bluuebunny/crossref_metadata_2025 huggingface dataset:
Forty million years of mutualism: Evidence for Eocene origin of the yucca-yucca moth association
Authors: Pellmyr Olle , Leebens-Mack James
Published: July 2002

The obligate mutualism between yuccas and yucca moths is a major model system for the study of coevolving species interactions. Exploration of the processes that have generated current diversity and associations within this mutualism requires robust phylogenies and timelines for both moths and yuccas. Here we establish a molecular clock for the moths based on mtDNA and use it to estimate the time of major life history events within the yucca moths. Colonization of yuccas had occurred by 41.5 ± 9.8 million years ago (Mya), with rapid life history diversification and the emergence of pollinators within 0–6 My after yucca colonization. A subsequent burst of diversification 3.2 ± 1.8 Mya coincided with evolution of arid habitats in western North America. Derived nonpollinating cheater yucca moths evolved 1.26 ± 0.96 Mya. The estimated age of the moths far predates the host fossil record, but is consistent with suggested host age based on paleobotanical, climatological, biogeographical, and geological data, and a tentative estimation from an
rbcL
-based molecular clock for yuccas. The moth data are used to establish three alternative scenarios of how the moths and plants have coevolved. They yield specific predictions that can be tested once a robust plant phylogeny becomes available.
Tissue-specific and developmental stage-specific DNA binding by a mammalian SWI/SNF complex associated with human fetal-to-adult globin gene switching
Authors: O’Neill David , Yang John , Erdjument-Bromage Hediye , Bornschlegel Katherine , Tempst Paul , Bank Arthur
Published: July 2002

SWI/SNF complexes in yeast and higher eukaryotes are thought to facilitate gene activation and transcription factor binding by disrupting repressive chromatin structures. Little is known, however, about how these complexes target specific genes for activation. We now have purified a specialized SWI/SNF-related complex (PYR complex) from murine erythroleukemia (MEL) cell nuclear extract that binds pyrimidine-rich elements at the human and murine β-globin loci. PYR complex DNA-binding activity is restricted to definitive hematopoietic cells and is both DNA sequence- and length-dependent. Mass spectrometric identification of purified peptides and antibody supershift assays indicate that PYR complex contains at least four known mammalian SWI/SNF subunits: BAF57, INI1, BAF60a, and BAF170. PYR complex broadly footprints a 250-bp pyrimidine-rich element between the human fetal and adult β-globin genes. A short intergenic deletion that removes this element from a human globin locus cosmid construct results in delayed human fetal-to-adult globin gene switching in transgenic mice. Taken together, the data suggest that PYR complex may act through this intergenic element to facilitate human fetal-to-adult globin gene switching, presumably by opening the locus in the region of the adult genes to permit the binding of β-globin transcriptional activators.
Postgastrulation
Smad2-
deficient embryos show defects in embryo turning and anterior morphogenesis
Authors: Heyer Joerg , Escalante-Alcalde Diana , Lia Marie , Boettinger Erwin , Edelmann Winfried , Stewart Colin L. , Kucherlapati Raju
Published: July 2002

SMAD2 is a member of the transforming growth factor β and activin-signaling pathway. To examine the role of
Smad2
in postgastrulation development, we independently generated mice with a null mutation in this gene.
Smad2-
deficient embryos die around day 7.5 of gestation because of failure of gastrulation and failure to establish an anterior–posterior (A-P) axis. Expression of the homeobox gene
Hex
(the earliest known marker of the A-P polarity and the prospective head organizer) was found to be missing in
Smad2-
deficient embryos. Homozygous mutant embryos and embryonic stem cells formed mesoderm derivatives revealing that mesoderm induction is SMAD2 independent. In the presence of wild-type extraembryonic tissues,
Smad2-
deficient embryos developed beyond 7.5 and up to 10.5 days postcoitum, demonstrating a requirement for SMAD2 in extraembryonic tissues for the generation of an A-P axis and gastrulation. The rescued postgastrulation embryos showed malformation of head structures, abnormal embryo turning, and cyclopia. Our results show that
Smad2
expression is required at several stages during embryogenesis.
Octamer-based genome scanning distinguishes a unique subpopulation of
Escherichia coli
O157:H7 strains in cattle
Authors: Kim Jaehyoung , Nietfeldt Joseph , Benson Andrew K.
Published: July 2002

