: July 2002

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an
in vitro
protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems.
Targeted disruption of p70s6kdefines its role in protein synthesis and rapamycin sensitivity
Authors: Kawasome Hideki , Papst Philip , Webb Saiphone , Keller Gordon M. , Johnson Gary L. , Gelfand Erwin W. , Terada Naohiro
Published: July 2002

Here, we disrupted the p70 S6 kinase (p70s6k) gene in murine embryonic stem cells to determine the role of this kinase in cell growth, protein synthesis, and rapamycin sensitivity. p70s6k−/−cells proliferated at a slower rate than parental cells, suggesting that p70s6khas a positive influence on cell proliferation but is not essential. In addition, rapamycin inhibited proliferation of p70s6k−/−cells, indicating that other events inhibited by the drug, independent of p70s6k, also are important for both cell proliferation and the action of rapamycin. In p70s6k−/−cells, which exhibited no ribosomal S6 phosphorylation, translation of mRNA encoding ribosomal proteins was not increased by serum nor specifically inhibited by rapamycin. In contrast, rapamycin inhibited phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1), general mRNA translation, and overall protein synthesis in p70s6k−/−cells, indicating that these events proceed independently of p70s6kactivity. This study localizes the function of p70s6kto ribosomal biogenesis by regulating ribosomal protein synthesis at the level of mRNA translation.
AtPCS1, a phytochelatin synthase fromArabidopsis: Isolation andin vitroreconstitution
Authors: Vatamaniuk Olena K. , Mari Stéphane , Lu Yu-Ping , Rea Philip A.
Published: July 2002

Phytochelatins, a class of posttranslationally synthesized peptides, play a pivotal role in heavy metal, primarily Cd2+, tolerance in plants and fungi by chelating these substances and decreasing their free concentrations. Derived from glutathione and related thiols by the action of γ-glutamylcysteine dipeptidyl transpeptidases (phytochelatin synthases; EC2.3.2.15), phytochelatins consist of repeating units of γ-glutamylcysteine followed by a C-terminal Gly, Ser, or β-Ala residue [poly-(γ-Glu-Cys)n-Xaa]. Here we report the suppression cloning of a cDNA (AtPCS1) fromArabidopsis thalianaencoding a 55-kDa soluble protein that enhances heavy-metal tolerance and elicits Cd2+-activated phytochelatin accumulation when expressed inSaccharomyces cerevisiae. On the basis of these properties and the sufficiency of immunoaffinity-purified epitope-tagged AtPCS1 polypeptide for high rates of Cd2+-activated phytochelatin synthesis from glutathionein vitro, AtPCS1 is concluded to encode the enzyme phytochelatin synthase.
Constitutive cell surface association between CD4 and CCR5
Authors: Xiao Xiaodong , Wu Lijun , Stantchev Tzanko S. , Feng Yan-Ru , Ugolini Sophie , Chen Hong , Shen Zhimin , Riley James L. , Broder Christopher C. , Sattentau Quentin J. , Dimitrov Dimiter S.
Published: July 2002

HIV-1 entry into cells involves formation of a complex between gp120 of the viral envelope glycoprotein (Env), a receptor (CD4), and a coreceptor. For most strains of HIV, this coreceptor is CCR5. Here, we provide evidence that CD4 is specifically associated with CCR5 in the absence of gp120 or any other receptor-specific ligand. The amount of CD4 coimmunoprecipitated with CCR5 was significantly higher than that with the other major HIV coreceptor, CXCR4, and in contrast to CXCR4 the CD4–CCR5 coimmunoprecipitation was not significantly increased by gp120. The CD4–CCR5 interaction probably takes place via the second extracellular loop of CCR5 and the first two domains of CD4. It can be inhibited by CCR5- and CD4-specific antibodies that interfere with HIV-1 infection, indicating a possible role in virus entry. These findings suggest a possible pathway of HIV-1 evolution and development of immunopathogenicity, a potential new target for antiretroviral drugs and a tool for development of vaccines based on Env–CD4–CCR5 complexes. The constitutive association of a seven-transmembrane-domain G protein-coupled receptor with another receptor also indicates new possibilities for cross-talk between cell surface receptors.
Retinoid X receptors are essential for early mouse development and placentogenesis
Authors: Wendling Olivia , Chambon Pierre , Mark Manuel
Published: July 2002

