bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of growth factor receptor function by isoform heterodimerization.
Authors: Chang W P , Clevenger C V
Published: July 2002

Activation of prolactin (PRL)-dependent signaling occurs as the result of ligand-induced dimerization of receptor (PRLr). Although three PRLr isoforms (short, intermediate, and long) have been characterized and are variably coexpressed in PRL-responsive tissues, the functional effects of ligand-induced PRLr isoform heterodimerization have not been examined. To determine whether heterodimeric PRLr complexes were capable of ligand-induced signaling and cellular proliferation, chimeras consisting of the extracellular domain of either the alpha or beta subunit of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFr) and the intracellular domain of the rat intermediate or short PRLr isoforms (PRLr-I or PRLr-S) were synthesized. Because high affinity binding of GM-CSF is mediated by the extracellular domain of one alpha and beta GM-CSFr pair, use of GM-CSFr/PRLr chimera specifically directed the dimerization of the PRLr intracellular domains within ligand-receptor complexes. Stable transfection of these constructs into the Ba/F3 line was demonstrated by Northern blot and immunoprecipitation analyses. Flow cytometry revealed specific binding of a phycoerythrin-conjugated human GM-CSF to the transfectants, confirming cell surface expression of the chimeric receptors. When tested for their ability to proliferate in response to GM-CSF, only chimeric transfectants expressing GM-CSFr/PRLr-I homodimers demonstrated significant [3H]thymidine incorporation. GM-CSF stimulation of transfectants expressing either GM-CSFr/PRLr-S homodimers or GM-CSFr/PRLr-S+1 heterodimers failed to induce proliferation. Consistent with these data, the GM-CSF-induced activation of two phosphotyrosine kinases, Jak2 and Fyn, was observed only in homodimeric GM-CSFr/PRLr-I transfectants. These results show that the PRLr-S functions as a dominant negative isoform, down-regulating both signaling and proliferation mediated by the receptor complex. Thus, structural motifs necessary for Jak2 and Fyn activation within the carboxy terminus of the PRLr-I, absent in the PRLr-S, are required in each member of the dimeric PRLr complex.
Loss of oncogenic ras expression does not correlate with loss of tumorigenicity in human cells.
Authors: Plattner R , Anderson M J , Sato K Y , Fasching C L , Der C J , Stanbridge E J
Published: July 2002

ras oncogenes are mutated in at variety of human tumors, which suggests that they play an important role in human carcinogenesis. To determine whether continued oncogenic ras expression is necessary to maintain the malignant phenotype, we studied the human fibrosarcoma cell line, HT1080, which contains one mutated and one wild-type N-ras allele. We isolated a variant of this cell line that no longer contained the mutated copy of the N-ras gene. Loss of mutant N-ras resulted in cells that displayed a less transformed phenotype characterized by a flat morphology, decreased growth rate, organized actin stress fibers, and loss of anchorage-independent growth. The transformed phenotype was restored following reintroduction of mutant N-ras. Although loss of the oncogenic N-ras drastically affected in vitro growth parameters, the variant remained tumorigenic in nude mice indicating that mutated N-ras expression is not necessary for maintenance of the tumorigenic phenotype. We confirmed this latter observation in colon carcinoma cell lines that have lost activated K-ras expression via targeted knockout of the mutant K-ras gene.
Molecular identity and cellular distribution of advanced glycation endproduct receptors: relationship of p60 to OST-48 and p90 to 80K-H membrane proteins.
Authors: Li Y M , Mitsuhashi T , Wojciechowicz D , Shimizu N , Li J , Stitt A , He C , Banerjee D , Vlassara H
Published: July 2002

Advanced glycation endproducts (AGEs) are derivatives of nonenzymatic reactions between sugars and protein or lipids, and together with AGE-specific receptors are involved in numerous pathogenic processes associated with aging and hyperglycemia. Two of the known AGE-binding proteins isolated from rat liver membranes, p60 and p90, have been partially sequenced. We now report that the N-terminal sequence of p60 exhibits 95% identity to OST-48, a 48-kDa member of the oligosaccharyltransferase complex found in microsomal membranes, while sequence analysis of p90 revealed 73% and 85% identity to the N-terminal and internal sequences, respectively, of human 80K-H, a 80- to 87-kDa protein substrate for protein kinase C. AGE-ligand and Western analyses of purified oligosaccharyltransferase complex, enriched rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membranes from rat liver or RAW 264.7 macrophages yielded a single protein of approximately 50 kDa recognized by both anti-p60 and anti-OST-48 antibodies, and also exhibited AGE-specific binding. Immunoprecipitated OST-48 from rat rough endoplasmic reticulum fractions exhibited both AGE binding and immunoreactivity to an anti-p60 antibody. Immune IgG raised to recombinant OST-48 and 80K-H inhibited binding of AGE-bovine serum albumin to cell membranes in a dose-dependent manner. Immunostaining and flow cytometry demonstrated the surface expression of OST-48 and 80K-H on numerous cell types and tissues, including mononuclear, endothelial, renal, and brain neuronal and glial cells. We conclude that the AGE receptor components p60 and p90 are identical to OST-48, and 80K-H, respectively, and that they together contribute to the processing of AGEs from extra- and intracellular compartments and in the cellular responses associated with these pathogenic substances.
Two contact regions between Stat1 and CBP/p300 in interferon γ signaling
Authors: Zhang Jue J. , Vinkemeier Uwe , Gu Wei , Chakravarti Debabrata , Horvath Curt M. , Darnell James E.
Published: July 2002

