bluuebunny/crossref_metadata_2025 huggingface dataset:
A gene encoding a protein modified by the phytohormone indoleacetic acid
Authors: Walz Alexander , Park Seijin , Slovin Janet P. , Ludwig-Müller Jutta , Momonoki Yoshie S. , Cohen Jerry D.
Published: July 2002

We show that the expression of an indole-3-acetic acid (IAA)-modified protein from bean seed, IAP1, is correlated to the developmental period of rapid growth during seed development. Moreover, this protein undergoes rapid degradation during germination. The gene for IAP1, the most abundant protein covalently modified by IAA (
iap1,
GenBank accession no.
AF293023
) was isolated and cloned from bush bean (
Phaseolus vulgaris
) seeds. The 957-bp sequence encodes a 35-kDa polypeptide. IAA-modified proteins represent a distinct class of conjugated phytohormones and appear in bean to be the major form of auxin in seeds. IAA proteins also are found at other stages of development in bean plants. Our immunological and analytical data suggest that auxin modification of a small class of proteins may be a feature common to many plants.
Differential regulation by multiple promoters of the gene encoding the neuron-restrictive silencer factor
Authors: Koenigsberger Carol , Chicca John J. , Amoureux Marie-Claude , Edelman Gerald M. , Jones Frederick S.
Published: July 2002

NRSF/REST is a protein that silences transcription of a number of genes that contain a DNA element called the neuron-restrictive silencer element (NRSE). During embryogenesis, REST is expressed ubiquitously in nonneural cells, but is down-regulated during differentiation of neural progenitors into neurons. REST is also up-regulated in adult neurons by activity, suggesting a possible role for the protein in synaptic plasticity. To understand mechanisms that control expression of REST, we identified and characterized the promoter region of the mouse
REST
gene (
mREST
). A 4.5-kb DNA segment containing three exons (A, B, and C) that correspond to alternatively spliced 5′ untranslated regions (5′UTRs) was isolated and its DNA sequence was determined. Reverse transcription-PCR analyses of fibroblasts, astrocytes, and neural progenitors identified variants in which these exons were spliced to exon D, suggesting that exons A, B, and C may each have a promoter. Consistent with this hypothesis, primer extension and
in vitro
transcription experiments revealed clusters of RNA transcription initiation sites upstream of exons A, B, and C. Tests of
REST
/luciferase reporter constructs in Neuro2A and NIH 3T3 cells revealed promoters upstream of exons A and B that were active in both cell lines, and a promoter upstream of exon C that was weakly active only in NIH 3T3 cells. Six enhancer and two repressor regions were found to overlap each of the three promoters, and some of these were found to be cell type-specific. Combinatorial arrangements of these promoters with enhancer and repressor regions may allow modulation of
REST
expression in particular contexts.
Rapid topology mapping of
Escherichia coli
inner-membrane proteins by prediction and PhoA/GFP fusion analysis
Authors: Drew David , Sjöstrand Dan , Nilsson Johan , Urbig Thomas , Chin Chen-ni , de Gier Jan-Willem , von Heijne Gunnar
Published: July 2002

We present an approach that allows rapid determination of the topology of
Escherichia coli
inner-membrane proteins by a combination of topology prediction and limited fusion-protein analysis. We derive new topology models for 12 inner-membrane proteins: MarC, PstA, TatC, YaeL, YcbM, YddQ, YdgE, YedZ, YgjV, YiaB, YigG, and YnfA. We estimate that our approach should make it possible to arrive at highly reliable topology models for roughly 10% of the ≈800 inner-membrane proteins thought to exist in
E. coli
.
The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins
Authors: Egan James E. , Hall Amy B. , Yatsula Bogdan A. , Bar-Sagi Dafna
Published: July 2002

Signal transduction through epidermal growth factor receptors (EGFRs) is essential for the growth and development of multicellular organisms. A genetic screen for regulators of EGFR signaling has led to the identification of Sprouty, a cell autonomous inhibitor of EGF signaling that is transcriptionally induced by the pathway. However, the molecular mechanisms by which Sprouty exerts its antagonistic effect remain largely unknown. Here we have used transient expression in human cells to investigate the functional properties of human Sprouty (hSpry) proteins. Ectopically expressed full-length hSpry1 and hSpry2 induce the potentiation of EGFR-mediated mitogen-activated protein (MAP) kinase activation. In contrast, truncation mutants of hSpry1 and hSpry2 containing the highly conserved carboxyl-terminal cysteine-rich domain inhibit EGF-induced MAP kinase activation. The potentiating effect of the full-length hSpry2 proteins on EGF signaling is mediated by the amino-terminal domain and results from the sequestration of c-Cbl, which in turn leads to the inhibition of EGFR ubiquitination and degradation. These results indicate that hSpry2 can function both as a negative and positive regulator of EGFR-mediated MAP kinase signaling in a domain-dependent fashion. A dual function of this kind could provide a mechanism for achieving proper balance between the activation and repression of EGFR signaling.
MYC overexpression imposes a nonimmunogenic phenotype on Epstein–Barr virus-infected B cells
Authors: Staege Martin S. , Lee Steven P. , Frisan Teresa , Mautner Josef , Scholz Siegfried , Pajic Alexander , Rickinson Alan B. , Masucci Maria G. , Polack Axel , Bornkamm Georg W.
Published: July 2002

