bluuebunny/crossref_metadata_2025 huggingface dataset:
The reverse transcriptase component of the
Tetrahymena
telomerase ribonucleoprotein complex
Authors: Collins Kathleen , Gandhi Leena
Published: July 2002

Telomerase is a eukaryotic reverse transcriptase that adds simple sequence repeats to chromosome ends by copying a template sequence within the RNA component of the enzyme. We describe here the identification of a
Tetrahymena
telomerase protein with reverse transcriptase motifs, p133. This subunit is associated with the previously identified
Tetrahymena
telomerase RNA and the telomerase proteins p80 and p95 in immunoprecipitation assays. Therefore, all four known
Tetrahymena
telomerase components are present in a single complex. Expressed in rabbit reticulocyte lysate, recombinant p133 and telomerase RNA alone catalyze a reverse transcriptase activity with some similarities to and some differences from native
Tetrahymena
telomerase. These experiments suggest a complexity of telomerase structure and function.
Hepatocyte injury in tyrosinemia type 1 is induced by fumarylacetoacetate and is inhibited by caspase inhibitors
Authors: Kubo Shuji , Sun Maosen , Miyahara Michio , Umeyama Kazuhiro , Urakami Ken-ichi , Yamamoto Tetsuro , Jakobs Cornelis , Matsuda Ichiro , Endo Fumio
Published: July 2002

Tyrosinemia type 1, caused by mutations in the fumarylacetoacetate hydrolase gene (
Fah
), is characterized by severe liver injury. We earlier developed a tyrosinemic mouse model with two genetic defects,
Fah
and 4-hydroxyphenylpyruvate dioxygenase (
Hpd
) deficiencies. Apoptosis of hepatocytes was induced and an acute onset of liver failure occurred after administration of homogentisic acid (HGA), the intermediate metabolite between the enzymes HPD and FAH. Cytochrome
c
was released from mitochondria prior to liver failure in the
Fah
−/−
Hpd
−/− double-mutant mice after the administration of HGA. In a cell-free system, the addition of fumarylacetoacetate induced the release of cytochrome
c
from the mitochondria. We also found that caspase inhibitors were highly effective in preventing the liver failure induced by HGA in the double-mutant mice. Therefore, fumarylacetoacetate apparently induces the release of cytochrome
c
, which in turn triggers activation of the caspase cascade in hepatocytes of subjects with hereditary tyrosinemia type 1.
Promoter-proximal stalling results from the inability to recruit transcription factor IIH to the transcription complex and is a regulated event
Authors: Kumar K. Prasanna , Akoulitchev Sasha , Reinberg Danny
Published: July 2002

Promoter-proximal stalling, a general phenomenon observed during the expression of many RNA polymerase II transcribed genes, is dependent on transcription factor IIH (TFIIH). Reactions lacking TFIIH initiated transcription, but the transcription complex encountered a block to elongation proximal to the promoter. The accumulation of promoter-proximal stalled complexes was reduced in the presence of TFIIH and efficient escape from this site also required an activator. Promoter-proximal stalled complexes could not be induced to resume elongation. Our results indicate that effective recruitment of TFIIH into transcription complexes is achieved during formation of the preinitiation complex at the promoter. The studies establish that promoter clearance is a regulated event that requires TFIIH.
Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3
Authors: Juang Y.-T. , Lowther W. , Kellum M. , Au W.-C. , Lin R. , Hiscott J. , Pitha P. M.
Published: July 2002

The family of interferon (IFN) regulatory factors (IRFs) encodes DNA-binding transcription factors, some of which function as modulators of virus-induced signaling. The IRF-3 gene is constitutively expressed in many tissues and cell types, and neither virus infection nor IFN treatment enhances its transcription. In infected cells, however, IRF-3 protein is phosphorylated at the carboxyl terminus, which facilitates its binding to the CBP/p300 coactivator. In the present study, we demonstrate that overexpression of IRF-3 significantly enhances virus-mediated transcription of the IFNA and IFNB genes in infected cells as well as IFN synthesis. IRF-3-mediated activation of IFN genes depends in part on carboxyl-terminal phosphorylation of a cluster of Ser/Thr residues, because a mutant with Ser/Thr to Ala substitutions activates the IFN promoter less efficiently. However, overexpression of IRF-3 in human 2FTGH cells alone results in the induction of an antiviral state, which depends on functional IFN signaling, because IRF-3 does not induce an antiviral state in mutant 2FTGH cells defective in either JAK-1 or p48 functions; also no antiviral effect of IRF-3 could be demonstrated in Vero cells that lack the IFNA and IFNB genes. This finding indicates that the observed antiviral activity of IRF-3 in 2FTGH cells results mainly from the induction of IFNs. Furthermore, E1A protein inhibited IRF-3-mediated stimulation of the IFNA4 promoter in transient expression assays; this inhibition could be reversed partially by overexpression of CBP/p300 and was not demonstrated with the mutant of E1A that does not bind p300. These results identify IRF-3 and CBP/p300 as integral components of the virus-induced complex that stimulates type 1 IFN gene transcription. The observation that adenovirus E1A antagonizes IRF-3 mediated activation suggests that E1A and IRF-3 may compete for binding to CBP/p300 and implicates a novel mechanism by which adenovirus may overcome the antiviral effects of the IFN pathway.
Identification of HLA class II-restricted determinants ofMycobacteriumtuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice
Authors: Geluk Annemieke , Taneja Veena , van Meijgaarden Krista E. , Zanelli Eric , Abou-Zeid Christiane , Thole Jelle E. R. , de Vries René R. P. , David Chella S. , Ottenhoff Tom H. M.
Published: July 2002

