bluuebunny/crossref_metadata_2025 huggingface dataset:
Genome-wide identification of tissue-specific enhancers in the
Ciona
tadpole
Authors: Harafuji Naoe , Keys David N. , Levine Michael
Published: July 2002

Less than 100 cis-regulatory DNAs have been characterized in the context of transgenic metazoan embryos. Here we investigate the feasibility of conducting a genome-wide search for tissue-specific enhancers in the ascidian
Ciona intestinalis
. A total of 138 random genomic DNA fragments with an average size of 1.7 kb were separately placed 5′ of a
lacZ
reporter gene. Eleven of the
lacZ
fusion genes displayed localized patterns of expression in tadpole-stage
Ciona
embryos. At least five of these transgenes appear to contain bona fide tissue-specific enhancers that direct expression in the cerebral vesicle, neural tube, primordial adhesive organ, notochord, and tail epidermis. One of the enhancers maps near
Distalless
(
Ci-Dll-A
) and recapitulates most aspects of the endogenous expression pattern, including localized expression in the anterior-most regions of the neurogenic ectoderm. We discuss the prospects of creating a regulatory atlas of the
Ciona
genome, whereby every enhancer is identified for every gene.
Testosterone attenuates expression of vascular cell adhesion molecule-1 by conversion to estradiol by aromatase in endothelial cells: Implications in atherosclerosis
Authors: Mukherjee Tapan K. , Dinh Hillary , Chaudhuri Gautam , Nathan Lauren
Published: July 2002

We previously reported that testosterone attenuated atherogenesis in LDLR
−/−
male mice, and that this effect of testosterone was most likely caused by its conversion to estradiol. Estradiol inhibits vascular cell adhesion molecule-1 (VCAM-1) expression, and expression of VCAM-1 is one of the early events in atherogenesis. We assessed the cellular mechanism(s) involved by which testosterone attenuates atherogenesis. We evaluated whether testosterone inhibited TNFα-induced VCAM-1 expression via its conversion to estradiol by the enzyme aromatase in human umbilical vein endothelial cells (HUVEC). Aromatase mRNA was dedected by reverse transcription–PCR in these cells. Testosterone (30 nM–1 μM) attenuated VCAM-1 mRNA expression in a concentration-dependent manner. The non aromatizable androgen, dihydrotestosterone, had no effect on VCAM-1 mRNA expression. Testosterone was less effective in attenuating VCAM-1 expression in the presence of anastrozole, an inhibitor of aromatase, indicating that testosterone inhibited VCAM-1 via conversion to estradiol. Estradiol also attenuated VCAM-1 mRNA expression, but this action was not abolished in the presence of anastrozole, indicating that anastrozole itself did not modulate VCAM-1 mRNA expression. The effect of testosterone on VCAM-1 mRNA expression was inhibited in the presence of the estrogen receptor antagonist, ICI-182780. Testosterone also attenuated TNFα-induced VCAM-1 protein expression, and this attenuation was abolished in the presence of anastrozole. In conclusion, testosterone inhibited VCAM-1 mRNA and protein expression in HUVEC by its conversion to estradiol via the enzyme aromatase present in the endothelial cells. Results from our study may help explain the mechanism by which testosterone may have beneficial effects on the cardiovascular system.
Aluminum-dependent regulation of intracellular silicon in the aquatic invertebrate
Lymnaea stagnalis
Authors: Desouky Mahmoud , Jugdaohsingh Ravin , McCrohan Catherine R. , White Keith N. , Powell Jonathan J.
Published: July 2002

Silicon is essential for some plants, diatoms, and sponges but, in higher animals, its endogenous regulation has not been demonstrated. Silicate ions may be natural ligands for aluminum and here we show that, in the freshwater snail (
Lymnaea stagnalis
), intracellular silicon seems specifically up-regulated in response to sublethal aluminum exposure. X-ray microanalysis showed that exposure of snails to low levels of aluminum led to its accumulation in lysosomal granules, accompanied by marked up-regulation of silicon. Increased lysosomal levels of silicon were a specific response to aluminum because cadmium and zinc had no such effect. Furthermore, intra-lysosomal sulfur from metallothionein and other sulfur-containing ligands was increased after exposure to cadmium and zinc but not aluminum. To ensure that these findings indicated a specific
in vivo
response, and not
ex vivo
formation of hydroxy-aluminosilicates (HAS) from added aluminum (555 μg/liter) and water-borne silicon (43 μg/liter), two further studies were undertaken. In a ligand competition assay the lability of aluminum (527 μg/liter) was completely unaffected by the presence of silicon (46 μg/liter), suggesting the absence of HAS. In addition, exogenous silicon (6.5 mg/liter), added to the water column to promote formation of HAS, caused a
decrease
in lysosomal aluminum accumulation, showing that uptake of HAS would not explain the loading of aluminum into lysosomal granules. These findings, and arguments on the stability, lability, and kinetics of aluminum–silicate interactions, suggest that a silicon-specific mechanism exists for the
in vivo
detoxification of aluminum, which provides regulatory evidence of silicon in a multicellular organism.
Nitric oxide inhibits tumor necrosis factor-α-induced apoptosis by reducing the generation of ceramide
Authors: De Nadai Céline , Sestili Piero , Cantoni Orazio , Lièvremont Jean-Philippe , Sciorati Clara , Barsacchi Rico , Moncada Salvador , Meldolesi Jacopo , Clementi Emilio
Published: July 2002