Lymphoblastoid cell lines, generated by immortalization of normal B cells by Epstein–Barr virus (EBV)in vitro, have strong antigen-presenting capacity, are sensitive to EBV-specific cytotoxic T cells, and are highly allostimulatory in mixed lymphocyte culture. By contrast, EBV-positive Burkitt lymphoma (BL) cells are poor antigen presenters, are not recognized by EBV-specific cytotoxic T cells, and are poorly allostimulatory, which raises the question of whether immunological pressure exerted during BL pathogenesisin vivohas selected for a ‘nonimmunogenic’ tumor phenotype. The present work addresses this question by examining the immunogenicity/antigenicity of cell lines, generated by conversion of a conditionally immortalized lymphoblastoid cell line to permanent growth independent of EBV-latent proteins by introduction of a constitutively active or tetracycline-regulated c-mycgene (A1 and P493–6 cells, respectively). Compared with its parental lymphoblastoid cell line, A1 cells showed many of the features of the nonimmunogenic BL phenotype, namely poor allostimulatory activity, poor antigen-presenting function associated with impaired proteasomal activity, down-regulation of peptide transporter, reduced HLA class I expression, and an inability to present endogenously expressed EBV-latent proteins to cytotoxic T cells. P493–6 cells, when grown in the presence of estrogen with the exogenous c-mycgene switched off, were strongly immunogenic. The cells had lost their immunogenic potential, however, when grown on a c-myc-driven proliferation program in the absence of estrogen. Deregulation of c-myc, a step central to the development of uncontrolled BL cell growthin vivo, can thus impose a nonimmunogenic phenotype on proliferating human B cells in the absence of any immune pressure.
HIV type 1 Gag virus-like particle budding from spheroplasts of
Saccharomyces
cerevisiae
Authors: Sakuragi Sayuri , Goto Toshiyuki , Sano Kouichi , Morikawa Yuko
Published: July 2002

Expression of retroviral Gag protein in yeast has previously shown Gag targeting to the plasma membrane but little or no production of Gag virus-like particles (VLPs). Here we show that, after removal of the cell wall, the expression of HIV type 1 Gag protein in
Saccharomyces cerevisiae
spheroplasts allowed simultaneous budding of VLPs from the plasma membrane. Our data show that (
i
) the VLPs released from yeast spheroplasts were spherical and had morphological features, such as membrane apposed electron-dense layers, characteristic of the immature form of HIV particles; (
ii
) the VLPs were completely enclosed in the plasma membrane derived from yeast, which is denser than that of higher eukaryotic cells; (
iii
) the VLP Gag shells remained intact after treatment of nonionic detergent; and (
iv
) the VLPs were released soon after removal of the cell wall and accumulated up to 300 μg/liter of culture. Our results also show that VLP production was abolished by amino acid substitution of the Gag N-terminal myristoylglycine and impaired when Gag C-terminal deletions were extended beyond the nucleocapsid domain. These results were consistent with those obtained previously in higher eukaryotic expression systems, suggesting that similar Gag domains were used for VLP assembly. We suggest that the system described here offers significant advantages for studying host factors required for VLP budding. The system also may be available for production of vector virus-free VLPs for practical applications such as vaccine development.
Gaussian fluctuations and linear response in an electron transfer protein
Authors: Simonson Thomas
Published: July 2002

In response to charge separation or transfer, polar liquids respond in a simple linear fashion. A similar linear response for proteins might be expected from the central limit theorem and is postulated in widely used theories of protein electrostatics, including the Marcus electron transfer theory and dielectric continuum theories. Although these theories are supported by a variety of experimental data, the exact validity of a linear protein dielectric response has been difficult to determine. Molecular dynamics simulations are presented that establish a linear dielectric response of both protein and surrounding solvent over the course of a biologically relevant electron transfer reaction: oxido-reduction of yeast cytochrome
c
in solution. Using an umbrella-sampling free energy approach with long simulations, an accurate treatment of long-range electrostatics and both classical and quantum models of the heme, good agreement is obtained with experiment for the redox potential relative to a heme–octapeptide complex. We obtain a reorganization free energy that is only half that for heme–octapeptide and is reproduced with a dielectric continuum model where the heme vicinity has a dielectric constant of only 1.1. This value implies that the contribution of protein reorganization to the electron transfer free energy barrier is reduced almost to the theoretical limit (a dielectric of one), and that the fluctuations of the electrostatic potential on the heme have a simple harmonic form, in accord with Marcus theory, even though the fluctuations of many individual protein groups (especially at the protein surface) are anharmonic.
Regulation of toxin synthesis in
Clostridium difficile
by an alternative RNA polymerase sigma factor
Authors: Mani Nagraj , Dupuy Bruno
Published: July 2002