Multilocus-genotyping methods have shown that
Escherichia coli
O157:H7 is a geographically disseminated clone. However, high-resolution methods such as pulse-field gel electrophoresis demonstrate significant genomic diversity among different isolates. To assess the genetic relationship of human and bovine isolates of
E. coli
O157:H7 in detail, we have developed an octamer-based genome-scanning methodology, which compares the distance between over-represented, strand-biased octamers that occur in the genome. Comparison of octamer-based genome-scanning products derived from >1 megabase of the genome demonstrated the existence of two distinct lineages of
E. coli
O157:H7 that are disseminated within the United States. Human and bovine isolates are nonrandomly distributed among the lineages, suggesting that one of these lineages may be less virulent for humans or may not be efficiently transmitted to humans from bovine sources. Restriction fragment length polymorphism analysis with lambdoid phage genomes indicates that phage-mediated events are associated with divergence of the lineages, thereby providing one explanation for the degree of diversity that is observed among
E. coli
O157:H7 by other molecular-fingerprinting methods.
Elimination of prions by branched polyamines and implications for therapeutics
Authors: Supattapone Surachai , Nguyen Hoang-Oanh B. , Cohen Fred E. , Prusiner Stanley B. , Scott Michael R.
Published: July 2002

We report that branched polyamines, including polyamidoamide dendimers, polypropyleneimine, and polyethyleneimine, are able to purge PrP
Sc
, the protease-resistant isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture. The removal of PrP
Sc
by these compounds depends on both the concentration of branched polymer and the duration of exposure. Chronic exposure of ScN2a cells to low noncytotoxic concentrations of branched polyamines for 1 wk reduced PrP
Sc
to an undetectable level, a condition that persisted at least 3 wk after removal of the compound. Structure–activity analysis revealed that a high surface density of primary amino groups is required for polyamines to eliminate PrP
Sc
effectively from cells. The removal of PrP
Sc
by branched polyamines is attenuated by chloroquine in living cells, and exposure of scrapie-infected brain extracts with branched polyamines at acidic pH rendered the PrP
Sc
susceptible to protease
in vitro
, suggesting that endosomes or lysozomes may be the site of action. Our studies suggest that branched polyamines might be useful therapeutic agents for treatment of prion diseases and perhaps a variety of other degenerative disorders.
Ozone depletion and UVB radiation: Impact on plant DNA damage in southern South America
Authors: Rousseaux M. Cecilia , Ballaré Carlos L. , Giordano Carla V. , Scopel Ana L. , Zima Ana M. , Szwarcberg-Bracchitta Mariela , Searles Peter S. , Caldwell Martyn M. , Díaz Susana B.
Published: July 2002

The primary motivation behind the considerable effort in studying stratospheric ozone depletion is the potential for biological consequences of increased solar UVB (280–315 nm) radiation. Yet, direct links between ozone depletion and biological impacts have been established only for organisms of Antarctic waters under the influence of the ozone “hole;” no direct evidence exists that ozone-related variations in UVB affect ecosystems of temperate latitudes. Indeed, calculations based on laboratory studies with plants suggest that the biological impact of ozone depletion (measured by the formation of cyclobutane pyrimidine dimers in DNA) is likely to be less marked than previously thought, because UVA quanta (315–400 nm) may also cause significant damage, and UVA is unaffected by ozone depletion. Herein, we show that the temperate ecosystems of southern South America have been subjected to increasingly high levels of ozone depletion during the last decade. We found that in the spring of 1997, despite frequent cloud cover, the passages of the ozone hole over Tierra del Fuego (55° S) caused concomitant increases in solar UV and that the enhanced ground-level UV led to significant increases in DNA damage in the native plant
Gunnera magellanica
. The fluctuations in solar UV explained a large proportion of the variation in DNA damage (up to 68%), particularly when the solar UV was weighted for biological effectiveness according to action spectra that assume a sharp decline in quantum efficiency with increasing wavelength from the UVB into the UVA regions of the spectrum.
Separation of a midlevel density current from the bottom of a continental slope
Authors: Stern M. E.
Published: July 2002