Apoptosis triggered by death receptors proceeds after defined signal-transduction pathways. Whether signaling at the receptor level is regulated by intracellular messengers is still unknown. We have investigated the role of two messengers, ceramide and nitric oxide (NO), on the apoptotic pathway activated in human monocytic U937 cells by tumor necrosis factor-α (TNF-α) working at its p55 receptor. Two transduction events, the receptor recruitment of the adapter protein, TRADD, and the activation of the initiator caspase, caspase 8, were investigated. When administered alone, neither of the messengers had any effect on these events. In combination with TNF-α, however, ceramide potentiated, whereas NO inhibited, TNF-α-induced TRADD recruitment and caspase 8 activity. The effect of NO, which was cGMP-dependent, was due to inhibition of the TNF-α-induced generation of ceramide. Our results identify a mechanism of regulation of a signal-transduction pathway activated by death receptors.
Identification of
in vivo
expressed vaccine candidate antigens from
Staphylococcus aureus
Authors: Etz Hildegard , Minh Duc Bui , Henics Tamás , Dryla Agnieszka , Winkler Birgit , Triska Christine , Boyd Aoife P. , Söllner Johannes , Schmidt Walter , von Ahsen Uwe , Buschle Michael , Gill Steven R. , Kolonay James , Khalak Hanif , Fraser Claire M. , von Gabain Alexander , Nagy Eszter , Meinke Andreas
Published: July 2002

For the design of potent subunit vaccines, it is of paramount importance to identify all antigens immunologically recognized by a patient population infected with a pathogen. We have developed a rapid and efficient procedure to identify such commonly recognized antigens, and here we provide a comprehensive
in vivo
antigenic profile of
Staphylococcus aureus
, an important human pathogen.
S. aureus
peptides were displayed on the surface of
Escherichia coli
via fusion to one of two outer membrane proteins (LamB and FhuA) and probed with sera selected for high Ab titer and opsonic activity. A total of 60 antigenic proteins were identified, most of which are located or predicted to be located on the surface of the bacterium or secreted. The identification of these antigens and their reactivity with individual sera from patients and healthy individuals greatly facilitate the selection of promising vaccine candidates for further evaluation. This approach, which makes use of whole genome sequence information, has the potential to greatly accelerate and facilitate the formulation of novel vaccines and is applicable to any pathogen that induces Abs in humans and/or experimental animals.
Ectopic D-type cyclin expression induces not only DNA replication but also cell division in
Arabidopsis
trichomes
Authors: Schnittger Arp , Schöbinger Ulrike , Bouyer Daniel , Weinl Christina , Stierhof York-Dieter , Hülskamp Martin
Published: July 2002

Although the mechanisms controlling the two cell-cycle checkpoints G
1
-S and G
2
-M are well studied, it remains elusive how they are linked in higher eukaryotes. In animals, D-type cyclins have been implicated in the control of cell-cycle progression in mitotic as well as in endoreduplicating cells. By contrast, we show that the expression of the D-type cyclin
CYCD3;1
in endoreduplicating
Arabidopsis
trichome cells not only induced DNA replication but also cell divisions.
Docosahexaenoic acid-containing phospholipid molecular species in brains of vertebrates
Authors: Farkas Tibor , Kitajka Klara , Fodor Elfrieda , Csengeri István , Lahdes Eila , Yeo Young K. , Krasznai Zoltán , Halver John E.
Published: July 2002

The fatty acid composition of phospholipids and the contents of docosahexaenoic acid (DHA)-containing diacyl phosphatidylcholine and diacyl phosphatidylethanolamine molecular species were determined from brains of five fresh-water fish species from a boreal region adapted to 5°C, five fresh-water fish species from a temperate region acclimated to 5°C, five fresh-water fish species from a temperate region acclimated to 20°C, and three fresh water fish species from a subtropic region adapted to 25–26°C, as well as six mammalian species and seven bird species. There was little difference in DHA levels of fish brains from the different thermal environments; mammalian and bird brain phospholipids contained a few percentage points less DHA than those of the fish investigated. Molecular species of 22:6/22:6, 22:6/20:5, 22:6/20:4, 16:0/22:6, 18:0/22:6, and 18:1/22:6 were identified from all brain probes, and 16:0/22:6, 18:0/22:6, and 18:1/22:6 were the dominating species. Cold-water fish brains were rich in 18:1/22:6 diacyl phosphatidylethanolamine (and, to a lesser degree, in diacyl phosphatidylcholine), and its level decreased with increasing environmental/body temperature. The ratio of 18:0/22:6 to 16:0/22:6 phosphatidylcholine and phosphatidylethanolamine was inversely related to body temperature. Phospholipid vesicles from brains of cold-acclimated fish were more fluid, as assessed by using a 1,6-diphenyl-1,3,5-hexatriene fluorescent probe, than those from bird brains, but the fluidities were almost equal at the respective body temperatures. It is concluded that the relative amounts of these molecular species and their ratios to each other are the major factors contributing to the maintenance of proper fluidity relationships throughout the evolutionary chain as well as helping to maintain important brain functions such as signal transduction and membrane permeability.
Assembly and activation of site-specific recombination complexes
Authors: Peña Carol E. A. , Kahlenberg J. Michelle , Hatfull Graham F.
Published: July 2002