Clostridium difficile
, a causative agent of
antibiotic-associated diarrhea and its potentially lethal form,
pseudomembranous colitis, produces two large protein toxins that are
responsible for the cellular damage associated with the disease. The
level of toxin production appears to be critical for determining the
severity of the disease, but the mechanism by which toxin synthesis is
regulated is unknown. The product of a gene,
txeR
, that
lies just upstream of the
tox
gene cluster was shown to
be needed for
tox
gene expression
in vivo
and to activate promoter-specific transcription of the
tox
genes
in vitro
in conjunction with
RNA polymerases from
C. difficile
,
Bacillus
subtilis
, or
Escherichia coli
. TxeR was shown to
function as an alternative sigma factor for RNA polymerase. Because
homologs of TxeR regulate synthesis of toxins and a bacteriocin in
other
Clostridium
species, TxeR appears to be a
prototype for a novel mode of regulation of toxin genes.
Strong DNA binding by covalently linked dimeric Lac headpiece: Evidence for the crucial role of the hinge helices
Authors: Kalodimos Charalampos G. , Folkers Gert E. , Boelens Rolf , Kaptein Robert
Published: July 2002

The combined structural and biochemical studies on Lac repressor
bound to operator DNA have demonstrated the central role of the hinge
helices in operator bending and the induction mechanism. We have
constructed a covalently linked dimeric Lac-headpiece that binds DNA
with four orders of magnitude higher affinity as compared with the
monomeric form. This enabled a detailed biochemical and structural
study of Lac binding to its cognate wild-type and selected DNA
operators. The results indicate a profound contribution of hinge
helices to the stability of the protein–DNA complex and highlight
their central role in operator recognition. Furthermore, protein–DNA
interactions in the minor groove appear to modulate hinge helix
stability, thus accounting for affinity differences and protein-induced
DNA bending among the various operator sites. Interestingly, the
in vitro
DNA-binding affinity of the reported dimeric
Lac construct can de readily modulated by simple adjustment of redox
conditions, thus rendering it a potential artificial gene regulator.
Combined effects of radiotherapy and angiostatin gene therapy in glioma tumor model
Authors: Griscelli Frank , Li Hong , Cheong Chiat , Opolon Paule , Bennaceur-Griscelli Annelise , Vassal Gilles , Soria Jeannette , Soria Claudine , Lu He , Perricaudet Michel , Yeh Patrice
Published: July 2002

The objective of the present study was to evaluate the antitumor effect of a defective adenovirus expressing a secretable angiostatin-like molecule (AdK3) in combination with radiotherapy in rat C6 gliomas s.c. preestablished into athymic mice.
In vitro
, the combination regimen was significantly (
P
< 0.001) more cytotoxic for human microcapillary endothelial cells than either treatment alone, whereas survival of C6 glioma cells was not affected in the conditions used. Radiotherapy and AdK3 gene delivery was then studied on well established C6 xenografts (165 ± 70 mm
3
). In these tumors, AdK3 intratumoral injections had only a marginal effect. Interestingly, when experimental radiotherapy was added, significantly higher (
P
< 0.005), and possibly synergistic, antitumoral effects were observed that tightly correlated a marked decrease of intratumoral vascularization. The combination of radiotherapy and AdK3 intratumoral injections also revealed a significant (
P
< 0.05) inhibition of tumor growth as compared with either treatment alone for larger tumors (467 ± 120 mm
3
). Altogether, these data emphasize the potential of combining a destructive strategy directed against the tumor cells with an anti-angiogenic approach to fight cancer.
A mechanistic basis for Mre11-directed DNA joining at microhomologies
Authors: Paull Tanya T. , Gellert Martin
Published: July 2002

Repair of DNA double-strand breaks in vertebrate cells occurs mainly by an end-joining process that often generates junctions with sequence homologies of a few nucleotides. Mre11 is critical for this mode of repair in budding yeast and has been implicated in the microhomology-based joining. Here, we show that Mre11 exonuclease activity is sensitive to the presence of heterologous DNA, and to the structure and sequence of its ends. Addition of mismatched DNA ends stimulates degradation of DNA by Mre11, whereas cohesive ends strongly inhibit it. Furthermore, if a sequence identity is revealed during the course of degradation, it causes Mre11 nuclease activity to pause, thus stabilizing the junction at a site of microhomology. A nuclease-deficient Mre11 mutant that still binds DNA can also stimulate degradation by wild-type Mre11, suggesting that Mre11-DNA complexes may interact to bridge DNA ends and facilitate DNA joining.
Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase
Authors: Dillingham Mark S. , Soultanas Panos , Wiley Paul , Webb Martin R. , Wigley Dale B.
Published: July 2002

Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues. We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity. We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA. Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.
Deficient long-term memory and long-lasting long-term potentiation in mice with a targeted deletion of neurotrophin-4 gene
Authors: Xie Cui-Wei , Sayah David , Chen Qi-Sheng , Wei Wei-Zheng , Smith Desmond , Liu Xin
Published: July 2002