Embryos carrying null mutations of both retinoid X receptors α and β (RXRα
−/−
/RXRβ
−/−
mutants) were generated. These mutant embryos die between 9.5 and 10.5 days of gestation and display a wide range of abnormalities. The cause of the lethality appears to be the lack of formation of the labyrinthine zone of the chorioallantoic placenta. In a thorough analysis of mutant conceptuses, we establish that RXRs, through heterodimerization with retinoic acid receptors, are essential for postimplantation embryonic development before placentogenesis. RXRs are also essential for the formation of the chorioallantoic placenta, most probably through RXR/peroxisomal proliferator-activated receptor-γ heterodimers. Interestingly, as a RXR ligand appears dispensable, placentogenesis must be controlled by a yet unknown hormonal ligand(s) activating the heterodimerization partner(s) of RXRs.
Lpsd/Ran of endotoxin-resistant C3H/HeJ mice is defective in mediating lipopolysaccharide endotoxin responses
Authors: Wong Peter M. C. , Kang Anthony , Chen Hong , Yuan Quan , Fan Peidong , Sultzer Barnet M. , Kan Yuet Wai , Chung Siu-Wah
Published: July 2002

C3H/HeJ inbred mice are defective in that they are highly resistant to endotoxic shock as compared with normal responder mice. Their B cells and macrophages do not respond significantly when exposed to lipopolysaccharide (LPS), whereas cells from the responder mice do. Using a functional assay, we previously isolated a cDNA, which encodes for Ran/TC4 GTPase. We now show that this gene is mutated in C3H/HeJ mice, which accounts for their resistance to endotoxin stimulation. Sequence analysis of independent mutant Lpsd/Ran cDNAs isolated from splenic B cells of C3H/HeJ mice reveals a consistent single base substitution at position 870, where a thymidine is replaced with a cytidine.In situhybridization maps the Lpsd/Ran cDNA to mouse chromosome 4. By retroviral gene transfer, the wild-type Lpsn/Ran cDNA but not the mutant Lpsd/Ran cDNA can restore LPS responsiveness of C3H/HeJ cells. Adenoviral gene transferin vivowith the mutant Lpsd/Ran cDNA but not the wild-type Lpsn/Ran cDNA rescues endotoxin-sensitive mice from septic shock. Thus Lps/Ran is an important target for LPS-mediated signal transduction, and theLpsd/Rangene may be useful as a therapeutic sequence in gene therapy for endotoxemia and septic shock.
Monitoring the assembly of Ig light-chain amyloid fibrils by atomic force microscopy
Authors: Ionescu-Zanetti Cristian , Khurana Ritu , Gillespie Joel R. , Petrick Jay S. , Trabachino Lynne C. , Minert Lauren J. , Carter Sue A. , Fink Anthony L.
Published: July 2002

Aggregation of Ig light chains to form amyloid fibrils is a characteristic feature of light-chain amyloidosis, a light-chain deposition disease. A recombinant variable domain of the light chain SMA was used to form amyloid fibrilsin vitro. Fibril formation was monitored by atomic force microscopy imaging. Single filaments 2.4 nm in diameter were predominant at early times; protofibrils 4.0 nm in diameter were predominant at intermediate times; type I and type II fibrils 8.0 nm and 6.0 nm in diameter, respectively, were predominant at the endpoints. The increase in number of fibrils correlated with increased binding of the fluorescent dye thioflavin T. The fibrils and protofibrils showed a braided structure, suggesting that their formation involves the winding of protofibrils and filaments, respectively. These observations support a model in which two filaments combine to form a protofibril, two protofibrils intertwine to form a type I fibril, and three filaments form a type II fibril.
Role of the Y5 neuropeptide Y receptor in limbic seizures
Authors: Marsh Donald J. , Baraban Scott C. , Hollopeter Gunther , Palmiter Richard D.
Published: July 2002

Neuropeptide Y (NPY) is an inhibitory neuromodulator expressed abundantly in the central nervous system that is suspected of being an endogenous antiepileptic agent that can control propagation of limbic seizures. Electrophysiological and pharmacological data suggest that these actions of NPY are mediated by G protein-coupled NPY Y2 and NPY Y5 receptors. To determine whether the NPY Y5 receptor (Y5R) is required for normal control of limbic seizures, we examined hippocampal function and responsiveness to kainic acid-induced seizures in Y5R-deficient (
Y5R−/−
) mice. We report that
Y5R−/−
mice do not exhibit spontaneous seizure-like activity; however, they are more sensitive to kainic acid-induced seizures. Electrophysiological examination of hippocampal slices from mutant mice revealed normal function, but the antiepileptic effects of exogenously applied NPY were absent. These data demonstrate that Y5R has an important role in mediating NPY’s inhibitory actions in the mouse hippocampus and suggest a role for Y5R in the control of limbic seizures.
Localizing the cortical region mediating visual awareness of object identity
Authors: Bar Moshe , Biederman Irving
Published: July 2002