Interferon γ (IFN-γ) induces rapid tyrosine phosphorylation of
the latent cytoplasmic transcription factor, Stat1, which then forms
homodimers, translocates to the nucleus and participates in
IFN-γ-induced transcription. However, little is known of the
interactions between Stat1 and the general transcription machinery
during transcriptional activation. We show here that Stat1 can directly
interact with the CREB-binding protein (CBP)/p300 family of
transcriptional coactivators. Specifically, two interaction regions
were identified: the amino-terminal region of Stat1 interacts with the
CREB-binding domain of CBP/p300 and the carboxyl-terminal region of
Stat1 interacts with the domain of CBP/p300 that binds adenovirus E1A
protein. Transfection experiments suggest a role for these interactions
in IFN-γ-induced transcription. Because CBP/p300-binding is
required for the adenovirus E1A protein to regulate transcription of
many genes during viral replication and cellular transformation, it is
possible that the anti-viral effect of IFN-γ is based at least in
part on direct competition by nuclear Stat1 with E1A for CBP/p300
binding.
l-Selectin activates the Ras pathway via the tyrosine kinase p56lck
Authors: Brenner B. , Gulbins E. , Schlottmann K. , Koppenhoefer U. , Busch G. L. , Walzog B. , Steinhausen M. , Coggeshall K. M. , Linderkamp O. , Lang F.
Published: July 2002

Selectins mediate rolling, the initial step of leukocyte adhesion to endothelial cells [Springer, T. A. (1995)Annu. Rev. Physiol. 57, 827–872 and Butcher, E. C. (1991)Cell67, 1033–1036]. In this study we show thatl-selectin triggering of Jurkat cells using different antibodies or glycomimetics resulted in activation of the src-tyrosine kinase p56lck; tyrosine phosphorylation of intracellular proteins, in particular mitogen-activating protein kinase andl-selectin; and association of Grb2/Sos withl-selectin. This association correlated with an activation of p21Ras, mitogen-activating protein kinase, Rac2, and a transient increase of O2−synthesis. Stimulation of the Ras pathway byl-selectin requires functional p56lck, since p56lck-deficient Jurkat cells (JCaM1.6) do not show tyrosine phosphorylation, association ofl-selectin with Grb2/Sos, and activation of Ras uponl-selectin triggering. Transfection of JCaM1.6 cells with p56lckreconstitutes the observed signaling events. Genetic inhibition of Ras or Rac2 prevented Rac2 stimulation and O2−synthesis, respectively. The specificity and the physiological significance of the observed signaling cascade is indicated by stimulation ofl-selectin-transfected P815,l-selectin-positive CEM or peripheral blood lymphocytes resulting in the same activation events as in Jurkat cells. Our results point to a signaling cascade froml-selectin via p56lck, Grb2/Sos, Ras, and Rac2 to O2−.
Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy.
Authors: Black M E , Newcomb T G , Wilson H M , Loeb L A
Published: July 2002

Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer. This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 thymidine kinase. However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 thymidine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs. A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.
P-glycoprotein: a major determinant of rifampicin-inducible expression of cytochrome P4503A in mice and humans.
Authors: Schuetz E G , Schinkel A H , Relling M V , Schuetz J D
Published: July 2002