We examined the learning and memory of neurotrophin-4 (NT4)−/− mice by using fear conditioning. In both cue and context conditioning, we found significant deficits in the NT4 mutants at 2 and 24 h after training but not at 30 min. Hippocampal slices from the mutant mice showed normal basal synaptic transmission, short-term plasticity, and decremental long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses. These findings, together with the normal short-term memory, suggest that the hippocampal development of NT4−/− mice is largely unaffected. However, consistent with the long-term memory defects, the long-lasting LTP at the same synapses was attenuated significantly in the mutant mice. Our results suggest that NT4 plays a physiological role essential for hippocampus- and amygdala-dependent long-term memory and hippocampal long-lasting LTP and that NT4 may be useful in the therapy of acquired disorders of learning and memory.
Hexose permeation pathways in
Plasmodium falciparum
-infected erythrocytes
Authors: Woodrow Charles J. , Burchmore Richard J. , Krishna Sanjeev
Published: July 2002

Plasmodium falciparum
requires glucose as its energy source to multiply within erythrocytes but is separated from plasma by multiple membrane systems. The mechanism of delivery of substrates such as glucose to intraerythrocytic parasites is unclear. We have developed a system for robust functional expression in
Xenopus
oocytes of the
P. falciparum
asexual stage hexose permease, PfHT1, and have analyzed substrate specificities of PfHT1. We show that PfHT1 (a high-affinity glucose transporter,
K
m
≈ 1.0 mM) also transports fructose (
K
m
≈ 11.5 mM). Fructose can replace glucose as an energy source for intraerythrocytic parasites. PfHT1 binds fructose in a furanose conformation and glucose in a pyranose form. Fructose transport by PfHT1 is ablated by mutation of a single glutamine residue, Q169, which is predicted to lie within helix 5 of the hexose permeation pathway. Glucose transport in the Q169N mutant is preserved. Comparison in oocytes of transport properties of PfHT1 and human facilitative glucose transporter (GLUT)1, an archetypal mammalian hexose transporter, combined with studies on cultured
P. falciparum
, has clarified hexose permeation pathways in infected erythrocytes. Glucose and fructose enter erythrocytes through separate permeation pathways. Our studies suggest that both substrates enter parasites via PfHT1.
A genomewide survey of basic helix–loop–helix factors in
Drosophila
Authors: Moore Adrian W. , Barbel Sandra , Jan Lily Yeh , Jan Yuh Nung
Published: July 2002

The basic helix–loop–helix (bHLH) transcription factors play important roles in the specification of tissue type during the development of animals. We have used the information contained in the recently published genomic sequence of
Drosophila melanogaster
to identify 12 additional bHLH proteins. By sequence analysis we have assigned these proteins to families defined by Atonal, Hairy-Enhancer of Split, Hand, p48, Mesp, MYC/USF, and the bHLH-Per, Arnt, Sim (PAS) domain. In addition, one single protein represents a unique family of bHLH proteins. mRNA
in situ
analysis demonstrates that the genes encoding these proteins are expressed in several tissue types but are particularly concentrated in the developing nervous system and mesoderm.
Subretinal transplantation of genetically modified human cell lines attenuates loss of visual function in dystrophic rats
Authors: Lund Raymond D. , Adamson Peter , Sauvé Yves , Keegan David J. , Girman Sergej V. , Wang Shaomei , Winton Helen , Kanuga Naheed , Kwan Anthony S. L. , Beauchène Laurence , Zerbib Anne , Hetherington Len , Couraud Pierre-Olivier , Coffey Peter , Greenwood John
Published: July 2002

Royal College of Surgeons rats are genetically predisposed to
undergo significant visual loss caused by a primary dysfunction of
retinal pigment epithelial (RPE) cells. By using this model, we have
examined the efficacy of subretinal transplantation of two independent
human RPE cell lines each exhibiting genetic modifications that confer
long-term stability
in vitro
. The two cell lines, a
spontaneously derived cell line (ARPE19) and an extensively
characterized genetically engineered human RPE cell line (h1RPE7),
which expresses SV40 large T (tumor) antigen, were evaluated
separately. Both lines result in a significant preservation of visual
function as assessed by either behavioral or physiological techniques.
This attenuation of visual loss correlates with photoreceptor survival
and the presence of donor cells in the areas of rescued photoreceptors
at 5 months postgrafting (6 months of age). These results demonstrate
the potential of genetically modified human RPE cells for ultimate
application in therapeutic transplantation strategies for retinal
degenerative diseases caused by RPE dysfunction.
Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses toCryptococcus neoformans
Authors: Levitz Stuart M. , Nong Shu-hua , Mansour Michael K. , Huang Chao , Specht Charles A.
Published: July 2002