Presentations of pictures that are too brief to be recognized, or
even guessed above chance on a forced-choice test, nonetheless can
facilitate the recognition of the same pictures many trials later. This
subliminal visual priming was compared for images translated 4.8°
either
Within
or
Between
quadrants of the
visual field. Priming was evident only for images that remained within
the same quadrant in priming and test trials. Consequently, subliminal
visual priming is likely mediated by cortical areas in which cells have
receptive fields large enough to respond to both presentations of a
stimulus translated almost 5°, yet where the receptive fields are
confined to a single quadrant, namely, the human homologue of macaque
V4 or TEO (the posterior part of the inferior temporal cortex).
Awareness of object identity might therefore be associated exclusively
with activity at or beyond the anterior part of the inferior temporal
cortex, namely, area TE.
The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein β subunit
Authors: Makino Elina R. , Handy Jason W. , Li Tiansen , Arshavsky Vadim Y.
Published: July 2002

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein α subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Here we report that functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein β subunit (G
β
5L
). RGS9 and G
β
5L
also form a complex when coexpressed in cell culture. Our data are consistent with the recent observation that several RGS proteins, including RGS9, contain G protein γ-subunit like domain that can mediate their association with G
β
5
(Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. & Siderovski, D. P. (1998)
Proc. Natl. Acad. Sci. USA
95, 13307–13312). We report an example of such a complex whose cellular localization and function are clearly defined.
Regulation of G1progression by thePTENtumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway
Authors: Ramaswamy Shivapriya , Nakamura Noriaki , Vazquez Francisca , Batt David B. , Perera Sauni , Roberts Thomas M. , Sellers William R.
Published: July 2002

PTEN/MMAC1is a tumor suppressor gene located on chromosome 10q23. InheritedPTEN/MMAC1mutations are associated with a cancer predisposition syndrome known as Cowden’s disease. Somatic mutation of PTEN has been found in a number of malignancies, including glioblastoma, melanoma, and carcinoma of the prostate and endometrium. The protein product (PTEN) encodes a dual-specificity protein phosphatase and in addition can dephosphorylate certain lipid substrates. Herein, we show that PTEN protein induces a G1block when reconstituted inPTEN-null cells. APTENmutant associated with Cowden’s disease (PTEN;G129E) has protein phosphatase activity yet is defective in dephosphorylating inositol 1,3,4,5-tetrakisphosphatein vitroand fails to arrest cells in G1. These data suggest a link between induction of a cell-cycle block by PTEN and its ability to dephosphorylate,in vivo,phosphatidylinositol 3,4,5-trisphosphate. In keeping with this notion, PTEN can inhibit the phosphatidylinositol 3,4,5-trisphosphate-dependent Akt kinase, a downstream target of phosphatidylinositol 3-kinase, and constitutively active, but not wild-type, Akt overrides a PTEN G1arrest. Finally, tumor cells lacking PTEN contain high levels of activated Akt, suggesting that PTEN is necessary for the appropriate regulation of the phosphatidylinositol 3-kinase/Akt pathway.
Organization of human and mouse skeletal myosin heavy chain gene clusters is highly conserved
Authors: Weiss Allison , McDonough Debra , Wertman Brett , Acakpo-Satchivi Leslie , Montgomery Kate , Kucherlapati Raju , Leinwand Leslie , Krauter Kenneth
Published: July 2002

Myosin heavy chains (MyHCs) are highly conserved ubiquitous actin-based motor proteins that drive a wide range of motile processes in eukaryotic cells. MyHC isoforms expressed in skeletal muscles are encoded by a multigene family that is clustered on syntenic regions of human and mouse chromosomes 17 and 11, respectively. In an effort to gain a better understanding of the genomic organization of the skeletal MyHC genes and its effects on the regulation, function, and molecular genetics of this multigene family, we have constructed high-resolution physical maps of both human and mouse loci using PCR-based marker content mapping of P1-artificial chromosome clones. Genes encoding six MyHC isoforms have been mapped with respect to their linear order and transcriptional orientations within a 350-kb region in both human and mouse. These maps reveal that the order, transcriptional orientation, and relative intergenic distances of these genes are remarkably conserved between these species. Unlike many clustered gene families, this order does not reflect the known temporal expression patterns of these genes. However, the conservation of gene organization since the estimated divergence of these species (≈75–110 million years ago) suggests that the physical organization of these genes may be significant for their regulation and function.
Global curvature, thickness, and the ideal shapes of knots
Authors: Gonzalez Oscar , Maddocks John H.
Published: July 2002