T helper 1 cells play a major role in protective immunity against mycobacterial pathogens. Since the antigen (Ag) specificity of CD4+human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism. We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles. In these animals, all CD4+T cells are restricted by the HLA molecule. We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 ofMycobacterium tuberculosis, and only a single hsp65 epitope, p1–20. DR3.Ab0mice, immunized with bacillus Calmette–Guérin or hsp65, developed T cell responses toM. tuberculosis, and recognized the same hsp65 epitope, p1–20. Hsp65-immunized DQ8.Ab0mice mounted a strong response to bacillus Calmette–Guérin but not to p1–20. Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171–200, 311–340, and 411–440. DR3.Ab0mice immunized with a second majorM. tuberculosisprotein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51–70, that was identified in this study. Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0mice. Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity. Immunization of DR3.Ab0with the immunodominant peptides p1–20 and p51–70 induced T cell reactivity toM. tuberculosis. Thus, for two different Ags, T cells from DR3.Ab0mice and HLA-DR3+ humans recognize the same immunodominant determinants. Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.
High-affinity binding of hemimethylated
oriC
by
Escherichia coli
membranes is mediated by a multiprotein system that includes SeqA and a newly identified factor, SeqB
Authors: Shakibai Nader , Ishidate Kohei , Reshetnyak Elena , Gunji Shigemichi , Kohiyama Masamichi , Rothfield Lawrence
Published: July 2002

The binding of hemimethylated
oriC
to
Escherichia coli
membranes has been implicated in the prevention of premature reinitiation at newly replicated chromosomal origins in a reaction that involves the SeqA protein. We describe the resolution of the membrane-associated
oriC
-binding activity into two fractions, both of which are required for the high-affinity binding of hemimethylated
oriC
. The active component in one fraction is identified as SeqA. The active component of the second fraction is a previously undescribed protein factor, SeqB. The reconstituted system reproduced the salient characteristics of the membrane-associated binding activity, suggesting that the membrane-associated
oriC
-binding machinery of
E. coli
is likely to be a multiprotein system that includes the SeqA and SeqB proteins.
Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21
CIP1/WAF1
and cyclin D proteins
Authors: Yu Zhong-Kang , Gervais Jennifer L. M. , Zhang Hui
Published: July 2002

Deregulation of cell proliferation is a hallmark of cancer. In many transformed cells, the cyclin A/CDK2 complex that contains S-phase kinase associated proteins 1 and 2 (SKP1 and SKP2) is highly induced. To determine the roles of this complex in the cell cycle regulation and transformation, we have examined the composition of this complex. We report here that this complex contained an additional protein, human CUL-1, a member of the cullin/CDC53 family. The identification of CUL-1 as a member of the complex raises the possibility that the p19
SKP1
/p45
SKP2
/CUL-1 complex may function as the yeast SKP1-CDC53-F-box (SCF) protein complex that acts as a ubiquitin E3 ligase to regulate the G
1
/S transition. In mammalian cells, cyclin D, p21
CIP1/WAF1
, and p27
KIP1
are short-lived proteins that are controlled by ubiquitin-dependent proteolysis. To determine the potential
in vivo
targets of the p19
SKP1
/p45
SKP2
/CUL-1 complex, we have used the specific antisense oligodeoxynucleotides against either SKP1, SKP2, or CUL-1 RNA to inhibit their expression. Treatment of cells with these oligonucleotides caused the selective accumulation of p21 and cyclin D proteins. The protein level of p27 was not affected. These data suggest that the human p19
SKP1
/p45
SKP2
/CUL-1 complex is likely to function as an E3 ligase to selectively target cyclin D and p21 for the ubiquitin-dependent protein degradation. Aberrant expression of human p19
SKP1
/p45
SKP2
/CUL-1 complex thus may contribute to tumorigenesis by regulating the protein levels of G
1
cell cycle regulators.
Architectural DNA binding by a high-mobility-group/kinesin-like subunit in mammalian SWI/SNF-related complexes
Authors: Wang Weidong , Chi Tianhuai , Xue Yutong , Zhou Sharleen , Kuo Ann , Crabtree Gerald R.
Published: July 2002