Site-specific recombination is responsible for a broad range of biological phenomena, including DNA inversion, resolution of transposition intermediates, and the integration and excision of bacteriophage genomes. Integration of mycobacteriophage L5 is catalyzed by a phage-encoded integrase with recombination occurring between specific attachment sites on the phage and mycobacterial chromosomes (
attP
and
attB
, respectively). Although some site-specific recombination systems simply involve binding of the recombinase to the sites of strand exchange, synapsis, and recombination, phage systems typically require the assembly of higher-order structures within which the recombinational potential of integrase is activated. The requirement for these structures derives from the necessity to regulate the directionality of recombination—either integration or excision—which must be closely coordinated with other aspects of the phage growth cycles. We show herein that there are multiple pathways available for the assembly of L5 recombination complexes, including the early synapsis of the
attP
and
attB
DNAs. This process is in contrast to the model for lambda integration and illustrates the different usage of molecular machineries to accomplish the same biological outcome.
Toward a new generation of vaccines: The anti-cytokine therapeutic vaccines
Authors: Zagury Daniel , Burny Arsene , Gallo Robert C.
Published: July 2002

Pathological conditions, such as cancers, viral infections, and autoimmune diseases, are associated with abnormal cytokine production, and the morbidity associated with many medical disorders is often directly a result of cytokine production. Because of the absence of negative feedback control occurring in some pathophysiologic situations, a given cytokine may flood and accumulate in the extracellular compartment of tissues or tumors thereby impairing the cytokine network homeostasis and contributing to local pathogenesis. To evaluate whether the rise of anti-cytokine Abs by vaccination is an effective way to treat these pathological conditions without being harmful to the organism, we have analyzed each step of the cytokine process (involving cytokine production, target response, and feedback regulation) and have considered them in the local context of effector–target cell microenvironment and in the overall context of the macroenvironment of the immune system of the organism. In pathologic tissues, Abs of high affinity, as raised by anti-cytokine vaccination, should neutralize the pool of cytokines ectopically accumulated in the extracellular compartment, thus counteracting their pathogenic effects. In contrast, the same Abs should not interfere with cytokine processes occurring in normal tissues, because under physiologic conditions cytokine production by effector cells (induced by activation but controlled by negative feedback regulation) does not accumulate in the extracellular compartment. These concepts are consistent with results showing that following animal and human anti-cytokine vaccination, induction of high-affinity Abs has proven to be safe and effective and encourages this approach as a pioneering avenue of therapy.
Immunosuppression by CD4+regulatory T cells induced by chronic retroviral infection
Authors: Iwashiro Michihiro , Messer Ronald J. , Peterson Karin E. , Stromnes Ingunn M. , Sugie Tomoharu , Hasenkrug Kim J.
Published: July 2002

Normal levels of CD4+regulatory T cells are critical for the maintenance of immunological homeostasis and the prevention of autoimmune diseases. However, we now show that the expansion of CD4+regulatory T cells in response to a chronic viral infection can lead to immunosuppression. Mice persistently infected with Friend retrovirus develop approximately twice the normal percentage of splenic CD4+regulatory T cells and lose their ability to reject certain tumor transplants. The role of CD4+regulatory T cells was demonstrated by the transmission of immunosuppression to uninfected mice by adoptive transfers of CD4+T cells. CD4+T cells from chronically infected mice were also immunosuppressivein vitro, inhibiting the generation of cytolytic T lymphocytes in mixed lymphocyte cultures. Inhibition occurred at the level of blast-cell formation through a mechanism or mechanisms involving transforming growth factor-β and the cell surface molecule CTLA-4 (CD152). These results suggest a possible explanation for HIV- and human T cell leukemia virus-I-induced immunosuppression in the absence of T cell depletion.
Retrograde signaling at central synapses
Authors: Tao Huizhong W. , Poo Mu-ming
Published: July 2002

Transcellular retrograde signaling from the postsynaptic target
cell to the presynaptic neuron plays critical roles in the formation,
maturation, and plasticity of synaptic connections. We here review
recent progress in our understanding of the retrograde signaling at
developing central synapses. Three forms of potential retrograde
signals—membrane-permeant factors, membrane-bound factors, and
secreted factors—have been implicated at both developing and mature
synapses. Although many of these signals may be active constitutively,
retrograde factors produced in association with activity-dependent
synaptic plasticity, e.g., long-term potentiation and long-term
depression, are of particular interest, because they may induce
modification of neuronal excitability and synaptic transmission,
functions directly related to the processing and storage of information
in the nervous system.
Subgenome chromosome walking in wheat: A 450-kb physical contig in
Triticum monococcum
L. spans the
Lr10
resistance locus in hexaploid wheat (
Triticum aestivum
L.)
Authors: Stein Nils , Feuillet Catherine , Wicker Thomas , Schlagenhauf Edith , Keller Beat
Published: July 2002