The fungusCryptococcus neoformansis a major cause of morbidity and mortality in patients with impaired CD4+T cell function, particularly those with AIDS. To identify cryptococcal antigens that could serve as vaccine candidates by stimulating T cell responses,C. neoformans-reactive CD4+T cell hybridomas were generated by immunization of C57BL/6 mice and fusion of splenocytes with thymoma cells. The antigen that stimulated one of the hybridomas, designated P1D6, to produce IL-2 was purified to homogeneity by sequential anion exchange chromatography, hydrophobic interaction chromatography, and SDS/PAGE. Based on its apparent molecular mass of 98 kDa and mannosylation, the antigen of interest was named MP98. MP98 was N terminal-sequenced, and the gene encoding the protein was cloned and sequenced. Recombinant MP98, expressed inSaccharomyces cerevisiae, stimulated P1D6 to produce IL-2. Analysis of the derived 458-aa sequence of MP98 reveals an N-terminal cleavable signal sequence, a polysaccharide deacetylase domain found in fungal chitin deacetylases, and a serine/threonine-rich C-terminal region. Overall, there were 103 serine/threonine residues serving as potential O-linked glycosylation sites as well as 12 possible N-linked glycosylation sites. Thus, aC. neoformansmannoprotein has been characterized that stimulates T cell responses and has molecular properties of a chitin deacetylase.
Shape anisotropy of a single random-walk polymer
Authors: Haber Charbel , Ruiz Sami Alom , Wirtz Denis
Published: July 2002

Random walks have been used to describe a wide variety of systems ranging from cell colonies to polymers. Sixty-five years ago, Kuhn [Kuhn, W. (1934)Kolloid-Z.68, 2–11] made the prediction, backed later by computer simulations, that the overall shape of a random-walk polymer is aspherical, yet no experimental work has directly tested Kuhn's general idea and subsequent computer simulations. By using fluorescence microscopy, we monitored the conformation of individual, long, random-walk polymers (fluorescently labeled DNA molecules) at equilibrium. We found that a polymer most frequently adopts highly extended, nonfractal structures with a strongly anisotropic shape. The ensemble-average ratio of the lengths of the long and short axes of the best-fit ellipse of the polymer was much larger than unity.
Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: Functional complementation of specific defects in gp120 and gp41
Authors: Salzwedel Karl , Berger Edward A.
Published: July 2002

The envelope glycoprotein (Env) of HIV-1 is displayed on the
surface of the virion or infected cell as an oligomer of multiple
gp120/gp41 complexes. We sought to unravel the relationships between
this oligomeric structure and the requirements for sequential
interactions with CD4 and coreceptor (CCR5 or CXCR4). We used a
quantitative cell fusion assay to examine the effects of coexpressing
pairs of Envs, each nonfunctional because of a specific defect in one
of the essential properties. We observed efficient fusion activity upon
coexpression of two Env variants, one containing a gp41 subunit with a
mutated fusion peptide and the other containing a gp120 subunit with a
mutated CD4 binding site or a mismatched coreceptor specificity. We
also observed fusion upon coexpression of two Env variants with
distinct gp120 defects, i.e., a CD4 binding site mutation and the
incorrect coreceptor specificity determinants. Coimmunoprecipitation
experiments verified the efficient formation of mixed oligomers,
suggesting that the observed fusion reflected subunit complementation
within the oligomeric complex. These results support a model in which
cooperative subunit interactions within the Env oligomer result in
concerted conformational changes upon receptor binding, resulting in
activation for fusion. The implications of these findings for Env
function and virus neutralization are discussed.
Position within the host intron is critical for efficient processing of box C/D snoRNAs in mammalian cells
Authors: Hirose Tetsuro , Steitz Joan A.
Published: July 2002

In mammalian cells, all small nucleolar RNAs (snoRNAs) that guide rRNA modification are encoded within the introns of host genes. A database analysis of human box C/D snoRNAs revealed conservation of their intronic location, with a preference for 70–80 nt upstream of the 3′ splice site. Transfection experiments showed that synthesis of
gas5
-encoded U75 and U76 snoRNAs dropped significantly for mutant constructs possessing longer or shorter spacers between the snoRNA and the 3′ splice site. However, the position of the snoRNA did not affect splicing of the host intron. Substitution mutations within the spacer indicated that the length, but not the specific sequence, is important. A
in vitro
system that couples pre-mRNA splicing and processing of U75 has been developed. U75 synthesis
in vitro
depends on its box C and D sequences and requires an appropriate spacer length. Further mutational analyses both
in vivo
and
in vitro
, with subsequent mapping of the branch points, revealed that the critical distance is from the snoRNA coding region to the branch point, suggesting synergy between splicing and snoRNA release.
A mutant allele of essential, general translation initiation factor
DED1
selectively inhibits translation of a viral mRNA
Authors: Noueiry Amine O. , Chen Jianbo , Ahlquist Paul
Published: July 2002