The SWI/SNF complex in yeast and
Drosophila
is thought to facilitate transcriptional activation of specific genes by antagonizing chromatin-mediated transcriptional repression. The mechanism by which it is targeted to specific genes is poorly understood and may involve direct DNA binding and/or interactions with specific or general transcription factors. We have previously purified a mammalian complex by using antibodies against BRG1, a human homologue of SWI2/SNF2. This complex is likely functionally related to the yeast SWI/SNF complex because all five subunits identified so far (referred to as BAFs, for BRG1-associated factors) are homologues of the yeast SWI/SNF subunits. However, we now describe the cloning of the 57-kDa subunit (BAF57), which is present only in higher eukaryotes but not in yeast. BAF57 is shared by all mammalian complexes and contains a high-mobility-group (HMG) domain adjacent to a kinesin-like region. Both recombinant BAF57 and the whole complex bind four-way junction (4WJ) DNA, which is thought to mimic the topology of DNA as it enters or exits the nucleosome. Surprisingly, complexes with mutations in the HMG domain of BAF57 can still bind 4WJ DNA and mediate ATP-dependent nucleosome disruption. Our work describes the first DNA binding subunit for SWI/SNF-like complexes and suggest that the mechanism by which mammalian and
Drosophila
SWI/SNF-like complexes interact with chromatin may involve recognition of higher-order chromatin structure by two or more DNA binding domains.
Major histocompatibility complex (MHC) class I KbDb−/− deficient mice possess functional CD8+ T cells and natural killer cells
Authors: Vugmeyster Yulia , Glas Rickard , Pérarnau Béatrice , Lemonnier François A. , Eisen Herman , Ploegh Hidde
Published: July 2002

We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb−/− mice express no detectable classical MHC classI-region associated (Ia) heavy chains, although β2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb−/− mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by β2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb−/− mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However,in vivopriming of KbDb−/− mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb−/− animals also have natural killer cells that retain their cytotoxic potential.
Protein fluctuations are sensed by stimulated infrared echoes of the vibrations of carbon monoxide and azide probes
Authors: Lim Manho , Hamm Peter , Hochstrasser Robin M.
Published: July 2002

The correlation functions of the fluctuations of vibrational frequencies of azide ions and carbon monoxide in proteins are determined directly from stimulated photon echoes generated with femtosecond infrared pulses. The asymmetric stretching vibration of azide bound to carbonic anhydrase II exhibits a pronounced evolution of its vibrational frequency distribution on the time scale of a few picoseconds, which is attributed to modifications of the ligand structure through interactions with the nearby Thr-199. When azide is bound in hemoglobin, a more complex evolution of the protein structure is required to interchange the different ligand configurations, as evidenced by the much slower relaxation of the frequency distribution in this case. The time evolution of the distribution of frequencies of carbon monoxide bound in hemoglobin occurs on the ≈10-ps time scale and is very nonexponential. The correlation functions of the frequency fluctuations determine the evolution of the protein structure local to the probe and the extent to which the probe can navigate those parts of the energy landscape where the structural configurations are able to modify the local potential energy function of the probe.
Bidirectional imprinting of a single gene:
GNAS1
encodes maternally, paternally, and biallelically derived proteins
Authors: Hayward Bruce E. , Moran Veronica , Strain Lisa , Bonthron David T.
Published: July 2002

The
GNAS1
gene encodes the α subunit of the guanine nucleotide-binding protein G
s
, which couples signaling through peptide hormone receptors to cAMP generation.
GNAS1
mutations underlie the hormone resistance syndrome pseudohypoparathyroidism type Ia (PHP-Ia), so the maternal inheritance displayed by PHP-Ia has raised suspicions that
GNAS1
is imprinted. Despite this suggestion, in most tissues G
s
α is biallelically encoded. In contrast, the large G protein XLαs, also encoded by
GNAS1
, is paternally derived. Because the inheritance of PHP-Ia predicts the existence of maternally, rather than paternally, expressed transcripts, we have investigated the allelic origin of other mRNAs derived from
GNAS1
. We find this gene to be remarkable in the complexity of its allele-specific regulation. Two upstream promoters, each associated with a large coding exon, lie only 11 kb apart, yet show opposite patterns of allele-specific methylation and monoallelic transcription. The more 5′ of these exons encodes the neuroendocrine secretory protein NESP55, which is expressed exclusively from the maternal allele. The NESP55 exon is 11 kb 5′ to the paternally expressed XLαs exon. The transcripts from these two promoters both splice onto
GNAS1
exon 2, yet share no coding sequences. Despite their structural unrelatedness, the encoded proteins, of opposite allelic origin, both have been implicated in regulated secretion in neuroendocrine tissues. Remarkably, maternally (NESP55), paternally (XLαs), and biallelically (G
s
α) derived proteins all are produced by different patterns of promoter use and alternative splicing of
GNAS1
, a gene showing simultaneous imprinting in both the paternal and maternal directions.
A translational repression assay procedure (TRAP) for RNA–protein interactions
in vivo
Authors: Paraskeva Efrosyni , Atzberger Ann , Hentze Matthias W.
Published: July 2002