For many agronomically important plant genes, only their position
on a genetic map is known. In the absence of an efficient transposon
tagging system, such genes have to be isolated by map-based cloning. In
bread wheat
Triticum aestivum
, the genome is hexaploid,
has a size of 1.6 × 10
10
bp, and contains more than
80% of repetitive sequences. So far, this genome complexity has not
allowed chromosome walking and positional cloning. Here, we demonstrate
that chromosome walking using bacterial artificial chromosome (BAC)
clones is possible in the diploid wheat
Triticum
monococcum
(A
m
genome). BAC end sequences were
mostly repetitive and could not be used for the first walking step. New
probes corresponding to rare low-copy sequences were efficiently
identified by low-pass DNA sequencing of the BACs. Two walking steps
resulted in a physical contig of 450 kb on chromosome 1A
m
S.
Genetic mapping of the probes derived from the BAC contig demonstrated
perfect colinearity between the physical map of
T.
monococcum
and the genetic map of bread wheat on chromosome
1AS. The contig genetically spans the
Lr10
leaf rust
disease resistance locus in bread wheat, with 0.13 centimorgans
corresponding to 300 kb between the closest flanking markers.
Comparison of the genetic to physical distances has shown large
variations within 350 kb of the contig. The physical contig can now be
used for the isolation of the orthologous regions in bread wheat. Thus,
subgenome chromosome walking in wheat can produce large physical
contigs and saturate genomic regions to support positional cloning.
Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner
Authors: Fraser Paul D. , Romer Susanne , Shipton Cathie A. , Mills Philippa B. , Kiano Joy W. , Misawa Norihiko , Drake Rachel G. , Schuch Wolfgang , Bramley Peter M.
Published: July 2002

Phytoene synthase from the bacterium
Erwinia uredovora
(
crt
B) has been overexpressed in tomato (
Lycopersicon esculentum
Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T
0
) were 2–4-fold higher than the controls, whereas phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T
1
and T
2
generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into phytoene) were not significantly altered by the presence of the bacterial phytoene synthase.
Antidiabetic thiazolidinediones inhibit leptin (ob) gene expression in 3T3-L1 adipocytes.
Authors: Kallen C B , Lazar M A
Published: July 2002

Lack of leptin (ob) protein causes obesity in mice. The leptin gene product is important for normal regulation of appetite and metabolic rate and is produced exclusively by adipocytes. Leptin mRNA was induced during the adipose conversion of 3T3-L1 cells, which are useful for studying adipocyte differentiation and function under controlled conditions. We studied leptin regulation by antidiabetic thiazolidinedione compounds, which are ligands for the adipocyte-specific nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) that regulates the transcription of other adipocyte-specific genes. Remarkably, leptin gene expression was dramatically repressed within a few hours after thiazolidinedione treatment. The ED50 for inhibition of leptin expression by the thiazolidinedione BRL49653 was between 5 and 50 nM, similar to its Kd for binding to PPARgamma. The relatively weak, nonthiazolidinedione PPAR activator WY 14,643 also inhibited leptin expression, but was approximately 1000 times less potent than BRL49653. These results indicate that antidiabetic thiazolidinediones down-regulate leptin gene expression with potencies that correlate with their abilities to bind and activate PPARgamma.
Effects of inefficient cleavage of the signal sequence of HIV-1 gp 120 on its association with calnexin, folding, and intracellular transport.
Authors: Li Y , Bergeron J J , Luo L , Ou W J , Thomas D Y , Kang C Y
Published: July 2002

The HIV-1 envelope glycoprotein gp120 displays inefficient intracellular transport, which is caused by its retention in the endoplasmic reticulum. Coexpression in insect cells (Sf9) of HIV-1 gp120 with calnexin has shown that their interaction was modulated by the signal sequence of HIV-1 gp120. gp120, with its natural signal sequence, showed a prolonged association with calnexin with a t1/2 of greater than 20 min. Replacement of the natural signal sequence with the signal sequence from mellitin led to a decreased time of association of gp120 with calnexin (t1/2 < 10 min). These different times of calnexin association coincided both with the folding of gp120 as measured by the ability of bind CD4 and with endoplasmic reticulum to Golgi transport as analyzed by the acquisition of partial endoglycosidase H resistance. Using a monospecific antibody to the HIV-1 gp120 natural signal peptide, we showed that calnexin associated with N-glycosylated but uncleaved gp120. Only after dissociation from calnexin was gp120 cleaved, but very inefficiently. Only the small proportion of signal-cleaved gp120 molecules acquired transport competence and were secreted. This is the first report demonstrating the effect of the signal sequence on calnexin association.
Muller's ratchet decreases fitness of a DNA-based microbe.
Authors: Andersson D I , Hughes D
Published: July 2002

