bluuebunny/crossref_metadata_2025 huggingface dataset:
Normal cerebellar development but susceptibility to seizures in mice lacking G protein-coupled, inwardly rectifying K
+
channel GIRK2
Authors: Signorini Stefano , Liao Y. Joyce , Duncan Stephen A. , Jan Lily Y. , Stoffel Markus
Published: July 2002

G protein-gated, inwardly rectifying K
+
channels (GIRK) are effectors of G protein-coupled receptors for neurotransmitters and hormones and may play an important role in the regulation of neuronal excitability. GIRK channels may be important in neurodevelopment, as suggested by the recent finding that a point mutation in the pore region of GIRK2 (G156S) is responsible for the weaver (
wv
) phenotype. The GIRK2 G156S gene gives rise to channels that exhibit a loss of K
+
selectivity and may also exert dominant-negative effects on G
βγ
-activated K
+
currents. To investigate the physiological role of GIRK2, we generated mutant mice lacking GIRK2. Unlike
wv
/
wv
mutant mice, GIRK2 −/− mice are morphologically indistinguishable from wild-type mice, suggesting that the
wv
phenotype is likely due to abnormal GIRK2 function. Like
wv
/
wv
mice, GIRK2 −/− mice have much reduced GIRK1 expression in the brain. They also develop spontaneous seizures and are more susceptible to pharmacologically induced seizures using a γ-aminobutyric acid antagonist. Moreover,
wv
/− mice exhibit much milder cerebellar abnormalities than
wv
/
wv
mice, indicating a dosage effect of the GIRK2 G156S mutation. Our results indicate that the weaver phenotypes arise from a gain-of-function mutation of GIRK2 and that GIRK1 and GIRK2 are important mediators of neuronal excitability
in vivo
.
Pleiotropic effects of a disrupted K+channel gene: Reduced body weight, impaired motor skill and muscle contraction, but no seizures
Authors: Ho Chi Shun , Grange Robert W. , Joho Rolf H.
Published: July 2002

To investigate the roles of K+channels in the regulation and fine-tuning of cellular excitability, we generated a mutant mouse carrying a disrupted gene for the fast activating, voltage-gated K+channel Kv3.1. Kv3.1−/−mice are viable and fertile but have significantly reduced body weights compared with their Kv3.1+/−littermates. Wild-type, heterozygous, and homozygous Kv3.1 channel-deficient mice exhibit similar spontaneous locomotor and exploratory activity. In a test for coordinated motor skill, however, homozygous Kv3.1−/−mice perform significantly worse than their heterozygous Kv3.1+/−or wild-type littermates. Both fast and slow skeletal muscles of Kv3.1−/−mice are slower to reach peak force and to relax after contraction, consequently leading to tetanic responses at lower stimulation frequencies. Both mutant muscles generate significantly smaller contractile forces during a single twitch and during tetanic conditions. Although Kv3.1−/−mutants exhibit a normal auditory frequency range, they show significant differences in their acoustic startle responses. Contrary to expectation, homozygous Kv3.1−/−mice do not have increased spontaneous seizure activity.
One gene in diamondback moth confers resistance to fourBacillus thuringiensis toxins
Authors: Tabashnik Bruce E. , Liu Yong-Biao , Finson Naomi , Masson Luke , Heckel David G.
Published: July 2002

Environmentally benign insecticides derived from the soil bacteriumBacillus thuringiensis(Bt) are the most widely used biopesticides, but their success will be short-lived if pests quickly adapt to them. The risk of evolution of resistance by pests has increased, because transgenic crops producing insecticidal proteins from Bt are being grown commercially. Efforts to delay resistance with two or more Bt toxins assume that independent mutations are required to counter each toxin. Moreover, it generally is assumed that resistance alleles are rare in susceptible populations. We tested these assumptions by conducting single-pair crosses with diamondback moth (Plutella xylostella), the first insect known to have evolved resistance to Bt in open field populations. An autosomal recessive gene conferred extremely high resistance to four Bt toxins (Cry1Aa, Cry1Ab, Cry1Ac, and Cry1F). The finding that 21% of the individuals from a susceptible strain were heterozygous for the multiple-toxin resistance gene implies that the resistance allele frequency was 10 times higher than the most widely cited estimate of the upper limit for the initial frequency of resistance alleles in susceptible populations. These findings suggest that pests may evolve resistance to some groups of toxins much faster than previously expected.
Ras-independent transformation by v-Src
Authors: Aftab Dana T. , Kwan Joanne , Martin G. Steven
Published: July 2002

