bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-nucleotide polymorphisms can cause different structural folds of mRNA
Authors: Shen Ling X. , Basilion James P. , Stanton Vincent P.
Published: July 2002

Single-nucleotide polymorphisms (SNPs) are the most common type of genetic variation in man. Genes containing one or more SNPs can give rise to two or more allelic forms of mRNAs. These mRNA variants may possess different biological functions as a result of differences in primary or higher order structures that interact with other cellular components. Here we report the observation of marked differences in mRNA secondary structure associated with SNPs in the coding regions of two human mRNAs: alanyl tRNA synthetase and replication protein A, 70-kDa subunit (RPA70). Enzymatic probing of SNP-containing allelic fragments of the mRNAs revealed pronounced allelic differences in cleavage pattern at sites 14 or 18 nt away from the SNP, suggesting that a single-nucleotide variation can give rise to different mRNA folds. By using phosphorothioate oligodeoxyribonucleotides complementary to the region of different allelic structures in the RPA70 mRNA, but not extending to the SNP itself, we find that the SNP exerts an allele-specific effect on the accessibility of its flanking site in the endogenous human RPA70 mRNA. This further supports the allele-specific structural features identified by enzymatic probing. These results demonstrate the contribution of common genetic variation to structural diversity of mRNA and suggest a broader role than previously thought for the effects of SNPs on mRNA structure and, ultimately, biological function.
Potent urea and carbamate inhibitors of soluble epoxide hydrolases
Authors: Morisseau Christophe , Goodrow Marvin H. , Dowdy Deanna , Zheng Jiang , Greene Jessica F. , Sanborn James R. , Hammock Bruce D.
Published: July 2002

The soluble epoxide hydrolase (sEH) plays a significant role in the biosynthesis of inflammation mediators as well as xenobiotic transformations. Herein, we report the discovery of substituted ureas and carbamates as potent inhibitors of sEH. Some of these selective, competitive tight-binding inhibitors with nanomolar
K
i
values interacted stoichiometrically with the homogenous recombinant murine and human sEHs. These inhibitors enhance cytotoxicity of
trans-
stilbene oxide, which is active as the epoxide, but reduce cytotoxicity of leukotoxin, which is activated by epoxide hydrolase to its toxic diol. They also reduce toxicity of leukotoxin
in vivo
in mice and prevent symptoms suggestive of acute respiratory distress syndrome. These potent inhibitors may be valuable tools for testing hypotheses of involvement of diol and epoxide lipids in chemical mediation
in vitro
or
in vivo
systems.
Rifins: A second family of clonally variant proteins expressed on the surface of red cells infected with
Plasmodium falciparum
Authors: Kyes Sue A. , Rowe J. Alexandra , Kriek Neline , Newbold Chris I.
Published: July 2002

Many pathogens evade the host immune response or adapt to their environment by expressing surface proteins that undergo rapid switching. In the case of
Plasmodium falciparum
, products of a multigene family known as
var
are expressed on the surface of infected red cells, where they undergo clonal antigenic variation and contribute to malaria pathogenesis by mediating adhesion to a variety of host endothelial receptors and to uninfected red blood cells by forming rosettes. Herein we show that a second gene family,
rif
, which is associated with
var
at subtelomeric sites in the genome, encodes clonally variant proteins (rifins) that are expressed on the infected red cell surface. Their high copy number, sequence variability, and red cell surface location indicate an important role for rifins in malaria host–parasite interaction.
Rice gibberellin-insensitive dwarf mutant gene
Dwarf 1
encodes the α-subunit of GTP-binding protein
Authors: Ashikari Motoyuki , Wu Jianzhong , Yano Masahiro , Sasaki Takuji , Yoshimura Atsushi
Published: July 2002

A rice
Dwarf 1
gene was identified by using a map-based cloning strategy. Its recessive mutant allele confers a dwarf phenotype. Linkage analysis revealed that a cDNA encoding the α-subunit of GTP-binding protein cosegregated with
d1
in 3,185
d1
segregants. Southern hybridization analysis with this cDNA as a probe showed different band patterns in several
d1
mutant lines. In at least four independent
d1
mutants, no gene transcript was observed by Northern hybridization analysis. Sequencing analysis revealed that an 833-bp deletion had occurred in one of the mutant alleles, which resulted in an inability to express GTP-binding protein. A transgenic
d1
mutant with GTP-binding protein gene restored the normal phenotype. We conclude that the rice
Dwarf 1
gene encodes GTP-binding protein and that the protein plays an important role in plant growth and development. Because the
d1
mutant is classified as gibberellin-insensitive, we suggest that the GTP-binding protein might be associated with gibberellin signal transduction.
Adenovirus-mediated regulable target gene expression
in vivo
Authors: Burcin Mark M. , Schiedner Gudrun , Kochanek Stefan , Tsai Sophia Y. , O’Malley Bert W.
Published: July 2002