Muller proposed that an asexual organism will inevitably accumulate deleterious mutations, resulting in an increase of the mutational load and an inexorable, ratchet-like, loss of the least mutated class [Muller, H.J. (1964) Mutat. Res. 1, 2-9]. The operation of Muller's ratchet on real populations has been experimentally demonstrated only in RNA viruses. However, these cases are exceptional in that the mutation rates of the RNA viruses are extremely high. We have examined whether Muller's ratchet operates in Salmonella typhimurium, a DNA-based organism with a more typical genomic mutation rate. Cells were grown asexually under conditions expected to result in high genetic drift, and the increase in mutational load was determined. S. typhimurium accumulated mutations under these conditions such that after 1700 generations, 1% of the 444 lineages tested had suffered an obvious loss of fitness, as determined by decreased growth rate. These results suggest that in the absence of sex and with high genetic drift, genetic mechanisms, such as back or compensatory mutations, cannot compensate for the accumulation of deleterious mutations. In addition, we measured the appearance of auxotrophs, which allowed us to calculate an average spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base pair per generation. This rate is measured for the largest genetic target studied so far, a collection of about 200 genes.
Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli.
Authors: Eide L , Bjørås M , Pirovano M , Alseth I , Berdal K G , Seeberg E
Published: July 2002

One gene locus on chromosome I in Saccharomyces cerevisiae encodes a protein (YAB5_YEAST; accession no. P31378) with local sequence similarity to the DNA repair glycosylase endonuclease III from Escherichia coli. We have analyzed the function of this gene, now assigned NTG1 (endonuclease three-like glycosylase 1), by cloning, mutant analysis, and gene expression in E. coli. Targeted gene disruption of NTG1 produces a mutant that is sensitive to H2O2 and menadione, indicating that NTG1 is required for repair of oxidative DNA damage in vivo. Northern blot analysis and expression studies of a NTG1-lacZ gene fusion showed that NTG1 is induced by cell exposure to different DNA damaging agents, particularly menadione, and hence belongs to the DNA damage-inducible regulon in S. cerevisiae. When expressed in E. coli, the NTG1 gene product cleaves plasmid DNA damaged by osmium tetroxide, thus, indicating specificity for thymine glycols in DNA similarly as is the case for EndoIII. However, NTG1 also releases formamidopyrimidines from DNA with high efficiency and, hence, represents a glycosylase with a novel range of substrate recognition. Sequences similar to NTG1 from other eukaryotes, including Caenorhabditis elegans, Schizosaccharomyces pombe, and mammals, have recently been entered in the GenBank suggesting the universal presence of NTG1-like genes in higher organisms. S. cerevisiae NTG1 does not have the [4Fe-4S] cluster DNA binding domain characteristic of the other members of this family.
Binocular vision in insects: How mantids solve the correspondence problem
Authors: Rossel Samuel
Published: July 2002

Praying mantids use binocular cues to judge whether their prey is
in striking distance. When there are several moving targets within
their binocular visual field, mantids need to solve the correspondence
problem. They must select between the possible pairings of retinal
images in the two eyes so that they can strike at a single real target.
In this study, mantids were presented with two targets in various
configurations, and the resulting fixating saccades that precede the
strike were analyzed. The distributions of saccades show that mantids
consistently prefer one out of several possible matches. Selection is
in part guided by the position and the spatiotemporal features of the
target image in each eye. Selection also depends upon the binocular
disparity of the images, suggesting that insects can perform local
binocular computations. The pairing rules ensure that mantids tend to
aim at real targets and not at “ghost” targets arising from false
matches.
Mutation detection by highly sensitive methods indicates that p53 gene mutations in breast cancer can have important prognostic value.
Authors: Kovach J S , Hartmann A , Blaszyk H , Cunningham J , Schaid D , Sommer S S
Published: July 2002

Human cancer cells with a mutated p53 tumor-suppressor gene have a selective growth advantage and may exhibit resistance to ionizing radiation and certain chemotherapeutic agents. To examine the prognostic value of mutations in the p53 gene, a cohort of 90 Midwestern Caucasian breast cancer patients were analyzed with methodology that detects virtually 100% of all mutations. The presence of a p53 gene mutation was by far the single most predictive indicator for recurrence and death (relative risks of 4.7 and 23.2, respectively). Direct detection of p53 mutations had substantially greater prognostic value than immunohistochemical detection of p53 overexpression. Analysis of p53 gene mutations may permit identification of a subset of breast cancer patients who, despite lack of conventional indicators of poor prognosis, are at high risk of early recurrence and death.
ARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADP-ribosylation factor domain and a 46-kDa GTPase-activating domain.
Authors: Vitale N , Moss J , Vaughan M
Published: July 2002