The high-salinity water flowing out of the Mediterranean Sea
descends to mid depths in the density-stratified ocean, continues as a
narrow jet along the Iberian continental slope, and intermittently
detaches large-scale eddies (called “Meddies”). This process is
important because it maintains the relatively high mean salinity of a
major water mass (the “Mediterranean Intermediate Water”) in the
North Atlantic. Our simplified model of this jet consists of a moving
layer with intermediate density ρ
2
sandwiched between
motionless layers of density ρ
1
< ρ
2
and
ρ
3
> ρ
2
. The inshore (anticyclonic)
portion of the midlevel jet (in the “ρ
2
-water”)
rests on an inclined bottom (the continental slope), whereas the
(cyclonic) offshore portion rests on the density interface of the
stagnant deep (ρ
3
) layer. An inviscid,
steady
, and finite-amplitude longwave theory is used to
show that if the cross-stream topographic slope
increases
gradually in the downstream direction, then the
“ρ
2
-jet” is deflected off the bottom slope and onto
the upper density interface of the ρ
3
layer. The computed
magnitude of this separation effect is such as to produce an
essentially free jet which is removed from the
stabilizing
influence of the continental topography. It is therefore conjectured
that time-dependent effects (baroclinic instability) will produce
further amplification, causing an eddy to detach seaward from the
branch of the jet remaining on the slope.
Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B
Authors: Jiang Bing-Hua , Aoki Masahiro , Zheng Jenny Z. , Li Jian , Vogt Peter K.
Published: July 2002

The oncogene
p3k
, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation.
p53 regulates a G
2
checkpoint through cyclin B1
Authors: Innocente Steven A. , Abrahamson John L. A. , Cogswell John P. , Lee Jonathan M.
Published: July 2002

The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G
2
, we have derived a human ovarian cell that undergoes p53-dependent G
2
arrest at 32°C. We have found that p53 prevents G
2
/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter. Cyclin B1 is the regulatory subunit of the cdc2 kinase and is a protein required for mitotic initiation. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G
2
checkpoint has a role in preventing neoplastic transformation.
Molecular and biophysical characterization of TT virus: Evidence for a new virus family infecting humans
Authors: Mushahwar Isa K. , Erker James C. , Muerhoff A. Scott , Leary Thomas P. , Simons John N. , Birkenmeyer Larry G. , Chalmers Michelle L. , Pilot-Matias Tami J. , Dexai Suresh M.
Published: July 2002

The recent isolation of a novel DNA virus from the serum of a Japanese patient (T.T.) has provided the latest possible candidate virus associated with cryptogenic hepatitis. In the present study, we report the complete nucleotide sequence of this virus (TTV) isolated from the serum of a West African. Based on PCR studies designed to amplify overlapping regions of the viral genome and sensitivity to digestion with mung bean nuclease, the viral genome is circular and negative stranded, and comprises 3,852 nt, which is 113 nt longer than the prototype isolate from Japan. Cesium chloride density gradient centrifugation demonstrated banding of the virus at 1.31–1.34 g/ml; filtration studies indicated that TTV had a particle size of 30–50 nm. These results suggest that the virus is similar to the Circoviridae, viruses known to infect plants and vertebrates (e.g., birds and swine); however, sequence similarity searches of available databases did not reveal identity between TTV and other viruses. Phylogenetic analyses of a 260-nt region from 151 globally distributed isolates demonstrated the existence of three major TTV genotypes. Several individuals at high risk for infection with parenterally transmitted viruses were infected with more than one genotype. There was no correlation between genotype and geographic origin. Finally, intravenous inoculation of TTV-positive human serum into chimpanzees demonstrated that TTV can be transmitted to primates; no biochemical or histological evidence for hepatitis was obtained. The distinct biophysical and molecular characteristics of TTV suggest that it is a member of a new family of viruses, which we have tentatively named the Circinoviridae.
Crystal structure of human type II inosine monophosphate dehydrogenase: Implications for ligand binding and drug design
Authors: Colby Thomas D. , Vanderveen Kristen , Strickler Michael D. , Markham George D. , Goldstein Barry M.
Published: July 2002