To regulate expression of a transferred gene in response to an exogenous compound, we have combined a high capacity adenoviral vector devoid of all viral coding sequences with a regulatory system that can be used to express a target gene
in vivo
in a selected site and at a desired time. This system uses a chimeric transactivator, GLp65, which consists of a mutated progesterone receptor–ligand binding domain fused to the GAL4 DNA binding domain and part of the activation domain of the human p65 protein, a component of the NF-κB complex. In the presence of the antiprogestin mifepristone, this chimeric regulator binds to a target gene containing the 17-mer GAL4 binding site, resulting in an efficient ligand-inducible transactivation of the target gene. We inserted the regulator GLp65 and a regulable human growth hormone target gene containing the 17-mer GAL4 binding site into the same adenoviral vector. To obtain tissue-specific expression of the target gene, we coupled the regulator to a liver-specific promoter. Infection of HepG2 cells and experimental mice with the adenovirus resulted in consistently high induction levels of human growth hormone in the presence of mifepristone whereas the transgene expression was undetectable in the absence of the ligand. Taken together, our regulable adenoviral vector represents an important tool for transgene regulation that can be used for potentially diverse applications, ranging from tissue-specific gene expression in transgenic animals to human gene therapy.
Involvement of regulatory and catalytic subunits of phosphoinositide 3-kinase in NF-κB activation
Authors: Béraud Christophe , Henzel William J. , Baeuerle Patrick A.
Published: July 2002

Hypoxia, reoxygenation, and the tyrosine phosphatase inhibitor pervanadate activate the transcription factor NF-κB, involving phosphorylation of its inhibitor IκB-α on tyrosine 42. This modification does not lead to degradation of IκB by the proteasome/ubiquitin pathway, as is seen on stimulation of cells with proinflammatory cytokines. It is currently unknown how tyrosine-phosphorylated IκB is removed from NF-κB. Here we show that p85α, the regulatory subunit of PI3-kinase, specifically associates through its Src homology 2 domains with tyrosine-phosphorylated IκB-α
in vitro
and
in vivo
after stimulation of T cells with pervanadate. This association could provide a mechanism by which newly tyrosine-phosphorylated IκB is sequestered from NF-κB. Another mechanism by which PI3-kinase contributed to NF-κB activation in response to pervanadate appeared to involve its catalytic p110 subunit. This was evident from the inhibition of pervanadate-induced NF-κB activation and reporter gene induction by treatment of cells with nanomolar amounts of the PI3-kinase inhibitor wortmannin. The compound had virtually no effect on tumor necrosis factor- and interleukin-1-induced NF-κB activities. Wortmannin did not inhibit tyrosine phosphorylation of IκB-α or alter the stability of the PI3-kinase complex but inhibited Akt kinase activation in response to pervanadate. Our data suggest that both the regulatory and the catalytic subunit of PI3-kinase play a role in NF-κB activation by the tyrosine phosphorylation-dependent pathway.
A G
sα
mutant designed to inhibit receptor signaling through G
s
Authors: Iiri Taroh , Bell Sean M. , Baranski Thomas J. , Fujita Toshiro , Bourne Henry R.
Published: July 2002

Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein α subunit (Gα), thereby generating two potential signaling molecules, Gα–GTP and free Gβγ. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Gα mutation. Here we describe a mutant α subunit designed to inhibit receptor-mediated hormonal activation of G
s
, the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the α subunit (α
s
) of G
s
three separate mutations chosen because they impair α
s
function in complementary ways: the A
366
S mutant reduces affinity of α
s
for binding GDP, whereas the G
226
A and E
268
A mutations impair the protein’s ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) G
s
-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active α
s
mutant (α
s
–R
201
C) or to agonists for receptors that activate G
q
or G
i
. This α
s
triple mutant and cognate Gα mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.
Rapid production of specific vaccines for lymphoma by expression of the tumor-derived single-chain Fv epitopes in tobacco plants
Authors: McCormick Alison A. , Kumagai Monto H. , Hanley Kathleen , Turpen Thomas H. , Hakim Itzhak , Grill Laurence K. , Tusé Daniel , Levy Shoshana , Levy Ronald
Published: July 2002

Rapid production of protein-based tumor-specific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. InfectedNicotiana benthamianaplants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 μg/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin’s lymphoma.
A genetic method for dissecting the mechanism of transcriptional activator synergy by identical activators
Authors: Langdon Robert C. , Hochschild Ann
Published: July 2002

Pairs of transcriptional activators in prokaryotes have been shown to activate transcription synergistically from promoters with two activator binding sites. In some cases, such synergistic effects result from cooperative binding, but in other cases each DNA-bound activator plays a direct role in the activation process by interacting simultaneously with separate surfaces of RNA polymerase. In such cases, each DNA-bound activator must possess a functional activating region, the surface that mediates the interaction with RNA polymerase. When transcriptional activation depends on two or more identical activators, it is not straightforward to test the requirement of each activator for a functional activating region. Here we describe a method for directing a mutationally altered activator to either one or the other binding site, and we demonstrate the use of this method to examine the mechanism of transcriptional activator synergy by the
Escherichia coli
cyclic AMP receptor protein (CRP) working at an artificial promoter bearing two CRP-binding sites.
Sustained
in vivo
cardiac protection by a rationally designed peptide that causes ɛ protein kinase C translocation
Authors: Dorn Gerald W. , Souroujon Miriam C. , Liron Tamar , Chen Che-Hong , Gray Mary O. , Zhou Hui Zhong , Csukai Michael , Wu Guangyu , Lorenz John N. , Mochly-Rosen Daria
Published: July 2002