The P-glycoprotein (Pgp) efflux pump can influence the hepatocellular concentration of xenobiotics that are modulators and substrates of cytochrome P4503A (CYP3A). We tested the hypothesis that Pgp is a determinant of drug-inducible expression of CYP3A. The magnitude of CYP3A induction by rifampicin was compared in the human parental colon carcinoma cell line LS 180/WT (wild type) and in two derivative clones overexpressing the human multidrug resistance gene MDR1 (also designated PGY1) because of either drug selection (LS 180/ADR) or transfection with MDRI cDNA (LS 180/MDR). In both MDR1 cDNA-overexpressing clones, rifampicin induction of CYP3A mRNA and protein was decreased and required greater rifampicin concentrations compared with parental cells. The role of Pgp in regulation of CYP3A expression in vivo was analyzed in mice carrying a targeted disruption of the mdr1a mouse gene. Oral treatment with increasing doses of rifampicin resulted in elevated drug levels in the livers of mdr1a (-/-) mice compared with mdr1a (+/+) mice at all doses. Consistent with the enhanced accumulation of rifampicin in mdr1a (-/-) mice, lower doses of rifampicin were required for induction of CYP3A proteins, and the magnitude of CYP3A induction was greater at all doses of rifampicin in mdr1a (-/-) mice compared with mdr1a (+/+) mice. We conclude that Pgp-mediated transport is a critical element influencing the CYP3A inductive response.
On signal sequence polymorphisms and diseases of distribution.
Authors: Rosenblum J S , Gilula N B , Lerner R A
Published: July 2002

We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.
Head direction cells and episodic spatial information in rats without a hippocampus
Authors: Golob Edward J. , Taube Jeffrey S.
Published: July 2002

To successfully navigate through the environment animals rely on information concerning their directional heading and location. Many cells within the postsubiculum and anterior thalamus discharge as a function of the animal’s head direction (HD), while many cells in the hippocampus discharge in relation to the animal’s location. We placed lesions in the hippocampus and recorded from HD cells in the postsubiculum and anterior thalamus. Lesions of the hippocampus did not disrupt the HD cell signal in either brain area, indicating that the HD cell signal must be generated by structures external to the hippocampus. In addition, each cell’s preferred firing direction remained stable across days when the lesioned animal was placed into a novel environment. This stability appeared to weaken after several weeks of nonexposure to the new enclosure for two out of five animals, and subsequently recorded cells from these two animals established a new angular relationship between the familiar and novel environments. Our results suggest that extra-hippocampal structures are capable of creating and maintaining a novel representation of the animal’s environmental context. This representation shares features in common with mnemonic processes involving episodic memory that until now were assumed to require an intact hippocampus.
Cloning and characterization of
hOGG1
, a human homolog of the
OGG1
gene of
Saccharomyces cerevisiae
Authors: Radicella J. Pablo , Dherin Claudine , Desmaze Chantal , Fox Maurice S. , Boiteux Serge
Published: July 2002

The
OGG1
gene of
Saccharomyces cerevisiae
encodes a DNA glycosylase activity that is a functional analog of the Fpg protein from
Escherichia coli
and excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged DNA. The repair of this ubiquitous kind of oxidative damage is essential to prevent mutations both in bacteria and in yeast. A human cDNA clone carrying an ORF displaying homology to the yeast protein was identified. The predicted protein has 345 amino acids and a molecular mass of 39 kDa. This protein shares a 38% sequence identity with the yeast Ogg1 protein, adding this novel human gene product to the growing family of enzymes that the repair of oxidatively damaged bases and are related to the
E. coli
endonuclease III. Northern blot analysis indicates that this gene, localized to chromosome 3p25, is ubiquitously expressed in human tissues. The cloned coding sequence was expressed in an
E. coli
strain that carried a disrupted
fpg
gene, the bacterial functional analog of
OGG1
. Cell-free extracts from these cultures displayed a specific lyase activity on duplex DNA that carried an 8-oxoG/C base pair. The products of the reaction are consistent with an enzymatic activity like the one displayed by the yeast Ogg1. Analysis of the substrate specificity reveals a very strong preference for DNA fragments harboring 8-oxoG/C base pairs. The pattern of specificity correlates well with the one found for the yeast enzyme. Moreover, when the human coding sequence was expressed in a yeast strain mutant in
OGG1
it was able to complement the spontaneous mutator phenotype. These results make this novel gene (
hOGG1
) a strong candidate for the human homolog of the yeast
OGG1
and suggest an important role of its product in the protection of the genome from the mutagenic effects of the oxidatively damaged purines.
Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee
Authors: Yanagi Masayuki , Purcell Robert H. , Emerson Suzanne U. , Bukh Jens
Published: July 2002

We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV). We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. However, we found no evidence for HCV replication. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6–28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV.
The role of polyamine catabolism in polyamine analogue-induced programmed cell death
Authors: Ha Hyo Chol , Woster Patrick M. , Yager James D. , Casero Robert A.
Published: July 2002