Inosine monophosphate dehydrogenase (IMPDH) controls a key metabolic step in the regulation of cell growth and differentiation. This step is the NAD-dependent oxidation of inosine 5′ monophosphate (IMP) to xanthosine 5′ monophosphate, the rate-limiting step in the synthesis of the guanine nucleotides. Two isoforms of IMPDH have been identified, one of which (type II) is significantly up- regulated in neoplastic and differentiating cells. As such, it has been identified as a major target in antitumor and immunosuppressive drug design. We present here the 2.9-Å structure of a ternary complex of the human type II isoform of IMPDH. The complex contains the substrate analogue 6-chloropurine riboside 5′-monophosphate (6-Cl-IMP) and the NAD analogue selenazole-4-carboxamide adenine dinucleotide, the selenium derivative of the active metabolite of the antitumor drug tiazofurin. The enzyme forms a homotetramer, with the dinucleotide binding at the monomer–monomer interface. The 6 chloro-substituted purine base is dehalogenated, forming a covalent adduct at C6 with Cys-331. The dinucleotide selenazole base is stacked against the 6-Cl-IMP purine ring in an orientation consistent with the B-side stereochemistry of hydride transfer seen with NAD. The adenosine end of the ligand interacts with residues not conserved between the type I and type II isoforms, suggesting strategies for the design of isoform-specific agents.
Quantization of continuous arm movements in humans with brain injury
Authors: Krebs Hermano Igo , Aisen Mindy L. , Volpe Bruce T. , Hogan Neville
Published: July 2002

Segmentation of apparently continuous movement has been reported for over a century by human movement researchers, but the existence of primitive submovements has never been proved. In 20 patients recovering from a single cerebral vascular accident (stroke), we identified the apparent submovements that composed a continuous arm motion in an unloaded task. Kinematic analysis demonstrated a submovement speed profile that was invariant across patients with different brain lesions and provided experimental verification of the detailed shape of primitive submovements. The submovement shape was unaffected by its peak speed, and to test further the invariance of shape with speed, we analyzed movement behavior in a patient with myoclonus. This patient occasionally made involuntary shock-like arm movements, which occurred near the maximum capacity of the neuromuscular system, exhibited speed profiles that were comparable to those identified in stroke patients, and were also independent of speed.
Analysis of receptor signaling pathways by mass spectrometry: Identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors
Authors: Pandey Akhilesh , Podtelejnikov Alexandre V. , Blagoev Blagoy , Bustelo Xosé R. , Mann Matthias , Lodish Harvey F.
Published: July 2002

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR
in vivo
. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.
Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene
Authors: Goto Tomoaki , Nishi Toru , Tamura Takahiko , Dev Sukhendu B. , Takeshima Hideo , Kochi Masato , Yoshizato Kimio , Kuratsu Jun-ichi , Sakata Tsuneaki , Hofmann Gunter A. , Ushio Yukitaka
Published: July 2002

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by
in vivo
electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by
in vivo
EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the “A” fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.
Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide
Authors: Boger Dale L. , Sato Haruhiko , Lerner Aaron E. , Hedrick Michael P. , Fecik Robert A. , Miyauchi Hiroshi , Wilkie Gordon D. , Austin Bryce J. , Patricelli Matthew P. , Cravatt Benjamin F.
Published: July 2002

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12and oleoyl Δ9,10location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors withKis that drop below 200 pM and are 102–103times more potent than the corresponding trifluoromethyl ketones.
Permanence or change? The meaning of genetic variation
Authors: Salzano Francisco M.
Published: July 2002

Selected aspects of the evolutionary process and more specifically of the genetic variation are considered, with an emphasis in studies performed by my group. One key aspect of evolution seems to be the concomitant occurrence of dichotomic, contradictory (dialect) processes. Genetic variation is structured, and the dynamics of change at one level is not necessarily paralleled by that in another. The pathogenesis-related protein superfamily can be cited as an example in which permanence (the maintenance of certain key genetic features) coexists with change (modifications that led to different functions in different classes of organisms). Relationships between structure and function are exemplified by studies with hemoglobin Porto Alegre. The genetic structure of tribal populations may differ in important aspects from that of industrialized societies. Evolutionary histories also may differ when considered through the investigation of patrilineal or matrilineal lineages. Global evaluations taking into consideration all of these aspects are needed if we really want to understand the meaning of genetic variation.
A modular chitin-binding protease associated with hemocytes and hemolymph in the mosquito
Anopheles gambiae
Authors: Danielli Alberto , Loukeris Thanasis G. , Lagueux Marie , Müller Hans-Michael , Richman Adam , Kafatos Fotis C.
Published: July 2002