Brief periods of cardiac ischemia trigger protection from subsequent prolonged ischemia (preconditioning). ɛ Protein kinase C (ɛPKC) has been suggested to mediate preconditioning. Here, we describe an ɛPKC-selective agonist octapeptide, ψɛ receptor for activated C-kinase (ψɛRACK), derived from an ɛPKC sequence homologous to its anchoring protein, ɛRACK. Introduction of ψɛRACK into isolated cardiomyocytes, or its postnatal expression as a transgene in mouse hearts, increased ɛPKC translocation and caused cardio-protection from ischemia without any deleterious effects. Our data demonstrate that ɛPKC activation is required for protection from ischemic insult and suggest that small molecules that mimic this ɛPKC agonist octapeptide provide a powerful therapeutic approach to protect hearts at risk for ischemia.
Down-regulation of the expression of endothelial NO synthase is likely to contribute to glucocorticoid-mediated hypertension
Authors: Wallerath Thomas , Witte Klaus , Schäfer Stephan C. , Schwarz Petra M. , Prellwitz Winfried , Wohlfart Paulus , Kleinert Hartmut , Lehr Hans-Anton , Lemmer Björn , Förstermann Ulrich
Published: July 2002

Hypertension is a side effect of systemically administered glucocorticoids, but the underlying molecular mechanism remains poorly understood. Ingestion of dexamethasone by rats telemetrically instrumented increased blood pressure progressively over 7 days. Plasma concentrations of Na
+
and K
+
and urinary Na
+
and K
+
excretion remained constant, excluding a mineralocorticoid-mediated mechanism. Plasma NO
2
−
/NO
3
−
(the oxidation products of NO) decreased to 40%, and the expression of endothelial NO synthase (NOS III) was found down-regulated in the aorta and several other tissues of glucocorticoid-treated rats. The vasodilator response of resistance arterioles was tested by intravital microscopy in the mouse dorsal skinfold chamber model. Dexamethasone treatment significantly attenuated the relaxation to the endothelium-dependent vasodilator acetylcholine, but not to the endothelium-independent vasodilator S-nitroso-
N
-acetyl-
d
,
l
-penicillamine. Incubation of human umbilical vein endothelial cells, EA.hy 926 cells, or bovine aortic endothelial cells with several glucocorticoids reduced NOS III mRNA and protein expression to 60–70% of control, an effect that was prevented by the glucocorticoid receptor antagonist mifepristone. Glucocorticoids decreased NOS III mRNA stability and reduced the activity of the human NOS III promoter (3.5 kilobases) to ≈70% by decreasing the binding activity of the essential transcription factor GATA. The expressional down-regulation of endothelial NOS III may contribute to the hypertension caused by glucocorticoids.
Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes
Authors: Müllbacher Arno , Waring Paul , Tha Hla Ron , Tran Thao , Chin Seow , Stehle Thomas , Museteanu Crisan , Simon Markus M.
Published: July 2002

Analysis of perforin-deficient mice has identified the cytolytic pathway and perforin as the preeminent effector molecule in T cell-mediated control of virus infections. In this paper, we show that mice lacking both granzyme A (gzmA) and granzyme B (gzmB), which are, beside perforin, key constituents of cytolytic vesicles, are as incapable as are perforin-deficient mice of controlling primary infections by the natural mouse pathogen ectromelia, a poxvirus. Death of gzmA×gzmB double knockout mice occurred in a dose-dependent manner, despite the expression of functionally active perforin and the absence of an intrinsic defect to generate splenic cytolytic T cells. These results establish that both gzmA and gzmB are indispensable effector molecules acting in concert with perforin in granule exocytosis-mediated host defense against natural viral pathogens.
The
DNMT3B
DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome
Authors: Hansen R. Scott , Wijmenga Cisca , Luo Ping , Stanek Ann M. , Canfield Theresa K. , Weemaes Corry M. R. , Gartler Stanley M.
Published: July 2002