N1-ethyl-N11-[(cyclopropyl)methyl]-4,8,-diazaundecane (CPENSpm) is a polyamine analogue that represents a new class of antitumor agents that demonstrate phenotype-specific cytotoxic activity. However, the precise mechanism of its selective cytotoxic activity is not known. CPENSpm treatment results in the superinduction of the polyamine catabolic enzyme spermidine/spermineN1-acetyltransferase (SSAT) in sensitive cell types and has been demonstrated to induce programmed cell death (PCD). The catalysis of polyamines by the SSAT/polyamine oxidase (PAO) pathway produces H2O2as one product, suggesting that PCD produced by CPENSpm may be, in part, due to oxidative stress as a result of H2O2production. In the sensitive human nonsmall cell line H157, the coaddition of catalase significantly reduces high molecular weight (HMW) DNA (≥50 kb) and nuclear fragmentation. Important to note, specific inhibition of PAO byN,N′-bis(2,3-butadienyl)-1,4-butane-diamine results in a significant reduction of the formation of HMW DNA and nuclear fragmentation. In contrast, the coaddition of catalase or PAO inhibitor has no effect on reducing HMW DNA fragmentation induced byN1-ethyl-N11-[(cycloheptyl)methyl]-4,8,-diazaundecane, which does not induce SSAT and does not deplete intracellular polyamines. These results strongly suggest that H2O2production by PAO has a role in CPENSpm cytotoxicity in sensitive cells via PCD and demonstrate a potential basis for differential sensitivity to this promising new class of antineoplastic agents. Furthermore, the data suggest a general mechanism by which, under certain stimuli, cells can commit suicide through catabolism of the ubiquitous intracellular polyamines.
Protein–protein interactions among the Aux/IAA proteins
Authors: Kim Jungmook , Harter Klaus , Theologis Athanasios
Published: July 2002

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The
Aux/IAA
family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic βαα DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and
Arabidopsis
IAA1 and IAA2 proteins perform homo- and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers
in vitro.
Deletion analysis of PS-IAA4 indicates that the βαα containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an
Arabidopsis
λ-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the
Arabidopsis Aux/IAA
gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the
Aux/IAA
gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.
DNA methylation and genetic instability in colorectal cancer cells
Authors: Lengauer Christoph , Kinzler Kenneth W. , Vogelstein Bert
Published: July 2002

Apparent alterations in DNA methylation have been observed in many cancers, but whether such alterations represent a persistent alteration in the normal methylation process is not known. In this study, we report a striking difference in the expression of exogenously introduced retroviral genes in various colorectal cancer cell lines. Extinguished expression was associated with DNA methylation and could be reversed by treatment with the demethylating agent 5-azacytidine. A striking correlation between genetic instability and methylation capacity suggested that methylation abnormalities may play a role in chromosome segregation processes in cancer cells.
AP-1 transcriptional activity is regulated by a direct association between thioredoxin and Ref-1
Authors: Hirota Kiichi , Matsui Minoru , Iwata Satoshi , Nishiyama Akira , Mori Kenjiro , Yodoi Junji
Published: July 2002

Thioredoxin (TRX) is a pleiotropic cellular factor that has thiol-mediated redox activity and is important in regulation of cellular processes, including proliferation, apoptosis, and gene expression. The activity of several transcription factors is posttranslationally altered by redox modification(s) of specific cysteine residue(s). One such factor is nuclear factor (NF)-κB, whose DNA-binding activity is markedly augmented by TRX treatment
in vitro
. Similarly, the DNA-binding activity of activator protein 1 (AP-1) is modified by a DNA repair enzyme, redox factor 1 (Ref-1), which is identical to a DNA repair enzyme, AP endonuclease. Ref-1 activity is in turn modulated by various redox-active compounds, including TRX. We here report the molecular cascade of redox regulation of AP-1 mediated by TRX and Ref-1. Phorbol 12-myristate 13 acetate efficiently translocated TRX into the HeLa cell nucleus where Ref-1 preexists. This process seems to be essential for AP-1 activation by redox modification because co-overexpression of TRX and Ref-1 in COS-7 cells potentiated AP-1 activity only after TRX was transported into the nucleus by phorbol 12-myristate 13 acetate treatment. To prove the direct active site-mediated association between TRX and Ref-1, we generated a series of substitution-mutant cysteine residues of TRX. In both an
in vitro
diamide-induced cross-linking study and an
in vivo
mammalian two-hybrid assay we proved that TRX can associate directly with Ref-1 in the nucleus; also, we demonstrated the requirement of cysteine residues in the TRX catalytic center for the potentiation of AP-1 activity. This report presents an example of a cascade in cellular redox regulation.
Heteroclitic proliferative responses and changes in cytokine profile induced by altered peptides: Implications for autoimmunity
Authors: Nicholson Lindsay B. , Waldner Hanspeter , Carrizosa Ana M. , Sette Alessandro , Collins Mary , Kuchroo Vijay K.
Published: July 2002