Positive-strand RNA virus genomes are substrates for translation,
RNA replication, and encapsidation. To identify host factors involved
in these functions, we used the ability of brome mosaic virus (BMV) RNA
to replicate in yeast. We report herein identification of a mutation in
the essential yeast gene
DED1
that inhibited BMV RNA
replication but not yeast growth.
DED1
encodes a DEAD
(Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation
of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant
DED1
allele (
ded1–18
) resulted from
inhibited expression of viral polymerase-like protein 2a, encoded by
BMV RNA2. Inhibition of RNA2 translation was selective, with no effect
on general cellular translation or translation of BMV RNA1-encoded
replication factor 1a, and was independent of p20, a cellular
antagonist of
DED1
function in translation. Inhibition
of RNA2 translation in
ded1–18
yeast required the RNA2
5′ noncoding region (NCR), which also conferred a
ded1–18
-specific reduction in expression on a reporter
gene mRNA. Comparison of the similar RNA1 and RNA2 5′ NCRs identified a
31-nucleotide RNA2-specific region that was required for the
ded1–18
-specific RNA2 translation block and attenuated
RNA2 translation in wild-type yeast. Further comparisons and RNA
structure predictions suggest a modular arrangement of replication and
translation signals in RNA1 and RNA2 5′ NCRs that appears conserved
among bromoviruses. The 5′ attenuator and
DED1
dependence of RNA2 suggest that, despite its divided genome, BMV
regulates polymerase translation relative to other replication factors,
just as many single-component RNA viruses use translational
read-through and frameshift mechanisms to down-regulate polymerase. The
results show that a DEAD-box helicase can selectively activate
translation of a specific mRNA and may provide a paradigm for
translational regulation by other members of the ubiquitous DEAD-box
RNA helicase family.
Elasticity of MgO and a primary pressure scale to 55 GPa
Authors: Zha Chang-Sheng , Mao Ho-kwang , Hemley Russell J.
Published: July 2002

We have studied the elasticity and pressure–density equation of
state of MgO in diamond cells to 55 GPa and have doubled the previous
pressure limit of accurate elasticity determinations for crystals.
Integrating single-crystal velocity data from Brillouin scattering
measurements and density data from polycrystalline x-ray diffraction,
we obtained the three principal elastic tensor elements
(
C
11
,
C
12
, and
C
44
) and various secondary elasticity
parameters, including single-crystal elastic anisotropy, Cauchy
relation, aggregate sound velocities, and Poisson's ratio, as
functions of pressure. The present study also provides a direct
determination of pressure without recourse to any prior pressure
standard, thus creating a primary pressure scale. The commonly used
ruby fluorescence pressure scale has thus been improved to 1% accuracy
by the new MgO scale.
Dynamic methylation adjustment and counting as part of imprinting mechanisms.
Authors: Shemer R , Birger Y , Dean W L , Reik W , Riggs A D , Razin A
Published: July 2002

Monoallelic expression in diploid mammalian cells appears to be a widespread phenomenon, with the most studied examples being X-chromosome inactivation in eutherian female cells and genomic imprinting in the mouse and human. Silencing and methylation of certain sites on one of the two alleles in somatic cells is specific with respect to parental source for imprinted genes and random for X-linked genes. We report here evidence indicating that: (i) differential methylation patterns of imprinted genes are not simply copied from the gametes, but rather established gradually after fertilization; (ii) very similar methylation patterns are observed for diploid, tetraploid, parthenogenic, and androgenic preimplantation mouse embryos, as well as parthenogenic and androgenic mouse embryonic stem cells; (iii) haploid parthenogenic embryos do not show methylation adjustment as seen in diploid or tetraploid embryos, but rather retain the maternal pattern. These observations suggest that differential methylation in imprinted genes is achieved by a dynamic process that senses gene dosage and adjusts methylation similar to X-chromosome inactivation.
Presence of mRNA for glutamic acid decarboxylase in both excitatory and inhibitory neurons.
Authors: Cao Y , Wilcox K S , Martin C E , Rachinsky T L , Eberwine J , Dichter M A
Published: July 2002

Neurons in very low density hippocampal cultures that are physiologically identified as either GABAergic inhibitory or glutamatergic excitatory all contain mRNA for the gamma-aminobutyric acid (GABA) synthetic enzyme, glutamic acid decarboxylase (GAD), as detected by single cell mRNA amplification and PCR. However, consistent with the physiology, immunocytochemistry revealed that only a subset of the neurons stain for either GAD protein or GABA. A similar fraction hybridize with RNA probes for GAD65 and GAD67. Hippocampal CA1 pyramidal neurons in slice preparations, which are traditionally thought to be excitatory, also contain mRNA for GAD65 and GAD67. Hippocampal neurons in culture did not contain mRNA for two other neurotransmitter synthesizing enzymes, tyrosine hydroxylase, and choline acetyl transferase. These data suggest that in some neurons, presumably the excitatory neurons, GAD mRNA is selectively regulated at the level of translation. We propose that neurotransmitter phenotype may be posttranscriptionally regulated and neurons may exhibit transient phenotypic plasticity in response to environmental influences.
Evidence that DNA damage triggers interleukin 10 cytokine production in UV-irradiated murine keratinocytes.
Authors: Nishigori C , Yarosh D B , Ullrich S E , Vink A A , Bucana C D , Roza L , Kripke M L
Published: July 2002