The global radius of curvature of a space curve is introduced. This function is related to, but distinct from, the standard local radius of curvature and is connected to various physically appealing properties of a curve. In particular, the global radius of curvature function provides a concise characterization of the thickness of a curve, and of certain ideal shapes of knots as have been investigated within the context of DNA.
Human monoclonal antibodies reactive to oligodendrocytes promote remyelination in a model of multiple sclerosis
Authors: Warrington Arthur E. , Asakura Kunihiko , Bieber Allan J. , Ciric Bogoljub , Van Keulen Virginia , Kaveri Srini V. , Kyle Robert A. , Pease Larry R. , Rodriguez Moses
Published: July 2002

Promoting remyelination, a major goal of an effective treatment for demyelinating diseases, has the potential to protect vulnerable axons, increase conduction velocity, and improve neurologic deficits. Strategies to promote remyelination have focused on transplanting oligodendrocytes (OLs) or recruiting endogenous myelinating cells with trophic factors. Ig-based therapies, routinely used to treat a variety of neurological and autoimmune diseases, underlie our approach to enhance remyelination. We isolated two human mAbs directed against OL surface antigens that promoted significant remyelination in a virus-mediated model of multiple sclerosis. Four additional OL-binding human mAbs did not promote remyelination. Both human mAbs were as effective as human i.v. Ig, a treatment shown to have efficacy in multiple sclerosis, and bound to the surface of human OLs suggesting a direct effect of the mAbs on the cells responsible for myelination. Alternatively, targeting human mAbs to areas of central nervous system (CNS) pathology may facilitate the opsonization of myelin debris, allowing repair to proceed. Human mAbs were isolated from the sera of individuals with a form of monoclonal gammopathy. These individuals carry a high level of monoclonal protein in their blood without detriment, lending support to the belief that administration of these mAbs as a therapy would be safe. Our results are (
i
) consistent with the hypothesis that CNS-reactive mAbs, part of the normal Ig repertoire in humans, may help repair and protect the CNS from pathogenic immune injury, and (
ii
) further challenge the premise that Abs that bind OLs are necessarily pathogenic.
Bad bugs and beleaguered bladders: Interplay between uropathogenicEscherichia coliand innate host defenses
Authors: Mulvey Matthew A. , Schilling Joel D. , Martinez Juan J. , Hultgren Scott J.
Published: July 2002

Strains of uropathogenicEscherichia coli(UPEC) are the causative agents in the vast majority of all urinary tract infections. Upon entering the urinary tract, UPEC strains face a formidable array of host defenses, including the flow of urine and a panoply of antimicrobial factors. To gain an initial foothold within the bladder, most UPEC strains encode filamentous surface adhesive organelles called type 1 pili that can mediate bacterial attachment to, and invasion of, bladder epithelial cells. Invasion provides UPEC with a protective environment in which bacteria can either replicate or persist in a quiescent state. Infection with type 1-piliatedE. colican trigger a number of host responses, including cytokine production, inflammation, and the exfoliation of infected bladder epithelial cells. Despite numerous host defenses and even antibiotic treatments that can effectively sterilize the urine, recent studies demonstrate that uropathogens can persist within the bladder tissue. These bacteria may serve as a reservoir for recurrent infections, a common problem affecting millions each year.
Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor
Authors: Diamond Marc I. , Robinson Melissa R. , Yamamoto Keith R.
Published: July 2002

Spinobulbar muscular atrophy and Huntington's disease are caused by polyglutamine expansion in the androgen receptor and huntingtin, respectively, and their pathogenesis has been associated with abnormal nuclear localization and aggregation of truncated forms of these proteins. Here we show, in diverse cell types, that glucocorticoids can up- or down-modulate aggregation and nuclear localization of expanded polyglutamine polypeptides derived from the androgen receptor and huntingtin through specific regulation of gene expression. Wild-type glucocorticoid receptor (GR), as well as C-terminal deletion derivatives, suppressed the aggregation and nuclear localization of these polypeptides, whereas mutations within the DNA binding domain and N terminus of GR abolished this activity. Surprisingly, deletion of a transcriptional regulatory domain within the GR N terminus markedly increased aggregation and nuclear localization of the expanded polyglutamine proteins. Thus, aggregation and nuclear localization of expanded polyglutamine proteins are regulated cellular processes that can be modulated by a well-characterized transcriptional regulator, the GR. Our findings suggest approaches to study the molecular pathogenesis and selective neuronal degeneration of polyglutamine expansion diseases.