Signaling by a variety of receptor and nonreceptor tyrosine kinases is mediated by Ras, a membrane-associated GTPase. Expression of v-Src, a transforming nonreceptor tyrosine kinase, results in Ras activation, and inhibition of Ras function in NIH 3T3 cells suppresses transformation by v-Src, indicating that in these cells Ras-dependent signaling pathways are required for v-Src to exert its biological effects. However, we show here that Ras was not activated in Rat-2 fibroblasts transformed by wild-type v-Src, or in chicken embryo fibroblasts transformed by SRX5, a v-Src mutant with a linker insertion at the major site of autophosphorylation. Expression of a dominant-negative mutant of Ras completely inhibited the ability of v-Src to activate the mitogen-activated protein kinase ERK2, which is downstream of Ras. However, dominant-negative Ras did not suppress transformation by v-Src as judged by a variety of criteria. Thus, v-Src can transform at least some cell types in the absence of Ras activation or Ras-stimulated ERK2 activity, and in these cells activation of Ras-independent signaling pathways must therefore be sufficient for transformation.
Bcl-xLcan inhibit apoptosis in cells that have undergone Fas-induced protease activation
Authors: Boise Lawrence H. , Thompson Craig B.
Published: July 2002

Programmed cell death or apoptosis provides an irreversible mechanism for the elimination of excess or damaged cells. Several recent studies have implicated the activation of the interleukin 1β-converting enzyme/Ced-3 (ICE/Ced-3) family of proteases as the “point of no return” in apoptotic cell death, while others have suggested that loss of mitochondrial membrane potential (ΔΨm) is the ultimate determinant of cell death. The temporal relationship of these two events during apoptosis and the role of Bcl-2 proteins in inhibiting these steps has not been defined. To examine these issues, control and Bcl-xL-transfected Jurkat T cells were treated with Fas antibodies in the presence and absence of the ICE protease inhibitor zVAD-FMK. ICE/Ced-3 protease activity was monitored by following the cleavage of poly(ADP-ribose) polymerase (PARP) and ΔΨmwas followed by rhodamine 123 fluorescence. Although Bcl-xLexpression did not block Fas-induced protease activation, it substantially inhibited the subsequent loss of ΔΨmand cell death in Fas-treated cells. In contrast, zVAD-FMK blocked PARP cleavage as well as loss of ΔΨmand cell death. Together these data demonstrate that Bcl-xLcan maintain cell viability by preventing the loss of mitochondrial membrane potential that occurs as a consequence of ICE/Ced-3 protease activation.
Genomic mutation rates for lifetime reproductive output and lifespan in
Caenorhabditis elegans
Authors: Keightley Peter D. , Caballero Armando
Published: July 2002

Theory concerning the evolution of sex and recombination and mutation load relies on information on rates and distributions of effects of deleterious mutations. Direct information on the genomic mutation rate in
Drosophila
implies that an accumulation of mildly deleterious mutations reduces viability of populations by at least 1% per generation. We carried out an experiment to measure the deleterious mutation rate in
Caenorhabditis elegans
, in which independent sublines were maintained with one hermaphrodite parent per generation, conditions that minimize the opportunity for natural selection and lead to random fixation of deleterious mutations. After 60 generations of mutation accumulation, negligible changes in mean reproductive output and lifespan occurred, but the genetic variance increased at rates typical for life history traits in other species. The estimated deleterious mutation rate per haploid genome for fitness,
U
, was 0.0026, a figure two orders of magnitude smaller than previously measured for viability in
Drosophila
.
The role of habitat shift in the evolution of lizard morphology: evidence from tropical Tropidurus
Authors: Vitt Laurie J. , Caldwell Janalee P. , Zani Peter A. , Titus Tom A.
Published: July 2002

We compared morphology of two geographically close populations of the tropical lizardTropidurus hispidusto test the hypothesis that habitat structure influences the evolution of morphology and ecology at the population level.T. hispidusisolated on a rock outcrop surrounded by tropical forest use rock crevices for refuge and appear dorsoventrally compressed compared with those in open savanna. A principal components analysis revealed that the populations were differentially distributed along an axis representing primarily three components of shape: body width, body height, and hind-leg length. Morphological divergence was supported by a principal components analysis of size-free morphological variables. Mitochondrial DNA sequences of ATPase 6 indicate that these populations are closely related relative to otherT. hispidus, the rock outcrop morphology and ecology are derived withinT. hispidus, and morphological and ecological divergence has occurred more rapidly than genetic divergence. This suggests that natural selection can rapidly adjust morphology and ecology in response to a recent history of exposure to habitats differing in structure, a result heretofore implied from comparative studies among lizard species.
Deleterious mutation accumulation and the regeneration of genetic resources
Authors: Schoen Daniel J. , David Jacques L. , Bataillon Thomas M.
Published: July 2002

The accumulation of mildly deleterious mutations accompanying recurrent regeneration of plant germ plasm was modeled under regeneration conditions characterized by different amounts of selection and genetic drift. Under some regeneration conditions (sample sizes ≥75 individuals and bulk harvesting of seed) mutation accumulation was negligible, but under others (sample sizes <75 individuals or equalization of seed production by individual plants) mutation numbers per genome increased significantly during 25–50 cycles of regeneration. When mutations also are assumed to occur (at elevated rates) during seed storage, significant mutation accumulation and fitness decline occurred in 10 or fewer cycles of regeneration regardless of the regeneration conditions. Calculations also were performed to determine the numbers of deleterious mutations introduced and remaining in the genome of an existing variety after hybridization with a genetic resource and subsequent backcrossing. The results suggest that mutation accumulation has the potential to reduce the viability of materials held in germ plasm collections and to offset gains expected by the introduction of particular genes of interest from genetic resources.
Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants
Authors: Corson Laura B. , Strain Jeffrey J. , Culotta Valeria C. , Cleveland Don W.
Published: July 2002