UV irradiation interferes with the induction of T cell-mediated immune responses, in part by causing cells in the skin to produce immunoregulatory cytokines. Recent evidence implicates UV-induced DNA damage as a trigger for the cascade of events leading to systemic immune suppression in vivo. However, to date, there has been no direct evidence linking DNA damage and cytokine production in UV-irradiated cells. Here we provide such evidence by showing that treatment of UV-irradiated murine keratinocytes in vitro with liposomal T4 endonuclease V, which accelerates the repair of cyclobutylpyrimidine dimers in these cells, inhibits their production of immunosuppressive cytokines, including interleukin 10. Application of these liposomes to murine skin in vivo also reduced the induction of interleukin 10 by UV irradiation, whereas liposomes containing heat-inactivated T4 endonuclease V were ineffective. These results support our hypothesis that unrepaired DNA damage in the skin activates the production of cytokines that down-regulate immune responses initiated at distant sites.
c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.
Authors: Polack A , Hörtnagel K , Pajic A , Christoph B , Baier B , Falk M , Mautner J , Geltinger C , Bornkamm G W , Kempkes B
Published: July 2002

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.
A novel functional role for apolipoprotein B in male infertility in heterozygous apolipoprotein B knockout mice.
Authors: Huang L S , Voyiaziakis E , Chen H L , Rubin E M , Gordon J W
Published: July 2002

Male infertility, affecting as many as 10% of the adult population, is an extremely prevalent disorder. In most cases, the cause of the condition is unknown, and genetic factors that might affect male fertility, other than some sequences on the Y chromosome, have not been identified. We report here that male mice heterozygous for a targeted mutation of the apolipoprotein B (apo B) gene exhibit severely compromised fertility. Sperm from these mice failed to fertilize eggs both in vivo and in vitro. However, these sperm were able to fertilize eggs once the zona pellucida was removed but displayed persistent abnormal binding to the egg after fertilization. In vitro fertilization-related and other experiments revealed reduced sperm motility, survival time, and sperm count also contributed to the infertility phenotype. Recognition of the infertility phenotype led to the identification of apo B mRNA in the testes and epididymides of normal mice, and these transcripts were substantially reduced in the affected animals. Moreover, when the genomic sequence encoding human apo B was introduced into these animals, normal fertility was restored. These findings suggest that this genetic locus may have an important impact on male fertility and identify a previously unrecognized function for apo B.
Nocturnality and species survival.
Authors: Daily G C , Ehrlich P R
Published: July 2002

Surveys of butterfly and moth diversity in tropical forest fragments suggest that nocturnality confers a dispersal, and possibly a survival, advantage. The butterfly faunas of smaller fragments were depauperate; in contrast, the species richness of nocturnal moths was similar in all fragments and even in pasture. The lack of correlation between butterfly and moth species richness among fragments (r2 = 0.005) is best explained by movements of moths at night when ambient conditions in forest and pasture are most similar; butterflies face substantial daytime temperature, humidity, and solar radiation barriers. This interpretation is supported by information on birds, beetles, and bats.
Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.
Authors: Mingari M C , Schiavetti F , Ponte M , Vitale C , Maggi E , Romagnani S , Demarest J , Pantaleo G , Fauci A S , Moretta L
Published: July 2002

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class 1 molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR V beta-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V beta families were largely represented, and most of the other V beta s were barely detected. In addition, analysis of recombinant clones of the largely represented V beta families demonstrated that these V beta s were oligoclonally or monoclonally expanded.
The visual response of retinal ganglion cells is not altered by optic nerve transection in transgenic mice overexpressing Bcl-2
Authors: Porciatti V. , Pizzorusso T. , Cenni M. C. , Maffei L.
Published: July 2002

Attempts to rescue retinal ganglion cells from retrograde
degeneration have had limited success, and the residual function of
surviving neurons is not known. Recently, it has been found that
axotomized retinal ganglion cells die by apoptotic mechanisms.
We have used adult transgenic mice overexpressing the Bcl-2 protein, a
powerful inhibitor of apoptosis, as a model for preventing
injury-induced cell death
in vivo
. Several months after
axotomy, the majority of retinal ganglion cells survived and exhibited
normal visual responses. In control wild-type mice, the vast majority
of axotomized retinal ganglion cells degenerated, and the physiological
responses were abolished. These results suggest that strategies aimed
at increasing Bcl-2 expression, or mimicking its function, might
effectively counteract trauma-induced cell death in the central nervous
system. Neuronal survival is a necessary condition in the challenge for
promoting regeneration and eventually restoring neuronal function.
Somatic and germ cell parasitism in a colonial ascidian: Possible role for a highly polymorphic allorecognition system
Authors: Stoner Douglas S. , Weissman Irving L.
Published: July 2002