The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins (G proteins) hydrolyze GTP at a rate significantly higher than do most members of the Ras family of approximatelly 20-kDa GTP-binding proteins, which depend on a GTPase-activating protein (GAP) for acceleration of GTP hydrolysis. It has been demonstrated that an inserted domain in the G-protein alpha subunit, not present in the much smaller Ras-like proteins, is responsible for this difference [Markby, D. W., Onrust, R. & Bourne, H. R. (1993) Science 262, 1895-1900]. We report here that ARD1, a 64-kDa protein with an 18-kDa carboxyl-terminal ADP-ribosylation factor (ARF) domain, exhibited significant GTPase activity, whereas the ARF domain, expressed as a recombinant protein in Escherichia coli, did not. Addition of the 46-kDa amino-terminal extension (similarly synthesized in E. coli) to the GTP-binding ARF-domain of ARD1 enhanced GTPase activity and inhibited GDP dissociation. The kinetic properties of mixtures of the ARF and non-ARF domains were similar to those of an intact recombinant ARD1. Physical association of the two proteins was demonstrated directly by gel filtration and by using the immobilized non-ARF domain. Thus, like the alpha subunits of heterotrimeric G proteins, ARD1 appears to consist of two domains that interact to regulate the biological activity of the protein.
DNA sequence evidence for the segmental allotetraploid origin of maize
Authors: Gaut Brandon S. , Doebley John F.
Published: July 2002

It has long been suspected that maize is the product of an historical tetraploid event. Several observations support this possibility, including the fact that the maize genome contains duplicated chromosomal segments with colinear gene arrangements. Some of the genes from these duplicated segments have been sequenced. In this study, we examine the pattern of sequence divergence among 14 pairs of duplicated genes. We compare the pattern of divergence to patterns predicted by four models of the evolution of the maize genome—autotetraploidy, genomic allotetraploidy, segmental allotetraploidy, and multiple segmental duplications. Our analyses indicate that coalescent times for duplicated sequences fall into two distinct groups, corresponding to roughly 20.5 and 11.4 million years. This observation strongly discounts the possibility that the maize genome is the product of a genomic allotetraploid event, and it is also difficult to reconcile with either autotetraploidy or multiple independent segmental duplications. However, the presence of two (and only two) coalescent times is predicted by the segmental allotetraploid model. If the maize genome is the product of a segmental allotetraploid event, as these data suggest, then its two diploid progenitors diverged roughly 20.5 million years ago (Mya), and the allotetraploid event probably occurred approximately 11.4 Mya. Comparison of maize and sorghum sequences suggests that one of the two ancestral diploids shares a more recent common ancestor with sorghum than it does with the other ancestral diploid.
The evolution of plant nuclear genes
Authors: Clegg Michael T. , Cummings Michael P. , Durbin Mary L.
Published: July 2002

We analyze the evolutionary dynamics of three of the
best-studied plant nuclear multigene families. The data analyzed derive
from the genes that encode the small subunit of
ribulose-1,5-bisphosphate carboxylase (
rbc
S), the gene
family that encodes the enzyme chalcone synthase (
Chs
),
and the gene family that encodes alcohol dehydrogenases
(
Adh
). In addition, we consider the limited evolutionary
data available on plant transposable elements. New
Chs
and
rbc
S genes appear to be recruited at about 10 times
the rate estimated for
Adh
genes, and this is correlated
with a much smaller average gene family size for
Adh
genes. In addition, duplication and divergence in function appears to
be relatively common for
Chs
genes in flowering plant
evolution. Analyses of synonymous nucleotide substitution rates for
Adh
genes in monocots reject a linear relationship with
clock time. Replacement substitution rates vary with time in a complex
fashion, which suggests that adaptive evolution has played an important
role in driving divergence following gene duplication events. Molecular
population genetic studies of
Adh
and
Chs
genes reveal high levels of molecular diversity within species. These
studies also reveal that inter- and intralocus recombination are
important forces in the generation allelic novelties. Moreover,
illegitimate recombination events appear to be an important factor in
transposable element loss in plants. When we consider the recruitment
and loss of new gene copies, the generation of allelic diversity within
plant species, and ectopic exchange among transposable elements, we
conclude that recombination is a pervasive force at all levels of plant
evolution.
Brain-derived neurotrophic factor rapidly enhances phosphorylation of the postsynaptic
N
-methyl-
d
-aspartate receptor subunit 1
Authors: Suen Piin-Chau , Wu Kuo , Levine Eric S. , Mount Howard T. J. , Xu Jia-Ling , Lin Siang-Yo , Black Ira B.
Published: July 2002

Although neurotrophins have traditionally been regarded as neuronal survival factors, recent work has suggested a role for these factors in synaptic plasticity. In particular, brain-derived neurotrophic factor (BDNF) rapidly enhances synaptic transmission in hippocampal neurons through trkB receptor stimulation and postsynaptic phosphorylation mechanisms. Activation of trkB also modulates hippocampal long-term potentiation, in which postsynaptic
N
-methyl-
d
-aspartate glutamate receptors play a key role. However, the final common pathway through which BDNF increases postsynaptic responsiveness is unknown. We now report that BDNF, within 5 min of exposure, elicits a dose-dependent increase in phosphorylation of the
N
-methyl-
d
-aspartate receptor subunit 1. This acute effect occurred in hippocampal synaptoneurosomes, which contain pre- and postsynaptic elements, and in isolated hippocampal postsynaptic densities. Nerve growth factor, in contrast, caused no enhancement of phosphorylation. These results suggest a potential mechanism for trophin-induced potentiation of synaptic transmission.
The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for
Escherichia coli
behavior
Authors: Rebbapragada Anuradha , Johnson Mark S. , Harding Gordon P. , Zuccarelli Anthony J. , Fletcher Hansel M. , Zhulin Igor B. , Taylor Barry L.
Published: July 2002