Mutations in Cu, Zn superoxide dismutase (SOD1) cause the neurodegenerative disease familial amyotrophic lateral sclerosis from an as-yet-unidentified toxic property(ies). Analysis in
Saccharomyces cerevisiae
of a broad range of human familial amyotrophic lateral sclerosis–linked SOD1 mutants (A4V, G37R, G41D, H46R, H48Q, G85R, G93C, and I113T) reveals one property common to these mutants (including two at residues that coordinate the catalytic copper): Each does indeed bind copper and scavenge oxygen-free radicals
in vivo
. Neither decreased copper binding nor decreased superoxide scavenging activity is a property shared by all mutants. The demonstration that shows that all mutants tested do bind copper under physiologic conditions supports a mechanism of SOD1 mutant-mediated disease arising from aberrant copper-mediated chemistry catalyzed by less tightly folded (and hence less constrained) mutant enzymes. The mutant enzymes also are shown to acquire the catalytic copper
in vivo
through the action of CCS, a specific copper chaperone for SOD1, which in turn suggests that a search for inhibitors of this SOD1 copper chaperone may represent a therapeutic avenue.
Microvascular and tissue oxygen gradients in the rat mesentery
Authors: Tsai Amy G. , Friesenecker Barbara , Mazzoni Michelle C. , Kerger Heinz , Buerk Donald G. , Johnson Paul C. , Intaglietta Marcos
Published: July 2002

One of the most important functions of the blood circulation is O
2
delivery to the tissue. This process occurs primarily in microvessels that also regulate blood flow and are the site of many metabolic processes that require O
2
. We measured the intraluminal and perivascular pO
2
in rat mesenteric arterioles
in vivo
by using noninvasive phosphorescence quenching microscopy. From these measurements, we calculated the rate at which O
2
diffuses out of microvessels from the blood. The rate of O
2
efflux and the O
2
gradients found in the immediate vicinity of arterioles indicate the presence of a large O
2
sink at the interface between blood and tissue, a region that includes smooth muscle and endothelium. Mass balance analyses show that the loss of O
2
from the arterioles in this vascular bed primarily is caused by O
2
consumption in the microvascular wall. The high metabolic rate of the vessel wall relative to parenchymal tissue in the rat mesentery suggests that in addition to serving as a conduit for the delivery of O
2
the microvasculature has other functions that require a significant amount of O
2
.
Computerized polymorphic marker identification: Experimental validation and a predicted human polymorphism catalog
Authors: Fondon John W. , Mele Gina M. , Brezinschek Ruth I. , Cummings Donna , Pande Ashwini , Wren Jonathan , O’Brien Kevin M. , Kupfer Kenneth C. , Wei Ming-Hui , Lerman Michael , Minna John D. , Garner Harold R.
Published: July 2002

A computational system for the prediction of polymorphic loci directly and efficiently from human genomic sequence was developed and verified. A suite of programs, collectively called
pompous
(polymorphic marker prediction of ubiquitous simple sequences) detects tandem repeats ranging from dinucleotides up to 250 mers, scores them according to predicted level of polymorphism, and designs appropriate flanking primers for PCR amplification. This approach was validated on an approximately 750-kilobase region of human chromosome 3p21.3, involved in lung and breast carcinoma homozygous deletions. Target DNA from 36 paired B lymphoblastoid and lung cancer lines was amplified and allelotyped for 33 loci predicted by
pompous
to be variable in repeat size. We found that among those 36 predominately Caucasian individuals 22 of the 33 (67%) predicted loci were polymorphic with an average heterozygosity of 0.42. Allele loss in this region was found in 27/36 (75%) of the tumor lines using these markers.
pompous
provides the genetic researcher with an additional tool for the rapid and efficient identification of polymorphic markers, and through a World Wide Web site, investigators can use
pompous
to identify polymorphic markers for their research. A catalog of 13,261 potential polymorphic markers and associated primer sets has been created from the analysis of 141,779,504 base pairs of human genomic sequence in GenBank. This data is available on our Web site (
pompous.swmed.edu
) and will be updated periodically as GenBank is expanded and algorithm accuracy is improved.
Stable relativistic/charge-displacement channels in ultrahigh power density (≈10
21
W/cm
3
) plasmas
Authors: Borisov A. B. , Longworth J. W. , Boyer K. , Rhodes C. K.
Published: July 2002