RNA–protein interactions are central to many aspects of cellular metabolism, cell differentiation, and development as well as the replication of infectious pathogens. We have devised a versatile, broadly applicable
in vivo
system for the analysis of RNA–protein interactions in yeast. TRAP (translational repression assay procedure) is based on the translational repression of a reporter mRNA encoding green fluorescent protein by an RNA-binding protein for which a cognate binding site has been introduced into the 5′ untranslated region. Because protein binding to the 5′ untranslated region can sterically inhibit ribosome association, expression of the cognate binding protein causes significant reduction in the levels of green fluorescent protein fluorescence. By using RNA–protein interactions with affinities in the micromolar to nanomolar range, we demonstrate the specificity of TRAP as well as its ability to recover the cDNA encoding a specific RNA-binding protein, which has been diluted 500,000-fold with unrelated cDNAs, by using fluorescence-activated cell sorting. We suggest that TRAP offers a strategy to clone RNA-binding proteins for which little else than the binding site is known, to delineate RNA sequence requirements for protein binding as well as the protein domains required for RNA binding, and to study effectors of RNA–protein interactions
in vivo
.
Overexpression of
Escherichia coli
oxidoreductases increases recombinant insulin-like growth factor-I accumulation
Authors: Joly John C. , Leung W. S. , Swartz James R.
Published: July 2002

Transient overexpression of either DsbA or DsbC can double the yield of periplasmic insulin-like growth factor (IGF)-I in
Escherichia coli
to 8.5 g/liter. Strikingly, most of the overexpressed DsbA or DsbC is found in the reduced form, implying that enhanced disulfide isomerization is responsible for the substantial increase in IGF-I yield. All of the accumulated IGF-I has had the signal sequence removed, underscoring the secretion capacity of this organism as well as its utility for efficient production of polypeptide with the correct amino terminus. The overexpressed IGF-I constitutes approximately 30% of the total cell protein. Overproduction of active site mutants of DsbA instead of the wild-type gene do not produce this increase in yield. With wild-type levels of DsbA and DsbC, most of the secreted IGF-I is found in disulfide-linked aggregates, although 10% is soluble and about 5% is correctly folded. Contrary to expectations, overexpression of the disulfide oxidoreductases decreased the soluble fraction. Because the aggregated protein can be efficiently solubilized and refolded, the increased yield is a significant benefit for the production of IGF-I.
In vivo
functional discrimination between plant thioredoxins by heterologous expression in the yeast
Saccharomyces cerevisiae
Authors: Mouaheb Nabil , Thomas Dominique , Verdoucq Lionel , Monfort Patrick , Meyer Yves
Published: July 2002

Whereas vertebrates possess only two thioredoxin genes, higher plants present a much greater diversity of thioredoxins. For example,
Arabidopsis thaliana
has five cytoplasmic thioredoxins (type h) and at least as many chloroplastic thioredoxins. The abundance of plant thioredoxins leads to the question whether the various plant thioredoxins play a similar role or have specific functions. Because most of these proteins display very similar activities on artificial or biological substrates
in vitro
, we developed an
in vivo
approach to answer this question. The disruption of both of the two
Saccharomyces cerevisiae
thioredoxin genes leads to pleiotropic effects including methionine auxotrophy, H
2
O
2
hypersensitivity, altered cell cycle characteristics, and a limited ability to use methionine sulfoxide as source of methionine. We expressed eight plant thioredoxins (six cytoplasmic and two chloroplastic) in yeast
trx1, trx2
double mutant cells and analyzed the different phenotypes.
Arabidopsis
type h thioredoxin 2 efficiently restored sulfate assimilation whereas
Arabidopsis
type h thioredoxin 3 conferred H
2
O
2
tolerance. All thioredoxins tested could complement for reduction of methionine sulfoxide, whereas only type h thioredoxins were able to complement the cell cycle defect. These findings clearly indicate that specific interactions between plant thioredoxins and their targets occur
in vivo
.
The role of robustness and changeability on the origin and evolution of genetic codes
Authors: Maeshiro Tetsuya , Kimura Masayuki
Published: July 2002

We propose that an essential factor on the origin of genetic codes is a balanced accomplishment of robustness and changeability, two antithetical, but fundamental, properties for the survival and evolution of organisms. These measures are defined as the intrinsic properties of genetic codes. An evaluation of these properties explains the structural regularity of genetic codes, estimates the order of codon reassignment in deviant codes, and predicts the most probable deviant codes that exist. The enumeration of genetic codes that could have evolved from the standard genetic code under the selection pressure on robustness and changeability strongly limits the freedom of codon reassignments. The codon reassignments of all currently known deviant genetic codes belong to this predicted evolutionary path, and they generally give the highest improvements on robustness and changeability.
Stretching single-domain proteins: Phase diagram and kinetics of force-induced unfolding
Authors: Klimov D. K. , Thirumalai D.
Published: July 2002