Sp22D
, a modular serine protease encompassing chitin binding, low density lipoprotein receptor, and scavenger receptor cysteine-rich domains, was identified by molecular cloning in the malaria vector,
Anopheles gambiae
. It is expressed in multiple body parts and during much of development, most intensely in hemocytes. The protein appears to be posttranslationally modified. Its integral, putatively glycosylated form is secreted in the hemolymph, whereas a smaller form potentially generated by proteolytic processing is associated with the tissues. Bacterial challenge or wounding result in low-level RNA induction, but the protein does not bind to bacteria, nor is its processing affected by infection. However,
Sp22D
binds to chitin with high affinity and undergoes transient changes in processing during pupal to adult metamorphosis; it may respond to exposure to naked chitin during tissue remodeling or damage.
Spatial processing in the auditory cortex of the macaque monkey
Authors: Recanzone Gregg H.
Published: July 2002

The patterns of cortico-cortical and cortico-thalamic connections
of auditory cortical areas in the rhesus monkey have led to the
hypothesis that acoustic information is processed in series and in
parallel in the primate auditory cortex. Recent physiological
experiments in the behaving monkey indicate that the response
properties of neurons in different cortical areas are both functionally
distinct from each other, which is indicative of parallel processing,
and functionally similar to each other, which is indicative of serial
processing. Thus, auditory cortical processing may be similar to the
serial and parallel “what” and “where” processing by the
primate visual cortex. If “where” information is serially
processed in the primate auditory cortex, neurons in cortical areas
along this pathway should have progressively better spatial tuning
properties. This prediction is supported by recent experiments that
have shown that neurons in the caudomedial field have better spatial
tuning properties than neurons in the primary auditory cortex. Neurons
in the caudomedial field are also better than primary auditory cortex
neurons at predicting the sound localization ability across different
stimulus frequencies and bandwidths in both azimuth and elevation.
These data support the hypothesis that the primate auditory cortex
processes acoustic information in a serial and parallel manner and
suggest that this may be a general cortical mechanism for sensory
perception.
Correction of ornithine accumulation prevents retinal degeneration in a mouse model of gyrate atrophy of the choroid and retina
Authors: Wang Tao , Steel Gary , Milam Ann H. , Valle David
Published: July 2002

Deficiency of ornithine-δ-aminotransferase (OAT) in humans results in gyrate atrophy of the choroid and retina (GA), an autosomal recessive disorder characterized by ornithine accumulation and a progressive chorioretinal degeneration of unknown pathogenesis. To determine whether chronic, systemic reduction of ornithine can prevent this form of retinal degeneration, we used an arginine-restricted diet to maintain long term reduction of ornithine in a mouse model of OAT-deficiency (
Oat
−/−
) produced by gene targeting. We evaluated the mice over a 12-month period by measurement of plasma amino acids, electroretinograms, and retinal histologic and ultrastructural studies. We found that an arginine-restricted diet substantially reduces plasma ornithine levels and completely prevents retinal degeneration in
Oat
−/−
. This result indicates that ornithine accumulation is a necessary factor in the pathophysiology of the retinal degeneration in GA and that restoration of OAT activity in retina is not required for effective treatment of GA.
Impact of progesterone receptor on cell-fate decisions during mammary gland development
Authors: Shyamala G. , Yang X. , Cardiff R. D. , Dale E.
Published: July 2002