Robust stability is a chief characteristic of
relativistic/charge-displacement self-channeling. Theoretical
analysis of the dynamics of this stability (
i
) reveals a
leading role for the eigenmodes in the development of stable channels,
(
ii
) suggests a technique using a simple longitudinal
gradient in the electron density to extend the zone of stability into
the high electron density/high power density regime,
(
iii
) indicates that a situation approaching
unconditional stability can be achieved, (
iv
)
demonstrates the efficacy of the stable dynamics in trapping severely
perturbed beams in single uniform channels, and (
v
)
predicts that ≈10
4
critical powers can be trapped in a
single stable channel. The scaling of the maximum power density with
the propagating wavelength λ is shown to be proportional to
λ
−4
for a given propagating power and a fixed ratio of
the electron plasma density to the critical plasma density. An estimate
of the maximum power density that can be achieved in these channels
with a power of ≈2 TW at a UV (248 nm) wavelength gives a value of
≈10
21
W/cm
3
with a corresponding atomic
specific magnitude of ≈60 W/atom. The characteristic intensity
propagating in the channel under these conditions exceeds
10
21
W/cm
2
.
3,
N
4
-ethenocytosine, a highly mutagenic adduct, is a primary substrate for
Escherichia coli
double-stranded uracil-DNA glycosylase and human mismatch-specific thymine-DNA glycosylase
Authors: Saparbaev Murat , Laval Jacques
Published: July 2002

Exocyclic DNA adducts are generated in cellular DNA by various industrial pollutants such as the carcinogen vinyl chloride and by endogenous products of lipid peroxidation. The etheno derivatives of purine and pyrimidine bases 3,
N
4
-ethenocytosine (ɛC), 1,
N
6
-ethenoadenine (ɛA),
N
2
,3-ethenoguanine, and 1,
N
2
-ethenoguanine cause mutations. The ɛA residues are excised by the human and the
Escherichia coli
3-methyladenine-DNA glycosylases (ANPG and AlkA proteins, respectively), but the enzymes repairing ɛC residues have not yet been described. We have identified two homologous proteins present in human cells and
E. coli
that remove ɛC residues by a DNA glycosylase activity. The human enzyme is an activity of the mismatch-specific thymine-DNA glycosylase (hTDG). The bacterial enzyme is the double-stranded uracil-DNA glycosylase (dsUDG) that is the homologue of the hTDG. In addition to uracil and ɛC-DNA glycosylase activity, the dsUDG protein repairs thymine in a G/T mismatch. The fact that ɛC is recognized and efficiently excised by the
E. coli
dsUDG and hTDG proteins
in vitro
suggests that these enzymes may be responsible for the repair of this mutagenic lesion
in vivo
and be important contributors to genetic stability.
A family of fatty acid transporters conserved from mycobacterium to man
Authors: Hirsch David , Stahl Andreas , Lodish Harvey F.
Published: July 2002

Long chain fatty acids (LCFAs) are an important source of energy for most organisms. They also function as blood hormones, regulating key metabolic functions such as hepatic glucose production. Although LCFAs can diffuse through the hydrophobic core of the plasma membrane into cells, this nonspecific transport cannot account for the high affinity and specific transport of LCFAs exhibited by cells such as cardiac muscle, hepatocytes, and adipocytes. Transport of LCFAs across the plasma membrane is facilitated by fatty acid transport protein (FATP), a plasma membrane protein that increases LCFA uptake when expressed in cultured mammalian cells [Schaffer, J. E. & Lodish, H. F. (1994)
Cell
79, 427–436]. Here, we report the identification of four novel murine FATPs, one of which is expressed exclusively in liver and another only in liver and kidney. Both genes increase fatty acid uptake when expressed in mammalian cells. All five murine FATPs have homologues in humans in addition to a sixth
FATP
gene. FATPs are found in such diverse organisms as
Fugu rubripes
,
Caenorhabditis elegans
,
Drosophila melanogaster
,
Saccharomyces cerevisiae
, and
Mycobacterium tuberculosis
. The function of the
FATP
gene family is conserved throughout evolution as the
C. elegans
and mycobacterial FATPs facilitate LCFA uptake when overexpressed in COS cells or
Escherichia coli
, respectively. The identification of this evolutionary conserved fatty acid transporter family will allow us to gain a better understanding of the mechanisms whereby LCFAs traverse the lipid bilayer as well as yield insight into the control of energy homeostasis and its dysregulation in diseases such as diabetes and obesity.
Functional connectivity of the angular gyrus in normal reading and dyslexia
Authors: Horwitz B. , Rumsey J. M. , Donohue B. C.
Published: July 2002

The classic neurologic model for reading, based on studies of patients with acquired alexia, hypothesizes functional linkages between the angular gyrus in the left hemisphere and visual association areas in the occipital and temporal lobes. The angular gyrus also is thought to have functional links with posterior language areas (e.g., Wernicke’s area), because it is presumed to be involved in mapping visually presented inputs onto linguistic representations. Using positron emission tomography , we demonstrate in normal men that regional cerebral blood flow in the left angular gyrus shows strong within-task, across-subjects correlations (i.e., functional connectivity) with regional cerebral blood flow in extrastriate occipital and temporal lobe regions during single word reading. In contrast, the left angular gyrus is functionally disconnected from these regions in men with persistent developmental dyslexia, suggesting that the anatomical disconnection of the left angular gyrus from other brain regions that are part of the “normal” brain reading network in many cases of acquired alexia is mirrored by its functional disconnection in developmental dyslexia.
Social inhibition of song imitation among sibling male zebra finches
Authors: Tchernichovski Ofer , Nottebohm Fernando
Published: July 2002