Productive engagement of T cell receptors (TCRs) by cognate ligand (major histocompatibility complex plus peptide) leads to proliferation, differentiation, and the elaboration of effector functions. Altered peptides generated by single amino acid substitutions in the antigenic peptide have diverse effects on the outcome of the T cell response. We have generated an altered peptide (Q144) from an autoantigenic peptide of myelin proteolipid protein 139–151 by a single amino acid substitution (from tryptophan to glutamine) in the primary TCR contact at position 144 that is capable of inducing CD4+T cell responses in H-2smice. By using a Q144-specific T cell clone (Q1.1B6), we see a hierarchy in T cell proliferation and cytokine production with various position 144 substituted peptides and have identified a peptide (L144) that hyperstimulates this T cell clone. In contrast to Q144, L144 induces maximal proliferation at 7 logs lower antigen concentration, induces greater cell death at higher antigen dose, and induces the secretion of cytokines not detected following stimulation with the cognate ligand. This heteroclitic T cell response associated with changes in cytokine profile was observed with several other T cell clones of different specificities. The L144 peptide also induces costimulation independent proliferation and cytokine production from the Q1.1B6 T cell clone. We describe this as a superagonist response. Such responses may have a role in the initiation of autoimmunity by promoting a proinflammatory environment following ligation of a cross-reactive TCR on autoreactive T cells.
Multiple modes of cellular activation and virus transmission in HIV infection: A role for chronically and latently infected cells in sustaining viral replication
Authors: Grossman Zvi , Feinberg Mark B. , Paul William E.
Published: July 2002

CD4
+
T cell activation, required for virus replication in these cells, occurs in local microenvironmental domains in transient bursts. Thus, although most HIV originates from short-lived virus-producing cells, it is unlikely that chronic infection is generally sustained in rapid continuous cycles of productive infection as has been proposed. Such continuity of productive infection cycles would depend on efficient long-range transmission of HIV from one set of domains to another, in turn requiring the maintenance of sufficiently high concentrations of cell-free virus across lymphoid tissues at all times. By contrast, long-lived cellular sources of HIV maintain the capacity to infect newly activated cells at close range despite the temporal and spatial discontinuities of activation events. Such proximal activation and transmission (PAT) involving chronically and latently infected cells may be responsible for sustained infection, particularly when viral loads are low. Once CD4 cells are productively infected through PAT, they can infect other activated cells in their immediate vicinity. Such events propagate locally but generally do not spread systemically, unlike in the acute phase of the infection, because of the early establishment of protective anergy. Importantly, antiretroviral drug treatment is likely to differentially impact long-range transmission and PAT.
Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein δ
Authors: Sterneck Esta , Paylor Richard , Jackson-Lewis Vernice , Libbey Megan , Przedborski Serge , Tessarollo Lino , Crawley Jacqueline N. , Johnson Peter F.
Published: July 2002

CCAAT/enhancer binding protein δ (C/EBPδ) is a transcriptional regulator implicated in the hepatic acute phase response and in adipogenic and myeloid cell differentiation. We found that C/EBPδ is widely expressed in the peripheral and central nervous systems, including neurons of the hippocampal formation, indicating a role in neural functions. To examine the role of C/EBPδ
in vivo
, we generated mice with a targeted deletion of the C/EBPδ gene. This mutation does not interfere with normal embryonic and postnatal development. Performance in a battery of behavioral tests indicates that basic neurological functions are normal. Furthermore, performance in a Morris water maze task suggests that C/EBPδ mutant mice have normal spatial learning. However, in the contextual and auditory-cue-conditioned fear task, C/EBPδ null mice displayed significantly more conditioned freezing to the test context than did wild-type controls, but equivalent conditioning to the auditory cue. These data demonstrate a selectively enhanced contextual fear response in mice carrying a targeted genomic mutation and implicate C/EBPδ in the regulation of a specific type of learning and memory.
Interleukin 18 stimulates HIV type 1 in monocytic cells
Authors: Shapiro Leland , Puren Adrian J. , Barton Hazel A. , Novick Daniela , Peskind Robert L. , Shenkar Robert , Gu Yong , Su Michael S.-S. , Dinarello Charles A.
Published: July 2002

The cytokine interleukin (IL) 18 (formerly interferon γ-inducing factor) induces the T helper type 1 response. In the present studies, IL-18 increased HIV type 1 (HIV-1) production from 5- to 30-fold in the chronically infected U1 monocytic cell line. Inhibition of tumor necrosis factor (TNF) activity by the addition of TNF-binding protein reduced IL-18-stimulated HIV-1 production by 48%. In the same cultures, IL-18-induced IL-8 was inhibited by 96%. Also, a neutralizing anti-IL-6 mAb reduced IL-18-induced HIV-1 by 63%. Stimulation of U1 cells with IL-18 resulted in increased production of IL-6, and exogenous IL-6 added to U1 cells increased HIV-1 production 4-fold over control. A specific inhibitor of the p38 mitogen-activated protein kinase reduced IL-18-induced HIV-1 by 73%, and a 50% inhibition was observed at 0.05 μM. In the same cultures, IL-8 was inhibited by 87%. By gel-shift and supershift analyses, increased binding activity of the transcription factor NF-κB was measured in nuclear extracts from U1 cells 1 h after exposure to IL-18. These results demonstrate induction of HIV-1 by IL-18 in a monocyte target associated with an intermediate role for TNF and IL-6, activation of p38 mitogen-activated protein kinase, and nuclear translocation of NF-κB.
Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2
Authors: Soucek Thomas , Yeung Raymond S. , Hengstschläger Markus
Published: July 2002