DNA methylation is an important regulator of genetic information in species ranging from bacteria to humans. DNA methylation appears to be critical for mammalian development because mice nullizygous for a targeted disruption of the
DNMT1
DNA methyltransferase die at an early embryonic stage. No DNA methyltransferase mutations have been reported in humans until now. We describe here the first example of naturally occurring mutations in a mammalian DNA methyltransferase gene. These mutations occur in patients with a rare autosomal recessive disorder, which is termed the ICF syndrome, for immunodeficiency, centromeric instability, and facial anomalies. Centromeric instability of chromosomes 1, 9, and 16 is associated with abnormal hypomethylation of CpG sites in their pericentromeric satellite regions. We are able to complement this hypomethylation defect by somatic cell fusion to Chinese hamster ovary cells, suggesting that the ICF gene is conserved in the hamster and promotes
de novo
methylation. ICF has been localized to a 9-centimorgan region of chromosome 20 by homozygosity mapping. By searching for homologies to known DNA methyltransferases, we identified a genomic sequence in the ICF region that contains the homologue of the mouse
Dnmt3b
methyltransferase gene. Using the human sequence to screen ICF kindreds, we discovered mutations in four patients from three families. Mutations include two missense substitutions and a 3-aa insertion resulting from the creation of a novel 3′ splice acceptor. None of the mutations were found in over 200 normal chromosomes. We conclude that mutations in the
DNMT3B
are responsible for the ICF syndrome.
Major recent and independent changes in levels and patterns of expression have occurred at the
b
gene, a regulatory locus in maize
Authors: Selinger David A. , Chandler Vicki L.
Published: July 2002

The
b
locus encodes a transcription factor that regulates the expression of genes that produce purple anthocyanin pigment. Different
b
alleles are expressed in distinct tissues, causing tissue-specific anthocyanin production. Understanding how phenotypic diversity is produced and maintained at the
b
locus should provide models for how other regulatory genes, including those that influence morphological traits and development, evolve. We have investigated how different levels and patterns of pigmentation have evolved by determining the phenotypic and evolutionary relationships between 18 alleles that represent the diversity of
b
alleles in
Zea mays
. Although most of these alleles have few phenotypic differences, five alleles have very distinct tissue-specific patterns of pigmentation. Superimposing the phenotypes on the molecular phylogeny reveals that the alleles with strong and distinctive patterns of expression are closely related to alleles with weak expression, implying that the distinctive patterns have arisen recently. We have identified apparent insertions in three of the five phenotypically distinct alleles, and the fourth has unique upstream restriction fragment length polymorphisms relative to closely related alleles. The insertion in
B-Peru
has been shown to be responsible for its unique expression and, in the other two alleles, the presence of the insertion correlates with the phenotype. These results suggest that major changes in gene expression are probably the result of large-scale changes in DNA sequence and/or structure most likely mediated by transposable elements.
Alternative splicing, muscle calcium sensitivity, and the modulation of dragonfly flight performance
Authors: Marden James H. , Fitzhugh Gail H. , Wolf Melisande R. , Arnold Kristina D. , Rowan Barry
Published: July 2002

Calcium sensitivity of myosin cross-bridge activation in striated muscles commonly varies during ontogeny and in response to alterations in muscle usage, but the consequences for whole-organism physiology are not well known. Here we show that the relative abundances of alternatively spliced transcripts of the calcium regulatory protein troponin T (TnT) vary widely in flight muscle of
Libellula pulchella
dragonflies, and that the mixture of TnT splice variants explains significant portions of the variation in muscle calcium sensitivity, wing-beat frequency, and an index of aerodynamic power output during free flight. Two size-distinguishable morphs differ in their maturational pattern of TnT splicing, yet they show the same relationship between TnT transcript mixture and calcium sensitivity and between calcium sensitivity and aerodynamic power output. This consistency of effect in different developmental and physiological contexts strengthens the hypothesis that TnT isoform variation modulates muscle calcium sensitivity and whole-organism locomotor performance. Modulating muscle power output appears to provide the ecologically important ability to operate at different points along a tradeoff between performance and energetic cost.
Crystal structure of a dimeric chymotrypsin inhibitor 2 mutant containing an inserted glutamine repeat
Authors: Chen Yu Wai , Stott Kelvin , Perutz Max F.
Published: July 2002

We have constructed mutants of chymotrypsin inhibitor 2
with short glutamine repeats inserted into its inhibitory loop. These
mutants oligomerize when expressed in
Escherichia
coli
. The dimer of a mutant with four glutamines now has
been crystallized, and its structure has been solved by molecular
replacement by using the wild-type monomer as a search model. The
structure of each half of the dimer is found to be the same as that of
the wild-type monomer, except around the glutamine insertion. It was
proposed that the components of the oligomers are held together by
hydrogen bonds between the main-chain and side-chain amides of the
glutamine repeats. Instead, they appear to form by swapping domains on
folding in
E. coli
, and the glutamine repeats connecting
the components of the dimers are disordered.
The retinitis pigmentosa GTPase regulator, RPGR, interacts with the delta subunit of rod cyclic GMP phosphodiesterase
Authors: Linari Marco , Ueffing Marius , Manson Forbes , Wright Alan , Meitinger Thomas , Becker Jörg
Published: July 2002