A male zebra finch,Taeniopygia guttata, kept with its father until adulthood develops an imitation of its father’s song motif. We report here that the completeness of this imitation was sensitive to the social or auditory context in which the bird grew up: the greater the number of male siblings in a clutch, the shorter the mean duration of the song motif and the fewer the mean number of song notes imitated from the father; the latter shortfall was not compensated by other, improvised notes. We call this effect fraternal inhibition. Fraternal inhibition was avoided by members of a clutch that developed the song first. To our surprise, this role commonly fell to one of the younger birds in the clutch. Early song learning may influence fitness since individuals that produced the most complete imitations also tended to induce more egg laying.
Age and rate of diversification of the Hawaiian silversword alliance (Compositae)
Authors: Baldwin Bruce G. , Sanderson Michael J.
Published: July 2002

Comparisons between insular and continental radiations have been hindered by a lack of reliable estimates of absolute diversification rates in island lineages. We took advantage of rate-constant rDNA sequence evolution and an “external” calibration using paleoclimatic and fossil data to determine the maximum age and minimum diversification rate of the Hawaiian silversword alliance (Compositae), a textbook example of insular adaptive radiation in plants. Our maximum-age estimate of 5.2 ± 0.8 million years ago for the most recent common ancestor of the silversword alliance is much younger than ages calculated by other means for the Hawaiian drosophilids, lobelioids, and honeycreepers and falls approximately within the history of the modern high islands (≤5.1 ± 0.2 million years ago). By using a statistically efficient estimator that reduces error variance by incorporating clock-based estimates of divergence times, a minimum diversification rate for the silversword alliance was estimated to be 0.56 ± 0.17 species per million years. This exceeds average rates of more ancient continental radiations and is comparable to peak rates in taxa with sufficiently rich fossil records that changes in diversification rate can be reconstructed.
Lectin-like oxidized low-density lipoprotein receptor 1 mediates phagocytosis of aged/apoptotic cells in endothelial cells
Authors: Oka Kozo , Sawamura Tatsuya , Kikuta Ken-ichiro , Itokawa Shigekazu , Kume Noriaki , Kita Toru , Masaki Tomoh
Published: July 2002

Recognition of the exposure of phosphatidylserine (PS) on the outer surface of plasma membrane has been implicated in the phagocytosis of aged/apoptotic cells. Because oxidized low-density lipoprotein (OxLDL) has been reported to block the phagocytosis, here we examined whether lectin-like OxLDL receptor 1 (LOX-1), the OxLDL receptor in endothelial cells, mediates phagocytosis of aged/apoptotic cells by endothelial cells. Cultured bovine aortic endothelial cells (BAE) and Chinese hamster ovary (CHO) cells expressing bovine LOX-1 (BLOX-1-CHO), but not wild-type CHO-K1 cells, bound aged red blood cells (RBC) and apoptotic cells, which were further phagocytosed. The binding of aged RBC and the phagocytosis of apoptotic cells were inhibited by OxLDL, acetyl LDL, and other LOX-1 ligands in both BAE and BLOX-1-CHO. mAb against LOX-1 blocked the binding of aged RBC to BAE, suggesting a role for LOX-1 in the recognition of aged cells. The recombinant soluble LOX-1 inhibited the interactions of aged/apoptotic cells with both BLOX-1-CHO and BAE and distinguished aged RBC from native RBC and apoptotic cells from native cells. PS liposome inhibited these LOX-1-mediated interactions with aged/apoptotic cells, suggesting LOX-1 recognizes PS of the apoptotic cells. PS exposed on the surface of apoptotic cells is known to be procoagulant. Accordingly, increased OxLDL may be one of the reasons for enhanced coagulation in atherosclerosis, inhibiting the removal of apoptotic cells.
Directed disruption of the tobacco
ndhB
gene impairs cyclic electron flow around photosystem I
Authors: Shikanai Toshiharu , Endo Tsuyoshi , Hashimoto Takashi , Yamada Yasuyuki , Asada Kozi , Yokota Akiho
Published: July 2002

To evaluate the physiological significance of cyclic electron flow around photosystem (PS) I, we used a reverse genetic approach to focus on 11 chloroplast genes that encode homologs of mitochondrial complex I subunits (
ndhA
-
K
). Since their discovery, the exact function of the respiratory components in plant chloroplasts has been a matter of discussion. We disrupted one of these genes (
ndhB
) in tobacco by chloroplast transformation. Analysis of the transient increase in chlorophyll fluorescence after actinic light illumination and the redox kinetics of P700 (reaction center chlorophylls of PS I) suggest that the cyclic electron flow around PS I is impaired in the
ndhB
-deficient transformants. Transformants grew normally in a greenhouse, suggesting that the cyclic electron flow around PS I mediated by
ndh
gene products is dispensable in tobacco under mild environmental conditions.
MLL, a mammalian
trithorax-
group gene, functions as a transcriptional maintenance factor in morphogenesis
Authors: Yu Benjamin D. , Hanson Robin D. , Hess Jay L. , Horning Susan E. , Korsmeyer Stanley J.
Published: July 2002