We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in
Escherichia coli
. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in
aer
and
tsr
, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide
E. coli
to environments where maximal energy is available for growth.
Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces
Authors: Mukhopadhyay Chinmay K. , Mazumder Barsanjit , Lindley Peter F. , Fox Paul L.
Published: July 2002

Free transition metal ions oxidize lipids and lipoproteinsin vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likelyin vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidationin vitroand that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426because CpH426Aalmost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.
Two hemoglobin genes in
Arabidopsis thaliana
: The evolutionary origins of leghemoglobins
Authors: Trevaskis Ben , Watts Richard. A. , Andersson Carol R. , Llewellyn Danny J. , Hargrove Mark S. , Olson John S. , Dennis Elizabeth S. , Peacock W. James
Published: July 2002

We cloned two hemoglobin genes from
Arabidopsis thaliana
. One gene,
AHB1
, is related in sequence to the family of nonsymbiotic hemoglobin genes previously identified in a number of plant species (class 1). The second hemoglobin gene,
AHB2
, represents a class of nonsymbiotic hemoglobin (class 2) related in sequence to the symbiotic hemoglobin genes of legumes and
Casuarina
. The properties of these two hemoglobins suggest that the two families of nonsymbiotic hemoglobins may differ in function from each other and from the symbiotic hemoglobins.
AHB1
is induced, in both roots and rosette leaves, by low oxygen levels. Recombinant AHB1 has an oxygen affinity so high as to make it unlikely to function as an oxygen transporter.
AHB2
is expressed at a low level in rosette leaves and is low temperature-inducible. AHB2 protein has a lower affinity for oxygen than AHB1 but is similar to AHB1 in having an unusually low, pH-sensitive oxygen off-rate.
Altered phosphorylation and intracellular distribution of a (CUG)

n

triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice
Authors: Roberts Robert , Timchenko Nikolaj A. , Miller Jill W. , Reddy Sita , Caskey C. Thomas , Swanson Maurice S. , Timchenko Lubov T.
Published: July 2002

Myotonic dystrophy (DM) is associated with expansion of CTG repeats
in the 3′-untranslated region of the myotonin protein kinase (DMPK)
gene. The molecular mechanism whereby expansion of the (CUG)

n

repeats in the 3′-untranslated region of DMPK gene induces DM is
unknown. We previously isolated a protein with specific binding to CUG
repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA
processing and/or transport. Here we present evidence that the
phosphorylation status and intracellular distribution of the RNA
CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are
altered in homozygous DM patient and that CUG-BP/hNab50 is a
substrate for DMPK both
in vivo
and
in
vitro
. Data from two biological systems with reduced levels of
DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK
regulates both phosphorylation and intracellular localization of the
CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM
patients and DMPK knockout mice led to the elevation of the
hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of
the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK
knockout mice and in homozygous DM patient. DMPK also interacts with
and phosphorylates CUG-BP/hNab50 protein
in vitro
.
DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic
reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of
which was observed in the nuclei of DMPK knockout mice. These data
suggest a feedback mechanism whereby decreased levels of DMPK could
alter phosphorylation status of CUG-BP/hNab50, thus facilitating
nuclear localization of CUG-BP/hNab50. Our results suggest that DM
pathophysiology could be, in part, a result of sequestration of
CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn,
affect processing and transport of specific subclass of mRNAs.
Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin
Authors: Kato Takashi , Oda Atsushi , Inagaki Yoshimasa , Ohashi Hideya , Matsumoto Atsushi , Ozaki Katsutoshi , Miyakawa Yoshitaka , Watarai Hiroshi , Fuju Kazumi , Kokubo Atsuko , Kadoya Toshihiko , Ikeda Yasuo , Miyazaki Hiroshi
Published: July 2002

A heterogeneity in the molecular weight (
M
r
) of thrombopoietin (TPO) has been reported. We found several thrombin cleavage sites in human, rat, murine, and canine TPOs, and also found that human TPO undergoes selective proteolysis by thrombin. Recombinant human TPO (rhTPO) was incubated with human platelets in the presence of calcium ions to allow the generation of thrombin, and was cleaved into low
M
r
peptide fragments. The cleavage was completely inhibited by hirudin, indicating that the proteolysis was mediated by thrombin. In a platelet-free system, analyses of thrombin cleavage by immunoblotting using anti-human TPO peptide antibodies revealed that the four major thrombin-cleaved peptide fragments were selectively generated depending on the digestion time. The amino acid sequences of the thrombin-polypeptides were further analyzed, and two major thrombin cleavage sites were determined. One of them was at AR
191
-T
192
in the C-terminal domain of TPO, and thrombin cleaved first at this site. The other site at GR
117
-T
118
in the N-terminal domain was subsequently cleaved by prolonged thrombin digestion. As a result, the biological activity of TPO was modulated. The generation of truncated forms of TPO by thrombin may be a notable event in view of the platelet-related metabolism of TPO.
β
3
integrins mediate the cellular entry of hantaviruses that cause respiratory failure
Authors: Gavrilovskaya Irina N. , Shepley Michael , Shaw Robert , Ginsberg Mark H. , Mackow Erich R.
Published: July 2002