Recently, the retinitis pigmentosa 3 (
RP3
) gene has
been cloned and named retinitis pigmentosa GTPase regulator
(
RPGR
). The amino-terminal half of RPGR is homologous to
regulator of chromosome condensation (RCC1), the nucleotide exchange
factor for the small GTP-binding protein Ran. In a yeast two-hybrid
screen we identified the delta subunit of rod cyclic GMP
phosphodiesterase (PDEδ) as interacting with the RCC1-like domain
(RLD) of RPGR (RPGR
392
). The interaction of RPGR with
PDEδ was confirmed by pull-down assays and plasmon surface resonance.
The binding affinity was determined to be 90 nM. Six missense mutations
at evolutionary conserved residues within the RLD, which were found in
RP3 patients, were analyzed by using the two-hybrid system. All
missense mutations showed reduced interaction with PDEδ. A
non-RP3-associated missense substitution outside the RLD, V36F, did not
abolish the interaction with PDEδ. PDEδ is widely expressed and
highly conserved across evolution and is proposed to regulate the
membrane insertion or solubilization of prenylated proteins, including
the catalytic subunits of the PDE holoenzyme involved in
phototransduction and small GTP-binding proteins of the Rab family.
These results suggest that
RPGR
mutations give rise to
retinal degeneration by dysregulation of intracellular processes that
determine protein localization and protein transport.
Covariation of activity in visual and prefrontal cortex associated with subjective visual perception
Authors: Lumer Erik D. , Rees Geraint
Published: July 2002

Visual areas of the occipitotemporal pathway are thought to be essential for the conscious perception of objects, but the contribution of other cortical regions and the neural mechanisms leading to the awareness of a visual stimulus remain unclear. By using functional MRI in humans exposed to bistable viewing conditions, subjective visual perception was related to covariation of activity in multiple extrastriate ventral, parietal, and prefrontal cortical areas. The coordination of activity among these regions was not linked to external sensory or motor events; rather, it reflected internal changes in perception and varied in strength with the frequency of perceptual events, suggesting that functional interactions between visual and prefrontal cortex may contribute to conscious vision. Because similar cortical systems have been implicated in short-term memory and motor planning, the results also imply that related neural processes may underlie visual awareness and the organization of voluntary behavior contingent on sensory cues.
Ectopic bone morphogenetic proteins 5 and 4 in the chicken forebrain lead to cyclopia and holoprosencephaly
Authors: Golden Jeffrey A. , Bracilovic Ana , McFadden Kathryn A. , Beesley Jacqueline S. , Rubenstein John L.R. , Grinspan Judith B.
Published: July 2002

Proper dorsal–ventral patterning in the developing central nervous system requires signals from both the dorsal and ventral portions of the neural tube. Data from multiple studies have demonstrated that bone morphogenetic proteins (BMPs) and Sonic hedgehog protein are secreted factors that regulate dorsal and ventral specification, respectively, within the caudal neural tube. In the developing rostral central nervous system Sonic hedgehog protein also participates in ventral regionalization; however, the roles of BMPs in the developing brain are less clear. We hypothesized that BMPs also play a role in dorsal specification of the vertebrate forebrain. To test our hypothesis we implanted beads soaked in recombinant BMP5 or BMP4 into the neural tube of the chicken forebrain. Experimental embryos showed a loss of the basal telencephalon that resulted in holoprosencephaly (a single cerebral hemisphere), cyclopia (a single midline eye), and loss of ventral midline structures.
In situ
hybridization using a panel of probes to genes expressed in the dorsal and ventral forebrain revealed the loss of ventral markers with the maintenance of dorsal markers. Furthermore, we found that the loss of the basal telencephalon was the result of excessive cell death and not a change in cell fates. These data provide evidence that BMP signaling participates in dorsal–ventral patterning of the developing brain
in vivo
, and disturbances in dorsal–ventral signaling result in specific malformations of the forebrain.
The tail domain of lamin Dm0binds histones H2A and H2B
Authors: Goldberg Michal , Harel Amnon , Brandeis Michael , Rechsteiner Thomas , Richmond Timothy J. , Weiss Aryeh M. , Gruenbaum Yosef
Published: July 2002

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlatedin vivo, and lamins bind specifically to chromatinin vitro. Deletion mutants ofDrosophilalamin Dm0were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0tail domain. The apparentKdof binding of the lamin Dm0tail domain was found to be approximately 1 μM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm0tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm0tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm0tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomesin vivo.
Mitotic recombination map of 13cen–13q14 derived from an investigation of loss of heterozygosity in retinoblastomas
Authors: Hagstrom Stephanie A. , Dryja Thaddeus P.
Published: July 2002

Loss of heterozygosity at tumor-suppressor loci is an important oncogenic mechanism first discovered in retinoblastomas. We explored this phenomenon by examining a set of matched retinoblastoma and leukocyte DNA samples from 158 patients informative for DNA polymorphisms. Loss of heterozygosity at the retinoblastoma locus (13q14) was observed in 101 cases, comprising 7 cases with a somatic deletion causing hemizygosity and 94 with homozygosity (isodisomy). Homozygosity was approximately equally frequent in tumors from male and female patients, among patients with a germ-line vs. somatic initial mutation, and among patients in whom the initial mutation occurred on the maternal vs. paternal allele. A set of 75 tumors exhibiting homozygosity was investigated with markers distributed in the interval 13cen–13q14. Forty-one tumors developed homozygosity at all informative marker loci, suggesting that homozygosity occurred through chromosomal nondisjunction. The remaining cases exhibited mitotic recombination. There was no statistically significant bias in apparent nondisjunction vs. mitotic recombination among male vs. female patients or among patients with germ-line vs. somatic initial mutations. We compared the positions of somatic recombination events in the analyzed interval with a previously reported meiotic recombination map. Although mitotic crossovers occurred throughout the assayed interval, they were more likely to occur proximally than a comparable number of meiotic crossovers. Finally, we observed four triple-crossover cases, suggesting negative interference for mitotic recombination, the opposite of what is usually observed for meiotic recombination.
Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53
Authors: Tao Weikang , Levine Arnold J.
Published: July 2002