Determinative events in vertebrate embryogenesis appear to require the continuous expression of spatial regulators such as the clustered homeobox genes. The mechanisms that govern long-term patterns of gene expression are not well understood. In
Drosophila
, active and silent states of developmentally regulated loci are maintained by
trithorax
and
Polycomb
group. We have examined the developmental role of a mammalian homolog of
trx
and putative oncogene,
Mll
. Knockout mice reveal that
Mll
is required for maintenance of gene expression early in embryogenesis. Downstream targets of
Mll
including
Hoxa7
are activated appropriately in the absence of
Mll
but require
Mll
for sustaining their expression. The
Mll
−/− phenotype manifests later in development and is characterized by branchial arch dysplasia and aberrant segmental boundaries of spinal ganglia and somites. Thus,
Mll
represents an essential mechanism of transcriptional maintenance in mammalian development, which functions in multiple morphogenetic processes.
A response regulator of cyanobacteria integrates diverse environmental signals and is critical for survival under extreme conditions
Authors: Schwarz Rakefet , Grossman Arthur R.
Published: July 2002

Microorganisms must sense their environment and rapidly tune their metabolism to ambient conditions to efficiently use available resources. We have identified a gene encoding a response regulator, NblR, that complements a cyanobacterial mutant unable to degrade its light-harvesting complex (phycobilisome), in response to nutrient deprivation. Cells of the
nblR
mutant (
i
) have more phycobilisomes than wild-type cells during nutrient-replete growth, (
ii
) do not degrade phycobilisomes during sulfur, nitrogen, or phosphorus limitation, (
iii
) cannot properly modulate the phycobilisome level during exposure to high light, and (
iv
) die rapidly when starved for either sulfur or nitrogen, or when exposed to high light. Apart from regulation of phycobilisome degradation, NblR modulates additional functions critical for cell survival during nutrient-limited and high-light conditions. NblR does not appear to be involved in acclimation responses that occur only during a specific nutrient limitation. In contrast, it controls at least some of the general acclimation responses; those that occur during any of a number of different stress conditions. NblR plays a pivotal role in integrating different environmental signals that link the metabolism of the cell to light harvesting capabilities and the activities of the photosynthetic apparatus; this modulation is critical for cell survival.
Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors
Authors: Levanon Ditsa , Goldstein Robert E. , Bernstein Yael , Tang Hua , Goldenberg Dalia , Stifani Stefano , Paroush Ze’ev , Groner Yoram
Published: July 2002

The mammalian AML/CBFα runt domain (RD) transcription factors regulate hematopoiesis and osteoblast differentiation. Like their
Drosophila
counterparts, most mammalian RD proteins terminate in a common pentapeptide, VWRPY, which serves to recruit the corepressor Groucho (Gro). Using a yeast two-hybrid assay,
in vitro
association and pull-down experiments, we demonstrate that Gro and its mammalian homolog TLE1 specifically interact with AML1 and AML2. In addition to the VWRPY motif, other C-terminal sequences are required for these interactions with Gro/TLE1. TLE1 inhibits AML1-dependent transactivation of the T cell receptor (TCR) enhancers α and β, which contain functional AML binding sites, in transfected Jurkat T cells. LEF-1 is an additional transcription factor that mediates transactivation of TCR enhancers. LEF-1 and its
Drosophila
homolog Pangolin (Pan) are involved in the Wnt/Wg signaling pathway through interactions with the coactivator β-catenin and its highly conserved fly homolog Armadillo (Arm). We show that TLE/Gro interacts with LEF-1 and Pan, and inhibits LEF-1:β-catenin-dependent transcription. These data indicate that, in addition to their activity as transcriptional activators, AML1 and LEF-1 can act, through recruitment of the corepressor TLE1, as transcriptional repressors in TCR regulation and Wnt/Wg signaling.
Nuclear-encoded proteins target to the plastid inToxoplasma gondiiandPlasmodium falciparum
Authors: Waller Ross F. , Keeling Patrick J. , Donald Robert G. K. , Striepen Boris , Handman Emanuela , Lang-Unnasch Naomi , Cowman Alan F. , Besra Gurdyal S. , Roos David S. , McFadden Geoffrey I.
Published: July 2002

A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or “apicoplast,” is indispensable, but the complete sequence of both thePlasmodium falciparumandToxoplasma gondiiapicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast inPlasmodiumandToxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), β-ketoacyl-ACP synthase III (FabH), and β-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the apicoplast in transgenicT. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition ofP. falciparumgrowth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.
Inhibiting transthyretin conformational changes that lead to amyloid fibril formation
Authors: Peterson Scott A. , Klabunde Thomas , Lashuel Hilal A. , Purkey Hans , Sacchettini James C. , Kelly Jeffrey W.
Published: July 2002