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of aberrant growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The tuberous sclerosis complex gene-2 (
TSC2
) on chromosome 16 encodes the tumor suppressor protein tuberin. We have shown earlier that loss of
TSC2
is sufficient to induce quiescent cells to enter the cell cycle. Here we show that
TSC2
-negative fibroblasts exhibit a shortened G
1
phase. Although the expression of cyclin E, cyclin A, p21, or Cdc25A is unaffected,
TSC2
-negative cells express much lower amounts of the cyclin-dependent kinase (CDK) inhibitor p27 because of decreased protein stability. In
TSC2
mutant cells the amount of p27 bound to CDK2 is diminished, accompanied with elevated kinase activity. Ectopic expression studies revealed that the aforementioned effects can be reverted by transfecting
TSC2
in
TSC2
-negative cells. High ectopic levels of p27 have cell cycle inhibitory effects in
TSC2
-positive cells but not in
TSC2
-negative counterparts, although the latter still depend on CDK2 activity. Loss of
TSC2
induces soft agar growth of fibroblasts, a process that cannot be inhibited by high levels of p27. Both phenotypes of
TSC2
-negative cells, their resistance to the activity of ectopic p27, and the instability of endogenous p27, could be explained by our observation that the nucleoprotein p27 is mislocated into the cytoplasm upon loss of
TSC2
. These findings provide insights into the molecular mechanism of how loss of
TSC2
induces cell cycle entry and allow a better understanding of its tumor suppressor function.
Aging, mating, and the evolution of mtDNA heteroplasmy in
Drosophila melanogaster
Authors: Kann Lisa M. , Rosenblum Erica B. , Rand David M.
Published: July 2002

Heteroplasmy, the presence of more than one type of mtDNA within cells, is common in animals and has been associated with aging and disease in humans. Changes in the frequencies of mtDNA variants between cell and animal generations thus bears on the evolution of mtDNA and the progression of diverse pathologies. We have used densitometry of Southern blots of individual heteroplasmic
Drosophila melanogaster
to study the effects of age, increased egg production after mating, and temperature on evolution of heteroplasmy within and between generations. The frequency of the longer mtDNA variant consistently increased between early and late cohorts of F
1
offspring derived from 18 independent heteroplasmic mothers as they aged. Neither temperature (flies maintained at 25°C and 18°C) nor the holding of flies as virgins for 10 days before mating had significant effects on transmission patterns. However, at the ends of their lives, flies that had laid eggs at 25°C had a greater frequency of the long mtDNA than did their siblings who had laid eggs at 18°C. The evolution of heteroplasmy within a generation was studied in samples of siblings that either were mated or held as virgins, and then scored for mtDNA haplotype frequencies at two different ages (day 2 and day 14). Mated flies showed a significantly greater increase in the frequency of the long mtDNA variant with age than did the unmated flies. This system provides a model for the joint analysis of generational and chronological age in the transmission dynamics of a molecular polymorphism.
Extracellular HIV-1 virus protein R causes a large inward current and cell death in cultured hippocampal neurons: Implications for AIDS pathology
Authors: Piller Sabine C. , Jans Patricia , Gage Peter W. , Jans David A.
Published: July 2002

The small HIV-1 accessory protein Vpr (virus protein R) is a multifunctional protein that is present in the serum and cerebrospinal fluid of AIDS patients. We previously showed that Vpr can form cation-selective ion channels across planar lipid bilayers, introducing the possibility that, if incorporated into the membranes of living cells, Vpr might form ion channels and consequently perturb the maintained ionic gradient. In this study, we demonstrate, by a variety of approaches, that Vpr added extracellularly to intact cells does indeed form ion channels. We use confocal laser scanning microscopy to examine the subcellular localization of fluorescently labeled Vpr. Plasmalemma depolarization and damage are examined using the anionic potential-sensitive dye bis(1,3-dibutylbarbituric acid) trimethine oxonol and propidium iodide (PI), respectively, and the effect of Vpr on whole-cell current is demonstrated directly by using the patch-clamp technique. We show that recombinant purified extracellular Vpr associates with the plasmalemma of hippocampal neurons to cause a large inward cation current and depolarization of the plasmalemma, eventually resulting in cell death. Thus, we demonstrate a physiological action of extracellular Vpr and present its mechanistic basis. These findings may have important implications for neuropathologies in AIDS patients who possess significant amounts of Vpr in the cerebrospinal fluid.
Highly resolved chemical imaging of living cells by using synchrotron infrared microspectrometry
Authors: Jamin Nadège , Dumas Paul , Moncuit Janine , Fridman Wolf-Herman , Teillaud Jean-Luc , Carr G. Lawrence , Williams Gwyn P.
Published: July 2002