The Hdm2 oncoprotein inhibits p53 functions by two means: (
i
) it blocks p53’s transactivation activity and (
ii
) it targets p53 for degradation in a proteasome-dependent manner. Recent data indicate that Hdm2 shuttles between the nucleus and the cytoplasm and that the regulation of p53 levels by Hdm2 requires its nuclear export activity. Two different models are consistent with these observations. In the first, Hdm2 binds to p53 in the nucleus and shuttles p53 from the nucleus to the cytoplasm, and then it targets p53 to the cytoplasmic proteasome. Alternatively, Hdm2 and p53 could be exported separately from the nucleus and then associate in the cytoplasm, where Hdm2 promotes the degradation of p53. To distinguish between these two models, several Hdm2 mutants were employed. Hdm2NLS lacks the ability to enter the nucleus, whereas Hdm2NES is deficient in nuclear export. Hdm2NLS, Hdm2NES, or the combination of both mutants were unable to promote p53 degradation in the cotransfected 2KO cells (which were null for both the
p53
and
mdm2
genes), although wild-type Hdm2 efficiently reduced p53 levels under the same conditions. This observation is not a result of the differences in expression levels or stability between Hdm2 and these mutants. Moreover, coexpression of these mutants had no effect on wild-type Hdm-2-induced p53 destabilization. Thus, Hdm2 must shuttle p53 from the nucleus to the cytoplasm to target it for degradation in the cytoplasm.
In vivo
proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells
Authors: Cheshier Samuel H. , Morrison Sean J. , Liao Xinsheng , Weissman Irving L.
Published: July 2002

A rare set of hematopoietic stem cells (HSC) must undergo a massive expansion to produce mature blood cells. The phenotypic isolation of HSC from mice offers the opportunity to determine directly their proliferation kinetics. We analyzed the proliferation and cell cycle kinetics of long-term self-renewing HSC (LT-HSC) in normal adult mice. At any one time, ≈5% of LT-HSC were in S/G
2
/M phases of the cell cycle and another 20% were in G
1
phase. BrdUrd incorporation was used to determine the rate at which different cohorts of HSC entered the cell cycle over time. About 50% of LT-HSC incorporated BrdUrd by 6 days and >90% incorporated BrdUrd by 30 days. By 6 months, 99% of LT-HSC had incorporated BrdUrd. We calculated that approximately 8% of LT-HSC asynchronously entered the cell cycle per day. Nested reverse transcription–PCR analysis revealed cyclin D2 expression in a high proportion of LT-HSC. Although ≈75% of LT-HSC are quiescent in G
0
at any one time, all HSC are recruited into cycle regularly such that 99% of LT-HSC divide on average every 57 days.
Assigning protein functions by comparative genome analysis: Protein phylogenetic profiles
Authors: Pellegrini Matteo , Marcotte Edward M. , Thompson Michael J. , Eisenberg David , Yeates Todd O.
Published: July 2002

Determining protein functions from genomic sequences is a central goal of bioinformatics. We present a method based on the assumption that proteins that function together in a pathway or structural complex are likely to evolve in a correlated fashion. During evolution, all such functionally linked proteins tend to be either preserved or eliminated in a new species. We describe this property of correlated evolution by characterizing each protein by its phylogenetic profile, a string that encodes the presence or absence of a protein in every known genome. We show that proteins having matching or similar profiles strongly tend to be functionally linked. This method of phylogenetic profiling allows us to predict the function of uncharacterized proteins.
Smads bind directly to the Jun family of AP-1 transcription factors
Authors: Liberati Nicole T. , Datto Michael B. , Frederick Joshua P. , Shen Xing , Wong Carolyn , Rougier-Chapman Elissa M. , Wang Xiao-Fan
Published: July 2002

Smad3 and Smad4 are sequence-specific DNA-binding factors that bind to their consensus DNA-binding sites in response to transforming growth factor β (TGFβ) and activate transcription. Recent evidence implicates Smad3 and Smad4 in the transcriptional activation of consensus AP-1 DNA-binding sites that do not interact with Smads directly. Here, we report that Smad3 and Smad4 can physically interact with AP-1 family members.In vitrobinding studies demonstrate that both Smad3 and Smad4 bind all three Jun family members: JunB, cJun, and JunD. The Smad interacting region of JunB maps to a C-terminal 20-amino acid sequence that is partially conserved in cJun and JunD. We show that Smad3 and Smad4 also associate with an endogenous form of cJun that is rapidly phosphorylated in response to TGFβ. Providing evidence for the importance of this interaction between Smad and Jun proteins, we demonstrate that Smad3 is required for the activation of concatamerized AP-1 sites in a reporter construct that has previously been characterized as unable to bind Smad proteins directly. Together, these data suggest that TGFβ-mediated transcriptional activation through AP-1 sites may involve a regulated interaction between Smads and AP-1 transcription factors.
Three proteins define a class of human histone deacetylases related to yeast Hda1p
Authors: Grozinger Christina M. , Hassig Christian A. , Schreiber Stuart L.
Published: July 2002