Single-molecule force spectroscopy reveals unfolding of domains in titin on stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single-domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively, whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great variations reflecting the heterogeneity of force-induced unfolding pathways. The approach to the stretched state occurs in a stepwise “quantized” manner. Unfolding dynamics and forces required to stretch proteins depend sensitively on topology. The unfolding rates increase exponentially with force
f
till an optimum value, which is determined by the barrier to unfolding when
f
= 0. A mapping of these results to proteins shows qualitative agreement with force-induced unfolding of Ig-like domains in titin. We show that single-molecule force spectroscopy can be used to map the folding free energy landscape of proteins in the absence of denaturants.
Feedback control of a master bacterial cell-cycle regulator
Authors: Domian Ibrahim J. , Reisenauer Ann , Shapiro Lucy
Published: July 2002

The transcriptional regulator CtrA controls several key cell-cycle events in
Caulobacter crescentus
, including the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems.
Caulobacter
goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. We report here that
ctrA
expression is under the control of two promoters: a promoter (P1) that is active only in the early predivisional cell and a stronger promoter (P2) that is active in the late predivisional cell. Both promoters exhibit CtrA-mediated feedback regulation: the early P1 promoter is negatively controlled by CtrA, and the late P2 promoter is under positive feedback control. The CtrA protein footprints conserved binding sites within the P1 and P2 promoters. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. The ensuing accumulation of CtrA results in the activation of the P2 promoter and the repression of the P1 promoter late in the cell cycle. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events.
Activation of erythropoietin receptor in the absence of hormone by a peptide that binds to a domain different from the hormone binding site
Authors: Naranda Tatjana , Wong Kenneth , Kaufman R. Ilene , Goldstein Avram , Olsson Lennart
Published: July 2002

Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin.
Developmental selection within the angiosperm style: Using gamete DNA to visualize interspecific pollen competition
Authors: Williams J. H. , Friedman W. E. , Arnold M. L.
Published: July 2002

Among flowering plants, females often have little control over the genetic relatedness of pollen deposited on stigmas. Thus, postpollination processes are of primary importance for mate discrimination. The ability to screen and select among male gametes during pollen tube growth within the female tissues of the stigma, style, and ovary is critical to the process of mate choice and reproductive isolation. However, direct evidence of the mechanistic/developmental processes associated with mate choice in flowering plants is sparse. We studied the contribution of postpollination, prefertilization processes to reproductive isolation in two wind-pollinated species of birch (Betula) that commonly hybridize in nature. We exploited the 3-fold difference in ploidy level between these two species to determine the paternity of individual pollen tubes growing within female reproductive tissues. We then tracked their developmental fate in conspecific, heterospecific, and mixed-species crosses. This design allowed a direct comparison of developmental processes responsible for reproductive isolation in both single-species and mixed-species pollinations. Our results have important implications for the population genetic outcome of hybridization dynamics in natural populations. Paternity analysis of progeny from mixed-species pollinations revealed that conspecific pollen tubes sired more than 98% of seedlings. Biased siring success was not the result of differential embryo abortion. We detected strong, early postpollination barriers such as pollen tube incompatibility, slower pollen tube growth, and delayed generative cell mitosis. Conspecific fertilization precedence was mediated by favorable or unfavorable male-female interactions, but there was no evidence for antagonistic male-male interactions.
T cell ignorance in mice to Borna disease virus can be overcome by peripheral expression of the viral nucleoprotein
Authors: Hausmann Jürgen , Hallensleben Wiebke , de la Torre Juan Carlos , Pagenstecher Axel , Zimmermann Christine , Pircher Hanspeter , Staeheli Peter
Published: July 2002

Infection of neonates with Borna disease virus (BDV) induces severe meningoencephalitis and neurological disorder in wild-type but not in β2-microglobulin-deficient mice of strain MRL (H-2k). Temporaryin vivodepletion of CD8+T cells delayed BDV-induced disease for several weeks. Depletion of CD4+T cells had a similar beneficial effect, indicating that the BDV-induced neurological disorder in mice is a CD4+T cell-dependent immunopathological process that is mediated by CD8+T cells. Lymphocytes prepared from brains of diseased mice were mainly from the CD8+T cell subset. They showed up-regulation of activation markers and exerted strong MHC I-restricted cytotoxic activity against target cells expressing the BDV nucleoprotein p40. Infection of B10.BR (H-2k) or congenic C57BL/10 (H-2b) mice resulted in symptomless, lifelong persistence of BDV in the brain. Superinfection with a recombinant vaccinia virus expressing BDV p40 but not with other vaccinia viruses induced severe neurological disease and encephalitis in persistently infected B10.BR mice but not in persistently infected C57BL/10 mice, indicating that the disease-inducing T cell response is restricted to the nucleoprotein of BDV in H-2kmice. Our results demonstrate that the cellular arm of the immune system may ignore the presence of a replicating virus in the central nervous system until proper antigenic stimulation at a peripheral site triggers the antiviral response.
Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants
Authors: Tanaka Yoshito , Gleason Catherine E. , Tran Phuong Oanh T. , Harmon Jamie S. , Robertson R. Paul
Published: July 2002