Mammary epithelium contains lineage-limited progenitors that give rise to cells that form distinct morphological structures, ducts vs. lobules, depending on the endocrine status of the female. Progesterone signaling through progesterone receptor (PR) is essential for lobulo-alveolar development that accompanies pregnancy, but not for ductal growth accompanying puberty. PR exists in two molecular forms, A and B, and an imbalance in the native ratio of the two isoforms can lead to alterations in PR signaling. Indeed, as we reported previously, in transgenic mice carrying additional A form of PR, mammary development is abnormal, characterized by excessive lateral ductal branching. This suggests that alterations in PR signaling may have important consequences to mammary development, particularly with regard to ductal vs. alveolar growth. To test this further, we created transgenic mice carrying additional B form of PR and report that mammary development in these mice is also abnormal, characterized by inappropriate alveolar growth. More importantly, these mammary glands, on serial transplantation, undergo a premature arrest in ductal growth without any alteration in the potential for lobulo-alveolar growth. Such an arrest in ductal growth does not occur with transgenics carrying additional A form of PR. These studies, therefore, provide strong evidence to indicate that PR signaling may be of paramount importance for appropriate cell-fate decisions during normal mammary development, and also that this requires a regulated expression of the two isoforms.
Elevated subsarcolemmal Ca
2+
in
mdx
mouse skeletal muscle fibers detected with Ca
2+
-activated K
+
channels
Authors: Mallouk Nora , Jacquemond Vincent , Allard Bruno
Published: July 2002

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca
2+
is increased in dystrophic muscle, leading to Ca
2+
accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca
2+
in muscle from
mdx
mice, an animal model of the human disease. In the present study, using plasma membrane Ca
2+
-activated K
+
channels as subsarcolemmal Ca
2+
probe, we tested the possibility of a Ca
2+
accumulation at the restricted subsarcolemmal level in
mdx
skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca
2+
-activated K
+
channels is significantly lower in
mdx
than in control muscle, suggesting a higher subsarcolemmal [Ca
2+
]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca
2+
-activated K
+
channel activation could correspond to a ≈3-fold increase in the subsarcolemmal Ca
2+
concentration in
mdx
muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in
mdx
skeletal muscle, leading to Ca
2+
overload at the subsarcolemmal level.
p53 associates with and targets ΔNp63 into a protein degradation pathway
Authors: Ratovitski Edward A. , Patturajan Meera , Hibi Kenji , Trink Barry , Yamaguchi Kengo , Sidransky David
Published: July 2002

A human p53 homologue, p63
(p40/p51/p73L/CUSP) that maps to the chromosomal
region 3q27–29 was found to produce a variety of transcripts that
encode DNA-binding proteins with and without a trans-activation domain
(TA- or ΔN-, respectively). The
p63
gene locus was
found to be amplified in squamous cell carcinoma, and overexpression of
ΔNp63 (p40) led to increased growth of transformed cells
in
vitro
and
in vivo
. Moreover,
p63
-null mice displayed abnormal epithelial development
and germ-line human mutations were found to cause ectodermal dysplasia.
We now demonstrate that certain p63 isotypes form complexes with p53.
p53 mutations R175H or R248W abolish the association of p53 with p63,
whereas V143A or R273H has no effect. Deletion studies suggest that the
DNA-binding domains of both p53 and p63 mediate the association.
Overexpression of wild type but not mutant (R175H) p53 results in the
caspase-dependent degradation of certain ΔNp63 proteins (p40 and
ΔNp63α). The association between p53 and ΔNp63 supports a
previously unrecognized role for p53 in regulation of ΔNp63
stability. The ability of p53 to mediate ΔNp63 degradation may
balance the capacity of ΔNp63 to accelerate tumorigenesis or to
induce epithelial proliferation.
Dispositional Optimism in Chinese People: What Does the Life Orientation Test Measure?
Authors: Cheng Sheung‐Tak , Hamid P. Nicholas
Published: July 2002

The Life Orientation Test was back‐translated to Chinese and administered to a sample of college students (N = 314) and a sample of normal adults (N = 306) along with measures of personality and symptoms. When subject to factor analysis, the positive items (representing optimism) and the negative items (representing pessimism) emerged as two separate factors. Empirically, optimism and pessimism were moderately correlated and had unique contributions to the explanation of personality and symptom measures, so that the total score of C‐LOT was a better predictor of criterion measures than either optimism or pessimism alone. However, C‐LOT's relationship with symptoms was eliminated after negative affectivity was controlled in the student sample, but not in the adult sample, thus suggesting some confounding with negative affectivity. Further research is required to improve the measurement of optimism in Chinese people and to clarify the relationship between optimism and pessimism.
Developmental Patterns of Free Recall and Frequency Judgement in Korean and American Students
Authors: Marx Melvin H. , Kim Yung Che , Henderson Bruce B.
Published: July 2002