Insoluble protein fibrils resulting from the self-assembly of a conformational intermediate are implicated as the causative agent in several severe human amyloid diseases, including Alzheimer’s disease, familial amyloid polyneuropathy, and senile systemic amyloidosis. The latter two diseases are associated with transthyretin (TTR) amyloid fibrils, which appear to form in the acidic partial denaturing environment of the lysosome. Here we demonstrate that flufenamic acid (Flu) inhibits the conformational changes of TTR associated with amyloid fibril formation. The crystal structure of TTR complexed with Flu demonstrates that Flu mediates intersubunit hydrophobic interactions and intersubunit hydrogen bonds that stabilize the normal tetrameric fold of TTR. A small-molecule inhibitor that stabilizes the normal conformation of a protein is desirable as a possible approach to treat amyloid diseases. Molecules such as Flu also provide the means to rigorously test the amyloid hypothesis, i.e., the apparent causative role of amyloid fibrils in amyloid disease.
Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosisin vivo
Authors: Zheng Timothy S. , Schlosser Stephan F. , Dao Tao , Hingorani Ravi , Crispe I. Nicholas , Boyer James L. , Flavell Richard A.
Published: July 2002

Both caspase-1- and caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. We assessed the contributions of these caspases to Fas signaling in hepatocyte cell deathin vitro. Although wild-type, caspase-1−/−, and caspase-3−/−hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3−/−hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1−/−apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation were seen within 6 hr, but neither event was observed for caspase-3−/−hepatocytes. We extended these studies to thymocytes and found that apoptotic caspase-3−/−thymocytes exhibited similar “abnormal” morphological changes and delayed DNA fragmentation observed in hepatocytes. Furthermore, the cleavage of various caspase substrates implicated in mediating apoptotic events, including gelsolin, fodrin, laminB, and DFF45/ICAD, was delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.
Toward controlling gene expression at will: Specific regulation of the
erbB
-
2
/
HER
-
2
promoter by using polydactyl zinc finger proteins constructed from modular building blocks
Authors: Beerli Roger R. , Segal David J. , Dreier Birgit , Barbas Carlos F.
Published: July 2002

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5′-GNN-3′ subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5′-(GNN)
6
-3′ family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human
erbB-2
gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5′-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16’s minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.
Dissociation among
in vitro
telomerase activity, telomere maintenance, and cellular immortalization
Authors: Counter Christopher M. , Hahn William C. , Wei Wenyi , Caddle Stephanie Dickinson , Beijersbergen Roderick L. , Lansdorp Peter M. , Sedivy John M. , Weinberg Robert A.
Published: July 2002

The immortalization of human cells is a critical step during tumorigenesis.
In vitro
, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.
Correction of murine galactosialidosis by bone marrow-derived macrophages overexpressing human protective protein/cathepsin A under control of the colony-stimulating factor-1 receptor promoter
Authors: Hahn Christopher N. , del Pilar Martin Maria , Zhou Xiao-Yan , Mann Linda W. , d’Azzo Alessandra
Published: July 2002

Galactosialidosis (GS) is a human neurodegenerative disease caused by a deficiency of lysosomal protective protein/cathepsin A (PPCA). The GS mouse model resembles the severe human condition, resulting in nephropathy, ataxia, and premature death. To rescue the disease phenotype, GS mice were transplanted with bone marrow from transgenic mice overexpressing human PPCA specifically in monocytes/macrophages under the control of the colony stimulating factor-1 receptor promoter. Transgenic macrophages infiltrated and resided in all organs and expressed PPCA at high levels. Correction occurred in hematopoietic tissues and nonhematopoietic organs, including the central nervous system. PPCA-expressing perivascular and leptomeningeal macrophages were detected throughout the brain of recipient mice, although some neuronal cells, such as Purkinje cells, continued to show storage and died. GS mice crossed into the transgenic background reflected the outcome of bone marrow-transplanted mice, but the course of neuronal degeneration was delayed in this model. These studies present definite evidence that macrophages alone can provide a source of corrective enzyme for visceral organs and may be beneficial for neuronal correction if expression levels are sufficient.
Molecular recognition of angiogenesis inhibitors fumagillin and ovalicin by methionine aminopeptidase 2
Authors: Griffith Eric C. , Su Zhuang , Niwayama Satomi , Ramsay Cameron A. , Chang Yie-Hwa , Liu Jun O.
Published: July 2002

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer and other human diseases. Fumagillin and ovalicin compose a class of structurally related natural products that potently inhibit angiogenesis by blocking endothelial cell proliferation. A synthetic analog of fumagillin, TNP-470, is currently undergoing clinical trials for treatment of a variety of cancers. A common target for fumagillin and ovalicin recently was identified as the type 2 methionine aminopeptidase (MetAP2). These natural products bind MetAP2 covalently, inhibiting its enzymatic activity. The specificity of this binding is underscored by the lack of inhibition of the closely related type 1 enzyme, MetAP1. The molecular basis of the high affinity and specificity of these inhibitors for MetAP2 has remained undiscovered. To determine the structural elements of these inhibitors and MetAP2 that are involved in this interaction, we synthesized fumagillin analogs in which each of the potentially reactive epoxide groups was removed either individually or in combination. We found that the ring epoxide in fumagillin is involved in the covalent modification of MetAP2, whereas the side chain epoxide group is dispensable. By using a fumagillin analog tagged with fluorescein, His-231 in MetAP2 was identified as the residue that is covalently modified by fumagillin. Site-directed mutagenesis of His-231 demonstrated its importance for the catalytic activity of MetAP2 and confirmed that the same residue is covalently modified by fumagillin. These results, in agreement with a recent structural study, suggest that fumagillin and ovalicin inhibit MetAP2 by irreversible blockage of the active site.
Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor β receptor
Authors: Lai Char-Chang , Henningson Carl , DiMaio Daniel
Published: July 2002