Chronic exposure of pancreatic islets to supraphysiologic concentrations of glucose causes adverse alterations in β cell function, a phenomenon termed glucose toxicity and one that may play a secondary pathogenic role in type 2 diabetes. However, no mechanism of action has been definitively identified for glucose toxicity in β cells. To ascertain whether chronic oxidative stress might play a role, we chronically cultured the β cell line, HIT-T15, in medium containing 11.1 mM glucose with and without the antioxidants,
N-
acetyl-
l
-cysteine (NAC) or aminoguanidine (AG). Addition of NAC or AG to the culture medium at least partially prevented decreases in insulin mRNA, insulin gene promoter activity, DNA binding of two important insulin promoter transcription factors (PDX-1/STF-1 and RIPE-3b1 activator), insulin content, and glucose-induced insulin secretion. These findings suggested that one mechanism of glucose toxicity in the β cell may be chronic exposure to reactive oxygen species, i.e., chronic oxidative stress. To ascertain the effects of these drugs on diabetes, NAC or AG was given to Zucker diabetic fatty rats, a laboratory model of type 2 diabetes, from 6 through 12 weeks of age. Both drugs prevented a rise in blood oxidative stress markers (8-hydroxy-2′-deoxyguanosine and malondialdehyde + 4-hydroxy-2-nonenal), and partially prevented hyperglycemia, glucose intolerance, defective insulin secretion as well as decrements in β cell insulin content, insulin gene expression, and PDX-1 (STF-1) binding to the insulin gene promoter. We conclude that chronic oxidative stress may play a role in glucose toxicity, which in turn may worsen the severity of type 2 diabetes.
Isolation and expression of a
Pax-6
gene in the regenerating and intact Planarian
Dugesia(G)tigrina
Authors: Callaerts P. , Munoz-Marmol A. M. , Glardon S. , Castillo E. , Sun H. , Li W.-H. , Gehring W. J. , Salo E.
Published: July 2002

The
Pax-6
gene encodes a transcription factor containing both a paired and a homeodomain and is highly conserved among Metazoa. In both vertebrates and invertebrates,
Pax-6
is required for eye morphogenesis, development of parts of the central nervous system, and, in some phyla, for the development of olfactory sense organs. Ectopic expression of
Pax-6
from insects, mammals, cephalopods, and ascidians induces ectopic eyes in
Drosophila,
suggesting that
Pax-6
may be a universal master control gene for eye morphogenesis. Platyhelminthes are an ancient phylum, originating from the base of spiralian protostomes, that bear primitive eyes, consisting of a group of rhabdomeric photoreceptor cells enclosed in a cup of pigment cells. The analysis of
Pax-6
and its expression pattern should provide insights into the ancestral function of
Pax-6
in eye morphogenesis. We have identified the
Pax-6
gene of the planarian
Dugesia(G)tigrina
(Platyhelminthes; Turbellaria; Tricladida). This gene shares significant sequence identity and conserved genomic organization with Pax-6 proteins from other phyla. Phylogenetic analysis indicates that it clusters with the other
Pax-6
genes, but in the most basal position.
DtPax-6
is expressed as a single transcript in both regenerating and fully grown eyes, and electron microscopy studies show strong expression in the perykarion of both photoreceptor and pigment cells. Very low levels of expression also are detectable in other body regions. Because a bona fide
Pax-6
homolog so far has not been detected in diploblastic animals, we speculate that
Pax-6
may be typical for triploblasts and that the appearance of additional Pax genes may have coincided with increasingly complex body plans.
Selection of antibodies for intracellular function using a two-hybridin vivosystem
Authors: Visintin Michela , Tse Eric , Axelson Hakan , Rabbitts Terence H. , Cattaneo Antonino
Published: July 2002

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed anin vivoassay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigenin vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis forde novoselection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.
Attenuation of virulence in
Mycobacterium tuberculosis
expressing a constitutively active iron repressor
Authors: Manabe Yukari C. , Saviola Beatrice J. , Sun Li , Murphy John R. , Bishai William R.
Published: July 2002

Iron is an essential nutrient for the survival of most organisms and has played a central role in the virulence of many infectious disease pathogens. Mycobacterial IdeR is an iron-dependent repressor that shows 80% identity in the functional domains with its corynebacterial homologue, DtxR (diphtheria to
x
in repressor). We have transformed
Mycobacterium tuberculosis
with a vector expressing an iron-independent, positive dominant, corynebacterial
dtxR
hyperrepressor, DtxR(E175K). Western blots of whole-cell lysates of
M. tuberculosis
expressing the
dtxR
(E175K) gene revealed the stable expression of the mutant protein in mycobacteria. BALB/c mice were infected by tail vein injection with 2 × 10
5
organisms of wild type or
M. tuberculosis
transformed with the
dtxR
mutant. At 16 weeks, there was a 1.2 log reduction in bacterial survivors in both spleen (
P
= 0.0002) and lungs (
P
= 0.006) with
M. tuberculosis
DtxR(E175K). A phenotypic difference in colonial morphology between the two strains also was noted. A computerized search of the
M. tuberculosis
genome for the palindromic consensus sequence to which DtxR and IdeR bind revealed six putative “iron boxes” within 200 bp of an ORF. Using a gel-shift assay we showed that purified DtxR binds to the operator region of five of these boxes. Attenuation of
M. tuberculosis
can be achieved by the insertion of a plasmid containing a constitutively active, iron-insensitive repressor, DtxR(E175K), which is a homologue of IdeR. Our results strongly suggest that IdeR controls genes essential for virulence in
M. tuberculosis.
Functional analysis of CNK in RAS signaling
Authors: Therrien Marc , Wong Allan M. , Kwan Elaine , Rubin Gerald M.
Published: July 2002