Gene expression is in part controlled by chromatin remodeling factors and the acetylation state of nucleosomal histones. The latter process is regulated by histone acetyltransferases and histone deacetylases (HDACs). Previously, three human and five yeast HDAC enzymes had been identified. These can be categorized into two classes: the first class represented by yeast Rpd3-like proteins and the second by yeast Hda1-like proteins. Human HDAC1, HDAC2, and HDAC3 proteins are members of the first class, whereas no class II human HDAC proteins had been identified. The amino acid sequence of Hda1p was used to search the GenBank/expressed sequence tag databases to identify partial sequences from three putative class II human HDAC proteins. The corresponding full-length cDNAs were cloned and defined as HDAC4, HDAC5, and HDAC6. These proteins possess certain features present in the conserved catalytic domains of class I human HDACs, but also contain additional sequence domains. Interestingly, HDAC6 contains an internal duplication of two catalytic domains, which appear to function independently of each other. These class II HDAC proteins have differential mRNA expression in human tissues and possess
in vitro
HDAC activity that is inhibited by trichostatin A. Coimmunoprecipitation experiments indicate that these HDAC proteins are not components of the previously identified HDAC1 and HDAC2 NRD and mSin3A complexes. However, HDAC4 and HDAC5 associate with HDAC3
in vivo
. This finding suggests that the human class II HDAC enzymes may function in cellular processes distinct from those of HDAC1 and HDAC2.
Hippocampal neurogenesis in adult Old World primates
Authors: Gould Elizabeth , Reeves Alison J. , Fallah Mazyar , Tanapat Patima , Gross Charles G. , Fuchs Eberhard
Published: July 2002

The production of new hippocampal neurons in adulthood has been well documented in rodents. Recent studies have extended these findings to other mammalian species, such as tree shrews and marmoset monkeys. However, hippocampal neurogenesis has not been demonstrated in adult Old World primates. To investigate this possibility, we injected 11 adult Old World monkeys of different ages (5–23 years) with the thymidine analog bromodeoxyuridine and examined the fate of the labeled cells at different survival times by using neuronal and glial markers. In the young-adult and middle-aged monkeys, we found a substantial number of cells that incorporated bromodeoxyuridine and exhibited morphological and biochemical characteristics of immature and mature neurons. New cells located in the dentate gyrus expressed a marker of immature granule neurons, Turned On After Division 64 kDa protein, as well as markers of mature granule neurons including neuron specific enolase, neuronal nuclei, and the calcium-binding protein calbindin. Fewer new cells expressed the astroglial marker glial fibrillary acidic protein. Evidence of neurogenesis was observed in the oldest monkeys (23 years) as well, but it appeared to be less robust. These results indicate that the adult brains of Old World monkeys produce new hippocampal neurons. Adult macaque monkeys may provide a useful primate model for studying the functional significance of adult neurogenesis.
The Theory of Everything
Authors: Laughlin R. B. , Pines David
Published: July 2002

We discuss recent developments in our understanding of matter, broadly construed, and their implications for contemporary research in fundamental physics.
Prefrontal cortex and episodic memory retrieval mode
Authors: Lepage Martin , Ghaffar Omar , Nyberg Lars , Tulving Endel
Published: July 2002

A multistudy analysis of positron emission tomography data identified three right prefrontal and two left prefrontal cortical sites, as well as a region in the anterior cingulate gyrus, where neuronal activity is correlated with the maintenance of episodic memory retrieval mode (REMO), a basic and necessary condition of remembering past experiences. The right prefrontal sites were near the frontal pole [Brodmann's area (BA) 10], frontal operculum (BA 47/45), and lateral dorsal area (BA 8/9). The two left prefrontal sites were homotopical with the right frontal pole and opercular sites. The same kinds of REMO sites were not observed in any other cerebral region. Many previous functional neuroimaging studies of episodic memory retrieval have reported activations near the frontal REMO sites identified here, although their function has not been clear. Many of these, too, probably have signaled their involvement in REMO. We propose that REMO activations largely if not entirely account for the frontal hemispheric asymmetry of retrieval as described by the original hemispheric encoding retrieval asymmetry model.
Bcl-2 decreases the free Ca
2+
concentration within the endoplasmic reticulum
Authors: Foyouzi-Youssefi Reyhaneh , Arnaudeau Serge , Borner Christoph , Kelley William L. , Tschopp Jürg , Lew Daniel P. , Demaurex Nicolas , Krause Karl-Heinz
Published: July 2002