A colonial protochordate,
Botryllus schlosseri
,
undergoes a natural transplantation reaction in the wild that results
alternatively in colony fusion (chimera formation) or inflammatory
rejection. A single, highly polymorphic histocompatibility locus
(called Fu/HC) is responsible for rejection versus fusion. Gonads are
seeded and gametogenesis can occur in colonies well after fusion, and
involves circulating germ-line progenitors. Buss proposed that colonial
organisms might develop self/non-self histocompatibility systems to
limit the possibility of interindividual germ cell “parasitism”
(GCP) to histocompatible kin [Buss, L. W. (1982)
Proc
.
Natl. Acad. Sci. USA
79, 5337–5341 and Buss, L. W.
(1987)
The Evolution of Individuality
(Princeton Univ.
Press, Princeton]. Here we demonstrate in laboratory and field
experiments that both somatic cell and (more importantly) germ-line
parasitism are a common occurrence in fused chimeras. These experiments
support the tenet in Buss’s hypothesis that germ cell and somatic cell
parasitism can occur in fused chimeras and that a somatic appearance
may mask the winner of a gametic war. They also provide an interesting
challenge to develop formulas that describe the inheritance of
competing germ lines rather than competing individuals. The fact that
fused
B
.
schlosseri
have higher rates of
GCP than unfused colonies additionally provides a rational explanation
for the generation and maintenance of a high degree of Fu/HC
polymorphism, largely limiting GCP to sibling offspring.
Evolution of chlorophyll and bacteriochlorophyll: the problem of invariant sites in sequence analysis.
Authors: Lockhart P J , Larkum A W , Steel M , Waddell P J , Penny D
Published: July 2002

Competing hypotheses seek to explain the evolution of oxygenic and anoxygenic processes of photosynthesis. Since chlorophyll is less reduced and precedes bacteriochlorophyll on the modern biosynthetic pathway, it has been proposed that chlorophyll preceded bacteriochlorophyll in its evolution. However, recent analyses of nucleotide sequences that encode chlorophyll and bacteriochlorophyll biosynthetic enzymes appear to provide support for an alternative hypothesis. This is that the evolution of bacteriochlorophyll occurred earlier than the evolution of chlorophyll. Here we demonstrate that the presence of invariant sites in sequence datasets leads to inconsistency in tree building (including maximum-likelihood methods). Homologous sequences with different biological functions often share invariant sites at the same nucleotide positions. However, different constraints can also result in additional invariant sites unique to the genes, which have specific and different biological functions. Consequently, the distribution of these sites can be uneven between the different types of homologous genes. The presence of invariant sites, shared by related biosynthetic genes as well as those unique to only some of these genes, has misled the recent evolutionary analysis of oxygenic and anoxygenic photosynthetic pigments. We evaluate an alternative scheme for the evolution of chlorophyll and bacteriochlorophyll.
Potentiation of the bioavailability of daidzin by an extract of Radix puerariae.
Authors: Keung W M , Lazo O , Kunze L , Vallee B L
Published: July 2002

The dose effect of pure daidzin on the suppression of ethanol intake in Syrian golden hamsters was compared with that of crude daidzin contained in a methanol extract of Radix puerariae (RP). EC50 values estimated from the graded dose-response curves for pure daidzin and RP extract daidzin are 23 and 2.3 mg per hamster per day, respectively. Apparently the antidipsotropic activity of the RP extract cannot be accounted for solely by its daidzin content (22 mg/g). In addition to daidzin, six other isoflavones were identified in the RP extract and quantified--namely, puerarin (160 mg per g of extract), genistin (3.7 mg/g), daidzein (2.6 mg/g), daidzein-4',7-diglucoside (1.2 mg/g), genistein (0.2 mg/g), and formononetin (0.16 mg/g). None of these, administered either alone or combined, contributes in any significant way to the antidipsotropic activity of the extract. Plasma daidzin concentration-time curves determined in hamsters administered various doses of pure daidzin or RP extract by i.p.injection indicate that the crude extract daidzin has approximately 10 times greater bioavailability than the pure compound. Reconstruction of the dose-response effects for pure and crude daidzin using bioavailable daidzin rather than administered dose gives a single curve. Synthetic daidzin added to the RP extract acquires the bioavailability of the endogenous daidzin that exists naturally in the extract. These results show that (i) daidzin is the major active principle in methanol extracts of RP, and (ii) additional constituents in the methanol extract of RP assist uptake of daidzin in golden hamsters.
Cross-validated maximum likelihood enhances crystallographic simulated annealing refinement
Authors: Adams Paul D. , Pannu Navraj S. , Read Randy J. , Brünger Axel T.
Published: July 2002

Recently, the target function for crystallographic refinement has been improved through a maximum likelihood analysis, which makes proper allowance for the effects of data quality, model errors, and incompleteness. The maximum likelihood target reduces the significance of false local minima during the refinement process, but it does not completely eliminate them, necessitating the use of stochastic optimization methods such as simulated annealing for poor initial models. It is shown that the combination of maximum likelihood with cross-validation, which reduces overfitting, and simulated annealing by torsion angle molecular dynamics, which simplifies the conformational search problem, results in a major improvement of the radius of convergence of refinement and the accuracy of the refined structure. Torsion angle molecular dynamics and the maximum likelihood target function interact synergistically, the combination of both methods being significantly more powerful than each method individually. This is demonstrated in realistic test cases at two typical minimum Bragg spacings (
d
min
= 2.0 and 2.8 Å, respectively), illustrating the broad applicability of the combined method. In an application to the refinement of a new crystal structure, the combined method automatically corrected a mistraced loop in a poor initial model, moving the backbone by 4 Å.