Connector enhancer of KSR (CNK) is a multidomain protein required for RAS signaling. Its C-terminal portion (CNK
C-term
) directly binds to RAF. Herein, we show that the N-terminal portion of CNK (CNK
N-term
) strongly cooperates with RAS, whereas CNK
C-term
efficiently blocks RAS- and RAF-dependent signaling when overexpressed in the
Drosophila
eye. Two effector loop mutants of RAS
V12
, S35 and C40, which selectively activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase pathways, respectively, do not cooperate with CNK. However, a strong cooperation is observed between CNK and RAS
V12G37
, an effector loop mutant known in mammals to activate specifically the RAL pathway. We have identified two domains in CNK
N-term
that are critical for cooperation with RAS. Our results suggest that CNK functions in more than one pathway downstream of RAS. CNK
c-term
seems to regulate RAF, a component of the MAPK pathway, whereas CNK
N-term
seems to be involved in a MAPK-independent pathway.
Late Quaternary extinction of a tree species in eastern North America
Authors: Jackson Stephen T. , Weng Chengyu
Published: July 2002

Widespread species- and genus-level extinctions of mammals in North
America and Europe occurred during the last deglaciation
[16,000–9,000 yr B.P. (by
14
C)], a period of rapid and
often abrupt climatic and vegetational change. These extinctions are
variously ascribed to environmental change and overkill by human
hunters. By contrast, plant extinctions since the Middle Pleistocene
are undocumented, suggesting that plant species have been
able to respond to environmental changes of the past several
glacial/interglacial cycles by migration. We provide evidence from
morphological studies of fossil cones and anatomical studies of fossil
needles that a now-extinct species of spruce (
Picea
critchfieldii
sp. nov.) was widespread in eastern North America
during the Last Glacial Maximum.
P. critchfieldii
was
dominant in vegetation of the Lower Mississippi Valley, and extended at
least as far east as western Georgia.
P. critchfieldii
disappeared during the last deglaciation, and its extinction is not
directly attributable to human activities. Similarly widespread plant
species may be at risk of extinction in the face of future climate
change.
T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation
Authors: Yamashita Masakatsu , Kimura Motoko , Kubo Masato , Shimizu Chiori , Tada Tomio , Perlmutter Roger M. , Nakayama Toshinori
Published: July 2002

The central role of type-2 helper T (Th2) cells in the development of allergic responses and immune responses against helminthic parasites is well documented. The differentiation of Th2 cells from naive T cells requires both the recognition of antigen by T cell antigen receptors (TCR) and the activation of downstream signal-transduction molecules of the interleukin 4 receptor (IL-4R) pathway, including Jak1, Jak3, and STAT6. Little is known, however, about how these two distinct pathways cooperate with each other to induce Th2 cells. Here, we use a T cell-specific H-Ras-dominant-negative transgenic mouse to show that TCR-mediated activation of the Ras/mitogen-activated protein kinase pathway alters IL-4R function and is required for Th2 cell differentiation. The enhancement of IL-4R signaling seems to be a consequence of both direct “crosstalk” with the TCR signaling pathway and increased protein expression of downstream signaling molecules of the IL-4R pathway. Therefore, successful Th2 differentiation depends on the effectiveness of the TCR-mediated activation of the Ras/mitogen-activated protein kinase pathway in modifying the IL-4R-mediated signaling pathway.
Structural characteristics of supramolecular assemblies formed by guanidinium-cholesterol reagents for gene transfection
Authors: Pitard Bruno , Oudrhiri Noufissa , Vigneron Jean-Pierre , Hauchecorne Michelle , Aguerre Olivier , Toury Renée , Airiau Marc , Ramasawmy Rajen , Scherman Daniel , Crouzet Joël , Lehn Jean-Marie , Lehn Pierre
Published: July 2002

We have recently discovered that cationic cholesterol derivatives characterized by guanidinium polar headgroups are very efficient for gene transfection
in vitro
and
in vivo
. In spite of being based on some rationale at the molecular level, the development of these new synthetic vectors was nevertheless empirical. Indeed, the factors and processes underlying cationic lipid-mediated gene transfer are still poorly understood. Thus, to get a better insight into the mechanisms involved, we have examined the supramolecular structure of lipid/DNA aggregates obtained when using reagent bis(guanidinium)-tren-cholesterol (BGTC), either alone or as a liposomal formulation with the neutral phospholipid dioleoyl phosphatidylethanolamine (DOPE). We here report the results of cryotransmission electron microscopy studies and small-angle x-ray scattering experiments, indicating the presence of multilamellar domains with a regular spacing of 70 Å and 68 Å in BGTC/DOPE–DNA and BGTC–DNA aggregates, respectively. In addition, DNA lipoplexes with similar lamellar patterns were detected inside transfected HeLa cells by conventional transmission electron microscopy. These results suggest that DNA condensation by multivalent guanidinium-cholesterol cationic lipids involves the formation of highly ordered multilamellar domains, the DNA molecules being intercalated between the lipid bilayers. These results also invite further investigation of the intracellular fate of the internalized lipid/DNA structures during their trafficking toward the cell nucleus. The identification of the basic features of active complexes should indeed help in the design of improved guanidinium-based vectors.
X chromosome evidence for ancient human histories
Authors: Harris Eugene E. , Hey Jody
Published: July 2002