The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca
2+
storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca
2+
that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca
2+
store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca
2+
uptake. Direct measurements with ER-targeted Ca
2+
-sensitive fluorescent “cameleon” proteins revealed that Bcl-2 decreased the free Ca
2+
concentration within the lumen of the ER, [Ca
2+
]
ER
. Analysis of the kinetics of Ca
2+
store depletion in response to the Ca
2+
-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca
2+
concentration within the ER lumen by increasing the Ca
2+
permeability of the ER membrane. The increased ER Ca
2+
permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.
Differential expression and function of members of the
zfh-1
family of zinc finger/homeodomain repressors
Authors: Postigo Antonio A. , Dean Douglas C.
Published: July 2002

zfh-1
is a zinc finger/homeodomain transcriptional repressor in
Drosophila
that regulates differentiation of muscle and gonadal cells and is also expressed in the central nervous system (CNS). Binding sites for
zfh-1
overlap with those for
snail,
and like
snail
, it recruits the corepressor CtBP-1. The protein ZEB-1 appears to be a vertebrate homologue of
zfh-1
and is expressed in several tissues including muscle, CNS, and T lymphocytes, and during skeletal differentiation. Mutation of the ZEB-1 gene led to a severe T cell phenotype and skeletal defects but, interestingly, no defects were evident in other ZEB-1-expressing tissues. These results suggested that another ZEB-1-related factor may compensate for the loss of ZEB-1 in other tissues. Here, we characterize such a ZEB-1-related protein, which we have termed as ZEB-2. The overall organization of ZEB-2 is similar to ZEB-1 and
zfh-1
and it has similar biochemical properties: it binds E boxes and interacts with CtBP-1 to repress transcription. However, there are also differences between ZEB-1 and ZEB-2, both in activity and tissue distribution. Whereas ZEB-1 and ZEB-2 overlap in skeletal muscle and CNS (providing an explanation for why mutation of ZEB-1 alone has little effect in these tissues), they show a different pattern of expression in lymphoid cells. ZEB-1, but not ZEB-2, is expressed in T cells from the thymus ZEB-2 appears to be expressed on splenic B cells. Additionally, ZEB-2 inhibits a wider spectrum of transcription factors than ZEB-1.
Anopheles gambiae
pilot gene discovery project: Identification of mosquito innate immunity genes from expressed sequence tags generated from immune-competent cell lines
Authors: Dimopoulos George , Casavant Thomas L. , Chang Shereen , Scheetz Todd , Roberts Chad , Donohue Micca , Schultz Jörg , Benes Vladimir , Bork Peer , Ansorge Wilhelm , Soares Marcelo Bento , Kafatos Fotis C.
Published: July 2002

Together with AIDS and tuberculosis, malaria is at the top of the list of devastating infectious diseases. However, molecular genetic studies of its major vector,
Anopheles gambiae
, are still quite limited. We have conducted a pilot gene discovery project to accelerate progress in the molecular analysis of vector biology, with emphasis on the mosquito's antimalarial immune defense. A total of 5,925 expressed sequence tags were determined from normalized cDNA libraries derived from immune-responsive hemocyte-like cell lines. The 3,242 expressed sequence tag-containing cDNA clones were grouped into 2,380 clone clusters, potentially representing unique genes. Of these, 1,118 showed similarities to known genes from other organisms, but only 27 were identical to previously known mosquito genes. We identified 38 candidate genes, based on sequence similarity, that may be implicated in immune reactions including antimalarial defense; 19 of these were shown experimentally to be inducible by bacterial challenge, lending support to their proposed involvement in mosquito immunity.
Attentional modulation of effective connectivity from V2 to V5/MT in humans
Authors: Friston K. J. , Büchel C.
Published: July 2002

The nonlinear nature of integration among cortical brain areas renders the effective connectivity between them inherently dynamic and context-sensitive. One emerging architectural principle of functional brain organization, which rests explicitly on these nonlinear interactions, is that neuronal responses expressed at any level in a sensory hierarchy reflect an interaction between (
i
) bottom up “driving” afferents from lower cortical areas and (
ii
) backwards “modulatory” inputs from higher areas that mediate top-down contextual effects. A compelling example is attentional modulation of responses in functionally specialized sensory areas. The aim of this work was to demonstrate that parietal regions may mediate selective attention to motion by modulating the effective connectivity from early visual cortex to the motion-sensitive area V5/MT. Using functional magnetic resonance imaging, and an analysis of effective connectivity based on nonlinear system identification, we found that backwards modulatory influences from the posterior parietal cortex are sufficient to account for a significant component of attentional modulation of V5/MT responses to “driving” inputs from V2. By explicitly modeling interactions among inputs to V5/MT, we were able to make inferences about the influences of V2 inputs and their concomitant activity-dependent modulation by parietal afferents. The latter effects embody dynamic changes in effective connectivity that may underlie attentional mechanisms. These results speak to the context-sensitive nature of functional integration in the brain and provide empirical evidence that attentional effects may be mediated by backwards connections, of a modulatory sort, in humans.