The bovine papillomavirus E5 protein is a 44-aa transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) β receptor and induces constitutive tyrosine phosphorylation and activation of the receptor, resulting in cell transformation. The E5 protein does not resemble PDGF, but rather activates the receptor in a ligand-independent fashion, thus providing a unique system to examine activation of receptor tyrosine kinases. Here, we used a variety of approaches to explore the mechanism of receptor activation by the E5 protein. Chemical cross-linking experiments revealed that the E5 protein activated only a small fraction of the endogenous PDGF β receptor in transformed fibroblasts and suggested that this fraction was constitutively dimerized. Coimmunoprecipitation experiments using extracts of cells engineered to coexpress full-length and truncated PDGF β receptors confirmed that the E5 protein induced oligomerization of the receptor. Furthermore, in cells expressing the E5 protein, a kinase-active receptor was able to trans-phosphorylate a kinase-negative mutant receptor but was unable to catalyze intramolecular autophosphorylation. These results indicated that the E5 protein induced PDGF β receptor activation by forming a stable complex with the receptor, resulting in receptor dimerization and trans-phosphorylation.
Molecular evidence for an African origin of the Hawaiian endemicHesperomannia (Asteraceae)
Authors: Kim Hyi-Gyung , Keeley Sterling C. , Vroom Peter S. , Jansen Robert K.
Published: July 2002

Identification of the progenitors of plants endemic to oceanic islands often is complicated by extreme morphological divergence between island and continental taxa. This is especially true for the Hawaiian Islands, which are 3,900 km from any continental source. We examine the origin ofHesperomannia, a genus of three species endemic to Hawaii that always have been placed in the tribe Mutisieae of the sunflower family. Phylogenetic analyses of representatives from all tribes in this family using the chloroplast genendhF(where ndhF is the ND5 protein of chloroplast NADH dehydrogenase) indicate thatHesperomanniabelongs to the tribe Vernonieae. Phylogenetic comparisons within the Vernonieae using sequences of bothndhFand the internal transcribed spacer regions of nuclear ribosomal DNA reveal thatHesperomanniais sister to African species ofVernonia. Long-distance dispersal northeastward from Africa to southeast Asia and across the many Pacific Ocean island chains is the most likely explanation for this unusual biogeographic connection. The 17- to 26-million-year divergence time between AfricanVernoniaandHesperomanniaestimated by the DNA sequences predates the age of the eight existing Hawaiian Islands. These estimates are consistent with an hypothesis that the progenitor ofHesperomanniaarrived at one of the low islands of the Hawaiian-Emperor chain between the late Oligocene and mid-Miocene when these islands were above sea level. Subsequent to its arrival the southeast Pacific island chains served as steppingstones for dispersal to the existing Hawaiian Islands.
NewDrosophilaintrons originate by duplication
Authors: Tarrío Rosa , Rodríguez-Trelles Francisco , Ayala Francisco J.
Published: July 2002

We have analyzed the phylogenetic distribution of introns in the gene coding for xanthine dehydrogenase in 37 species, including 31 dipterans sequenced by us. We have discovered three narrowly distributed novel introns, one in the medflyCeratitis capitata, the second in thewillistoniandsaltansgroups ofDrosophila, and the third in two sibling species of thewillistonigroup. The phylogenetic distribution of these introns favors the “introns-late” theory of the origin of genes. Analysis of the nucleotide sequences indicates that all three introns have arisen by duplication of a preexisting intron, which is pervasive inDrosophilaand other dipterans (and has a homologous position as an intron found in humans and other diverse organisms).
Expression of
Xa1
, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation
Authors: Yoshimura Satomi , Yamanouchi Utako , Katayose Yuichi , Toki Seiichi , Wang Zi-Xuan , Kono Izumi , Kurata Nori , Yano Masahiro , Iwata Nobuo , Sasaki Takuji
Published: July 2002

The
Xa1
gene in rice confers resistance to Japanese race 1 of
Xanthomonas oryzae
pv.
oryzae
, the causal pathogen of bacterial blight (BB). We isolated the
Xa1
gene by a map-based cloning strategy. The deduced amino acid sequence of the
Xa1
gene product contains nucleotide binding sites (NBS) and a new type of leucine-rich repeats (LRR); thus,
Xa1
is a member of the NBS-LRR class of plant disease-resistance genes, but quite different from
Xa21
, another BB-resistance gene isolated from rice. Interestingly,
Xa1
gene expression was induced on inoculation with a bacterial pathogen and wound, unlike other isolated resistance genes in plants, which show constitutive expression. The induced expression may be involved in enhancement of resistance against the pathogen.