Newly emerged hantaviruses replicate primarily in the pulmonary endothelium, cause acute platelet loss, and result in hantavirus pulmonary syndrome (HPS). We now report that specific integrins expressed on platelets and endothelial cells permit the cellular entry of HPS-associated hantaviruses. Infection with HPS-associated hantaviruses, NY-1 and Sin Nombre virus (SNV), is inhibited by antibodies to β
3
integrins and by the β
3
-integrin ligand, vitronectin. In contrast, infection with the nonpathogenic (no associated human disease) Prospect Hill virus was inhibited by fibronectin and β
1
-specific antibodies but not by β
3
-specific antibodies or vitronectin. Transfection with recombinant α
IIb
β
3
or α
v
β
3
integrins rendered cells permissive to NY-1 and SNV but not Prospect Hill virus infection, indicating that α
IIb
β
3
and α
v
β
3
integrins mediate the entry of NY-1 and SNV hantaviruses. Furthermore, entry is divalent cation independent, not blocked by arginine-glycine-aspartic acid peptides and still mediated by, ligand-binding defective, α
IIb
β
3
-integrin mutants. Hence, NY-1 and SNV entry is independent of β
3
integrin binding to physiologic ligands. These findings implicate integrins as cellular receptors for hantaviruses and indicate that hantavirus pathogenicity correlates with integrin usage.
Activities of adenovirus virus-associated RNAs: Purification and characterization of RNA binding proteins
Authors: Liao Huey-Jane , Kobayashi Ryuji , Mathews Michael B.
Published: July 2002

Most human adenoviruses encode two virus-associated (VA) RNAs, VA RNA
I
and VA RNA
II
, that accumulate to high levels in the cytoplasm of infected cells. The function of VA RNA
I
in blocking the activation of the cellular kinase PKR is well known, but the role of VA RNA
II
is obscure. Herein we characterize and purify several human proteins that interact preferentially with VA RNA
II
in Northwestern blot assays. Two of these proteins were identified as RNA helicase A and NF90, a component of the heterodimeric nuclear factor of activated T cells (NFAT). They copurified with the smaller NFAT subunit, NF45, which did not bind VA RNA
II
, and with an unidentified protein, p97, which did bind VA RNA
II
. Both RNA helicase A and NF90 contain two copies of a double-stranded (ds) RNA binding motif and bind strongly to dsRNA. NF90 interacts with RNAs in the following order of affinity: dsRNA > VA RNA
II
> VA RNA
I
> single-stranded RNA. Furthermore, VA RNA
II
is more effective than VA RNA
I
as an inhibitor of RNA helicase activity. These data identify RNA helicase A and NF90 as cellular proteins with an affinity for dsRNA and other structured RNA molecules and suggest that their functions are subject to regulation by RNA ligands including VA RNA
II
.
hZAC
encodes a zinc finger protein with antiproliferative properties and maps to a chromosomal region frequently lost in cancer
Authors: Varrault A. , Ciani E. , Apiou F. , Bilanges B. , Hoffmann A. , Pantaloni C. , Bockaert J. , Spengler D. , Journot L.
Published: July 2002

We previously reported the identification of mZac, a novel mouse zinc finger protein that shared with p53 the ability to regulate concomitantly apoptosis and cell cycle progression. We describe here the isolation, chromosomal localization, and functional
in vitro
characterization of its human homolog. hZAC is a widely expressed zinc finger protein that reveals transactivation and DNA-binding activity. hZAC inhibits tumor cell growth through induction of apoptotic cell death and G
1
arrest. Thus hZAC, like its mouse counterpart, displays antiproliferative properties through pathways known to be central to the activity of p53. We mapped
hZAC
on chromosome 6q24-q25, a region frequently deleted in many solid tumors. Indeed, allelic loss at 6q24-q25 has been shown in breast and ovary cancers, melanomas, astrocytomas, and renal cell carcinomas. Furthermore, Abdollahi
et al.
[Abdollahi, A., Godwin, A. K., Miller, P. D., Getts, L. A., Schultz, D. C., Tagushi, T., Testa, J. R. & Hamilton, T. C. (1997)
Cancer Res.
57, 2029–2034] recently isolated
ZAC
through its loss of expression in a surface epithelial ovary tumor model and accordingly named it
Lot
for “lost on transformation.” In view of these observations, the functional properties we report here provide further arguments to consider
hZAC
as a tumor suppressor gene candidate.