Using synchrotron radiation as an ultra-bright infrared source, we have been able to map the distributions of functional groups such as proteins, lipids, and nucleic acids inside a single living cell with a spatial resolution of a few microns. In particular, we have mapped the changes in the lipid and protein distributions in both the final stages of cell division and also during necrosis.
Nerve growth factor abrogates the tumorigenicity of human small cell lung cancer cell lines
Authors: Missale Cristina , Codignola Agnese , Sigala Sandra , Finardi Alessandra , Paez-Pereda Marcelo , Sher Emanuele , Spano PierFranco
Published: July 2002

Nerve growth factor (NGF) has antiproliferative and differentiating effects on adenomas of neuroendocrine origin. Cell lines derived from small-cell lung carcinoma (SCLC), a very aggressive neuroendocrine tumor, express NGF receptors. The role of NGF in the control of proliferation and progression of this carcinoma, however, has never been investigated. Chronic exposure of NCI-N-592 and GLC8 SCLC cell lines to NGF remarkably inhibited their proliferation rate both
in vitro
and
in vivo
, prevented their anchorage-independent clonal growth in soft agar, impaired their invasive capacity
in vitro
, and abolished their tumorigenic potential in nude mice. The proliferative response of SCLC cell lines to nicotine was also remarkably impaired by
in vitro
NGF treatment. Furthermore, NGF treatment activates in SCLC cell lines the expression and secretion of NGF. NGF thus reverts SCLC cell lines to a noninvasive, nontumorigenic phenotype that does not respond to nicotine and produces NGF.
Ezrin, a plasma membrane–microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway
Authors: Gautreau Alexis , Poullet Patrick , Louvard Daniel , Arpin Monique
Published: July 2002

ERM (Ezrin–Radixin–Moesin) proteins function as plasma membrane–actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. We have investigated ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1. Here we show that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, we found that ezrin interacts with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway.
Diversity of Holocene life forms in fossil glacier ice
Authors: Willerslev Eske , Hansen Anders J. , Christensen Bent , Steffensen Jørgen Peder , Arctander Peter
Published: July 2002

Studies of biotic remains of polar ice caps have been limited to morphological identification of plant pollen and spores. By using sensitive molecular techniques, we now demonstrate a much greater range of detectable organisms; from 2000- and 4000-year-old ice-core samples, we obtained and characterized 120 clones that represent at least 57 distinct taxa and reveal a diversity of fungi, plants, algae, and protists. The organisms derive from distant sources as well as from the local arctic environment. Our results suggest that additional taxa may soon be readily identified, providing a plank for future studies of deep ice cores and yielding valuable information about ancient communities and their change over time.
High-field EPR detection of a disulfide radical anion in the reduction of cytidine 5′-diphosphate by the E441Q R1 mutant of
Escherichia coli
ribonucleotide reductase
Authors: Lawrence Christopher C. , Bennati Marina , Obias Honorio V. , Bar Galit , Griffin Robert G. , Stubbe Joanne
Published: July 2002

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster–tyrosyl radical (Y⋅) cofactor and R1 is the site of the conversion of nucleoside diphosphates to 2′-deoxynucleoside diphosphates. A mutant in the R1 subunit of
Escherichia coli
RNR, E441Q, was generated in an effort to define the function of E441 in the nucleotide-reduction process. Cytidine 5′-diphosphate was incubated with E441Q RNR, and the reaction was monitored by using stopped-flow UV-vis spectroscopy and high-frequency (140 GHz) time-domain EPR spectroscopy. These studies revealed loss of the Y⋅ and formation of a disulfide radical anion and present experimental mechanistic insight into the reductive half-reaction catalyzed by RNR. These results support the proposal that the protonated E441 is required for reduction of a 3′-ketodeoxynucleotide by a disulfide radical anion. On the minute time scale, a second radical species was also detected by high-frequency EPR. Its
g
values suggest that this species may be a 4′-ketyl radical and is not on the normal reduction pathway. These experiments demonstrate that high-field time-domain EPR spectroscopy is a powerful new tool for deconvolution of a mixture of radical species.
Dynamics of transient pores in stretched vesicles
Authors: Sandre Olivier , Moreaux Laurent , Brochard-Wyart Françoise
Published: July 2002