Diverse African and non-African samples of the X-linked PDHA1 (pyruvate dehydrogenaseE1α subunit) locus revealed a fixed DNA sequence difference between the two sample groups. The age of onset of population subdivision appears to be about 200 thousand years ago. This predates the earliest modern human fossils, suggesting the transformation to modern humans occurred in a subdivided population. The base of the PDHA1 gene tree is relatively ancient, with an estimated age of 1.86 million years, a late Pliocene time associated with early species ofHomo. PDHA1 revealed very low variation among non-Africans, but in other respects the data are consistent with reports from other X-linked and autosomal haplotype data sets. Like these other genes, but in conflict with microsatellite and mitochondrial data, PDHA1 does not show evidence of human population expansion.
Thyroid hormone, T3-dependent phosphorylation and translocation of Trip230 from the Golgi complex to the nucleus
Authors: Chen Yumay , Chen Phang-Lang , Chen Chi-Fen , Sharp Z. Dave , Lee Wen-Hwa
Published: July 2002

Trip230 is a novel coactivator of the thyroid hormone receptor that is negatively regulated by the retinoblastoma tumor-suppressor protein. In an examination of its subcellular distribution, Trip230 localized predominantly to the vicinity of the Golgi instead of the nucleus, as other nuclear hormone receptor coactivators. Using a series of deletion mutants, a critical region identified for Golgi area targeting coincided with a previously defined thyroid hormone receptor-binding domain of Trip230. During cell cycle progression, the expression level of Trip230 is constant and a significant portion is imported into the nucleus at S phase. Within an hour of treating cells with T3, Trip230 immunofluorescence transiently colocalized with TR in prominent subnuclear structures. T3-dependent nuclear import of Trip230 does not require new protein synthesis. Coincident with T3 treatment and nuclear import, newly phosphorylated residue(s) appeared in Trip230, suggesting that phosphorylation may be involved in its nuclear import. These findings provided a novel mechanism for the regulation of nuclear hormone transcription factors by hormone-responsive phosphorylation and nuclear import of cytoplasmically located coactivators.
Exercise-induced changes in expression and activity of proteins involved in insulin signal transduction in skeletal muscle: Differential effects on insulin-receptor substrates 1 and 2
Authors: Chibalin Alexander V. , Yu Mei , Ryder Jeffrey W. , Song Xiao Mei , Galuska Dana , Krook Anna , Wallberg-Henriksson Harriet , Zierath Juleen R.
Published: July 2002

Level of physical activity is linked to improved glucose homeostasis. We determined whether exercise alters the expression and/or activity of proteins involved in insulin-signal transduction in skeletal muscle. Wistar rats swam 6 h per day for 1 or 5 days. Epitrochlearis muscles were excised 16 h after the last exercise bout, and were incubated with or without insulin (120 nM). Insulin-stimulated glucose transport increased 30% and 50% after 1 and 5 days of exercise, respectively. Glycogen content increased 2- and 4-fold after 1 and 5 days of exercise, with no change in glycogen synthase expression. Protein expression of the glucose transporter GLUT4 and the insulin receptor increased 2-fold after 1 day, with no further change after 5 days of exercise. Insulin-stimulated receptor tyrosine phosphorylation increased 2-fold after 5 days of exercise. Insulin-stimulated tyrosine phosphorylation of insulin-receptor substrate (IRS) 1 and associated phosphatidylinositol (PI) 3-kinase activity increased 2.5- and 3.5-fold after 1 and 5 days of exercise, despite reduced (50%) IRS-1 protein content after 5 days of exercise. After 1 day of exercise, IRS-2 protein expression increased 2.6-fold and basal and insulin-stimulated IRS-2 associated PI 3-kinase activity increased 2.8-fold and 9-fold, respectively. In contrast to IRS-1, IRS-2 expression and associated PI 3-kinase activity normalized to sedentary levels after 5 days of exercise. Insulin-stimulated Akt phosphorylation increased 5-fold after 5 days of exercise. In conclusion, increased insulin-stimulated glucose transport after exercise is not limited to increased GLUT4 expression. Exercise leads to increased expression and function of several proteins involved in insulin-signal transduction. Furthermore, the differential response of IRS-1 and IRS-2 to exercise suggests that these molecules have specialized, rather than redundant, roles in insulin signaling in skeletal muscle.