Four experiments were conducted to compare developmental changes in free recall and frequency judgement. In Experiment 1, 1012 Korean students were shown a series of animal names and then asked to recall them and to estimate the frequency with which they had occurred. The poorest performance on both tasks was by primary-school students and the best by secondary-school students; college students were intermediate in performance. Essentially similar results were obtained in Experiment 2, with an additional 288 Korean students, except that secondary-school students did not perform better than college students. In this experiment, there was complete control of item specificity over frequency and any possible clustering effect was eliminated by using unrelated words rather than animal names. In Experiment 3, the developmental trends in frequency judgement were replicated with 193 American students. Those developmental trends were obtained with another 186 American students in Experiment 4 using relative frequency judgements. Retrospective reports about how frequency judgements were made suggested a developmental shift from more literal counting strategies to more intuitive strength impression judgements. The results are interpreted as suggesting the need for some modification of the Hasher and Zacks (1979, 1984) age-invariance proposition for frequency judgement.
Excessive alcohol use by non‐elite sportsmen
Authors: BLACK DEBORAH , LAWSON JAMES , FLEISHMAN SANDRA
Published: July 2002

This study was designed to first, provide a profile of alcohol consumption patterns of young Australian men aged 16 to 34 years who participate in non‐elite sport and secondly, to explore the factors associated with excessive alcohol consumption by this group. There were 1613 male participants in the study. The type of sport played, smoking, age, whether their sporting team had a licensed club and country of birth were associated with drinking above safe recommended levels. The excessive levels of alcohol consumption by some non‐elite sportsmen in this study suggest a need to educate sports administrators, coaches and participants about safe alcohol consumption. Men were more likely to drink excessively when socializing with sporting team mates compared to drinking on social occasions with other groups.
Alcohol, auditory functioning and deafness
Authors: CRAWFORD A.
Published: July 2002

Abstract This paper reviews studies of alcohol and auditory impairment and the treatment of deaf people with alcohol problems and concludes that: (a) alcohol affects auditory functioning, (b) prenatal exposure to alcohol is a risk factor for hearing impairment in FAS children, (c) there may be genetic factors involved in the transmission of auditory abnormalities to the children of problem drinkers and (d) there is very little research in the drinking patterns of or counselling services for deaf people in the UK.
Effects of acute and chronic ethanol exposure on fatty acid ethyl ester synthases in mouse cerebellar membranes
Authors: ZHENG ZHIHONG , HUNGUND BASALINGAPPA L.
Published: July 2002

Fatty acid ethyl esters (FAEE), the products of esterification of fatty acids with ethanol (EtOH), are shown to cause organ injury in chronic alcohol abusers. Their formation is catalysed by the enzyme FAEE synthase which is present in both animal and human brain. In the present study, we investigated the effects of acute and chronic EtOH exposure on FAEE synthase activity in crude cerebellar membrane preparation of mice, using oleic, linoleic and arachidonic acids as substrates. The results indicate that FAEE synthase activity exists in synaptosomal membranes and the activity of this enzyme varied with various fatty acid substrates. The synthase activity was optimal in the membranes from the animals exposed acutely to EtOH when oleic acid was used as a substrate. A 14% increase in the incorporation of oleic acid was observed in the membranes from animals exposed to acute dose of EtOH. However, there was a 10% reduction in the synthase activity when arachidonic acid was used as a substrate in the membrane preparations from mice exposed chronically to EtOH. The results suggest that substrate specificity for FAEE synthase varied with the duration of exposure to EtOH.
Chromosome Fragments Possessing Only One Kinetochore Can Congress to the Spindle Equator
Authors: Khodjakov Alexey , Cole Richard W. , McEwen Bruce F. , Buttle Karolyn F. , Rieder Conly L.
Published: July 2002