We image macroscopic transient pores in mechanically
stretched giant vesicles. Holes open above a critical radius

r
c1

, grow up to a radius

r
c2

, and close. We interpret the upper limit

r
c2

by a relaxation of the membrane tension as
the holes expand. The closing of the holes is caused by a further
relaxation of the surface tension when the internal liquid leaks out. A
dynamic model fits our data for the growth and closure of pores.
Platelet-derived growth factor β receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3′ kinase signaling
Authors: Heuchel Rainer , Berg Ansgar , Tallquist Michelle , Åhlén Karina , Reed Rolf K. , Rubin Kristofer , Claesson-Welsh Lena , Heldin Carl-Henrik , Soriano Philippe
Published: July 2002

Platelet-derived growth factor (PDGF) isoforms lead to mitogenic, survival, and chemotactic responses in a variety of mesenchymal cell types during development and in the adult. We have studied the importance of phosphatidylinositol-3′ kinase (PI3K) signaling in these responses by mutating the PI3K-binding sites in the PDGF-β receptor by gene targeting in embryonic stem cells. Homozygous mutant mice developed normally; however, cells derived from the mutants were less chemotactic and had largely lost their ability to contract collagen gels in response to PDGF. Injection of a mast cell degranulating agent in mice led to a decrease in interstitial fluid pressure resulting in edema formation. In contrast to wild-type mice, mutant mice were unable to normalize the pressure after treatment with PDGF. Taken together, these observations suggest a function for PDGF signaling through PI3K in interstitial fluid homeostasis by modulating the tension between cells and extracellular matrix structures.
Central representation of time during motor learning
Authors: Conditt Michael A. , Mussa-Ivaldi Ferdinando A.
Published: July 2002

This study stemmed from the observation that the brain of human as well as nonhuman primates is capable of forming and memorizing remarkably accurate internal representations of the dynamics of the arm. These dynamics establish a functional relation between applied force and ensuing arm motion, a relation that generally is quite complex and nonlinear. Current evidence shows that the motor control system is capable of adapting to perturbing forces that depend on motion variables such as position, velocity, and acceleration. The experiments we report here were aimed at establishing whether or not the motor system also may adapt to forces that depend explicitly on time rather than on motion variables. Surprisingly, the experiments suggest a negative answer. When asked to compensate for a predictable and repeated time-varying pattern of disturbing forces, subjects learned to counteract the disturbance by producing forces that did not depend on time but on the velocity and the position of the arm. We conclude from this evidence that time and time-dependent dynamics are not explicitly represented within the neural structures that are responsible for motor adaptation. Although our findings are not sufficient to rule out the presence of a timing structure within the central nervous system, they are consistent with other investigations that conspicuously failed to find evidence for such a central clock.
Bidirectional, experience-dependent regulation of
N-
methyl-
d
-aspartate receptor subunit composition in the rat visual cortex during postnatal development
Authors: Quinlan Elizabeth M. , Olstein Daniel H. , Bear Mark F.
Published: July 2002

In the visual cortex, as elsewhere,
N-
methyl-
d
-aspartate receptors (NMDARs) play a critical role in triggering long-term, experience-dependent synaptic plasticity. Modifications of NMDAR subunit composition alter receptor function, and could have a large impact on the properties of synaptic plasticity. We have used immunoblot analysis to investigate the effects of age and visual experience on the expression of different NMDAR subunits in synaptoneurosomes prepared from rat visual cortices. NMDARs at birth are comprised of NR2B and NR1 subunits, and, over the first 5 postnatal weeks, there is a progressive inclusion of the NR2A subunit. Dark rearing from birth attenuates the developmental increase in NR2A. Levels of NR2A increase rapidly (in <2 hr) when dark-reared animals are exposed to light, and decrease gradually over the course of 3 to 4 days when animals are deprived of light. These data reveal that NMDAR subunit composition in the visual cortex is remarkably dynamic and bidirectionally regulated by sensory experience. We propose that NMDAR subunit regulation is a mechanism for experience-dependent modulation of synaptic plasticity in the visual cortex, and serves to maintain synaptic strength within an optimal dynamic range.
Chromosomal aberrations in PARP
−/−
mice: Genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA
Authors: Simbulan-Rosenthal Cynthia M. , Haddad Bassem R. , Rosenthal Dean S. , Weaver Zoë , Coleman Allen , Luo RuiBai , Young Hannah M. , Wang Zhao-Qi , Ried Thomas , Smulson Mark E.
Published: July 2002

Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP
−/−
mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP
−/−
mice and immortalized PARP
−/−
fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP
−/−
cells stably transfected with PARP cDNA [PARP
−/−
(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP
−/−
cells, p53 expression was partially restored in PARP
−/−
(+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene
Rb-1
in PARP
−/−
mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene
Jun
was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.