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.
Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity
Authors: Cáceres Javier F. , Misteli Tom , Screaton Gavin R. , Spector David L. , Krainer Adrian R.
Published: July 2002

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.
Regulation of β-Catenin Levels and Localization by Overexpression of Plakoglobin and Inhibition of the Ubiquitin-Proteasome System
Authors: Salomon Daniela , Sacco Paula A. , Roy Sujata Guha , Simcha Inbal , Johnson Keith R. , Wheelock Margaret J. , Ben-Ze'ev Avri
Published: July 2002

β-Catenin and plakoglobin (γ-catenin) are closely related molecules of the armadillo family of proteins. They are localized at the submembrane plaques of cell–cell adherens junctions where they form independent complexes with classical cadherins and α-catenin to establish the link with the actin cytoskeleton. Plakoglobin is also found in a complex with desmosomal cadherins and is involved in anchoring intermediate filaments to desmosomal plaques. In addition to their role in junctional assembly, β-catenin has been shown to play an essential role in signal transduction by the Wnt pathway that results in its translocation into the nucleus. To study the relationship between plakoglobin expression and the level of β-catenin, and the localization of these proteins in the same cell, we employed two different tumor cell lines that express N-cadherin, and α- and β-catenin, but no plakoglobin or desmosomal components. Individual clones expressing various levels of plakoglobin were established by stable transfection. Plakoglobin overexpression resulted in a dose-dependent decrease in the level of β-catenin in each clone. Induction of plakoglobin expression increased the turnover of β-catenin without affecting RNA levels, suggesting posttranslational regulation of β-catenin. In plakoglobin overexpressing cells, both β-catenin and plakoglobin were localized at cell– cell junctions. Stable transfection of mutant plakoglobin molecules showed that deletion of the N-cadherin binding domain, but not the α-catenin binding domain, abolished β-catenin downregulation. Inhibition of the ubiquitin-proteasome pathway in plakoglobin overexpressing cells blocked the decrease in β-catenin levels and resulted in accumulation of both β-catenin and plakoglobin in the nucleus. These results suggest that (a) plakoglobin substitutes effectively with β-catenin for association with N-cadherin in adherens junctions, (b) extrajunctional β-catenin is rapidly degraded by the proteasome-ubiquitin system but, (c) excess β-catenin and plakoglobin translocate into the nucleus.
Distinct Intracellular Compartments Involved in Invariant Chain Degradation and Antigenic Peptide Loading of Major Histocompatibility Complex (MHC) Class II Molecules
Authors: Ferrari Giorgio , Knight Andrew M. , Watts Colin , Pieters Jean
Published: July 2002

Major histocompatibility complex (MHC) class II molecules are transported to intracellular MHC class II compartments via a transient association with the invariant chain (Ii). After removal of the invariant chain, peptides can be loaded onto class II molecules, a process catalyzed by human leukocyte antigen-DM (HLA-DM) molecules. Here we show that MHC class II compartments consist of two physically and functionally distinct organelles. Newly synthesized MHC class II/Ii complexes were targeted to endocytic organelles lacking HLA-DM molecules, where Ii degradation occurred. From these organelles, class II molecules were transported to a distinct organelle containing HLA-DM, in which peptides were loaded onto class II molecules. This latter organelle was not directly accessible via fluid phase endocytosis, suggesting that it is not part of the endosomal pathway. Uptake via antigen-specific membrane immunoglobulin resulted however in small amounts of antigen in the HLA-DM positive organelles. From this peptide-loading compartment, class II–peptide complexes were transported to the plasma membrane, in part after transit through endocytic organelles. The existence of two separate compartments, one involved in Ii removal and the other functioning in HLA-DM–dependent peptide loading of class II molecules, may contribute to the efficiency of antigen presentation by the selective recruitment of peptide-receptive MHC class II molecules and HLA-DM to the same subcellular location.
Caveolin-1 and -2 in the Exocytic Pathway of MDCK Cells
Authors: Scheiffele P. , Verkade P. , Fra A.M. , Virta H. , Simons K. , Ikonen E.
Published: July 2002

Abstract. We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti–caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.