bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of embryonal tumor initiation: Distinct roles for MycN expression and
MYCN
amplification
Authors: Hansford Loen M. , Thomas Wayne D. , Keating Joanna M. , Burkhart Catherine A. , Peaston Anne E. , Norris Murray D. , Haber Michelle , Armati Patricia J. , Weiss William A. , Marshall Glenn M.
Published: August 2004

The mechanisms causing persistence of embryonal cells that later give rise to tumors is unknown. One tumorigenic factor in the embryonal childhood tumor neuroblastoma is the
MYCN
protooncogene. Here we show that normal mice developed neuroblast hyperplasia in paravertebral ganglia at birth that completely regressed by 2 weeks of age. In contrast, ganglia from
MYCN
transgenic (
TH-MYCN
) mice demonstrated a marked increase in neuroblast hyperplasia and MycN expression during week 1. Regression of neuroblast hyperplasia was then delayed and incomplete before neuroblastoma tumor formation at 6 and 13 weeks in homo- and hemizygote mice, respectively. Paravertebral neuronal cells cultured from perinatal
TH-MYCN
mice exhibited 3- to 10-fold resistance to nerve growth factor (NGF) withdrawal, compared with normal mice. Both low- and high-affinity NGF receptors were expressed in perinatal neuroblast hyperplasia but not in neuroblastoma tumor tissue.
MYCN
transgene amplification was present at low levels in perinatal neuroblast hyperplasia from both homo- and hemizygote
TH-MYCN
mice. However, only in hemizygous mice did tumor formation correlate with a stepwise increase in the frequency of
MYCN
amplification. These data suggest that inappropriate perinatal MycN expression in paravertebral ganglia cells from
TH-MYCN
mice initiated tumorigenesis by altering the physiologic process of neural crest cell deletion. Persisting embryonal neural crest cells underwent further changes, such as
MYCN
amplification and repression of NGF receptor expression, during tumor progression. Our studies provide a model for studying perinatal factors influencing embryonal tumor initiation.
Insulin resistance in skeletal muscles of caveolin-3-null mice
Authors: Oshikawa Jin , Otsu Koji , Toya Yoshiyuki , Tsunematsu Takashi , Hankins Raleigh , Kawabe Jun-ichi , Minamisawa Susumu , Umemura Satoshi , Hagiwara Yasuko , Ishikawa Yoshihiro
Published: August 2004

Type 2 diabetes is preceded by the development of insulin resistance, in which the action of insulin is impaired, largely in skeletal muscles. Caveolin-3 (Cav3) is a muscle-specific subtype of caveolin, an example of a scaffolding protein found within membranes. Cav is also known as growth signal inhibitor, although it was recently demonstrated that the genetic disruption of Cav3 did not augment growth in mice. We found, however, that the lack of Cav3 led to the development of insulin resistance, as exemplified by decreased glucose uptake in skeletal muscles, impaired glucose tolerance test performance, and increases in serum lipids. Such impairments were markedly augmented in the presence of streptozotocin, a pancreatic β cell toxin, suggesting that the mice were susceptible to severe diabetes in the presence of an additional risk factor. Insulin-stimulated activation of insulin receptors and downstream molecules, such as IRS-1 and Akt, was attenuated in the skeletal muscles of Cav3 null mice, but not in the liver, without affecting protein expression or subcellular localization. Genetic transfer of Cav3 by needle injection restored insulin signaling in skeletal muscles. Our findings suggest that Cav3 is an enhancer of insulin signaling in skeletal muscles but does not act as a scaffolding molecule for insulin receptors.
Fabricating complex three-dimensional nanostructures with high-resolution conformable phase masks
Authors: Jeon Seokwoo , Park Jang-Ung , Cirelli Ray , Yang Shu , Heitzman Carla E. , Braun Paul V. , Kenis Paul J. A. , Rogers John A.
Published: August 2004

High-resolution, conformable phase masks provide a means to fabricate, in an experimentally simple manner, classes of 3D nanostructures that are technologically important but difficult to generate in other ways. In this approach, light passing through a phase mask that has features of relief comparable in dimension to the wavelength generates a 3D distribution of intensity that exposes a photopolymer film throughout its thickness. Developing this polymer yields a structure in the geometry of the intensity distribution, with feature sizes as small as 50 nm. Rigorous coupled-wave analysis reveals the fundamental aspects of the optics associated with this method; a broad-range 3D nanostructures patterned with it demonstrates its technical capabilities. A nanoporous filter element built inside a microfluidic channel represents one example of the many types of functional devices that can be constructed.
High-resolution structure of a picornaviral internal cis-acting RNA replication element (
cre
)
Authors: Thiviyanathan Varatharasa , Yang Yan , Kaluarachchi Kumaralal , Rijnbrand Rene , Gorenstein David G. , Lemon Stanley M.
Published: August 2004

Picornaviruses constitute a medically important family of RNA viruses in which genome replication critically depends on a small RNA element, the cis-acting replication element (
cre
), that templates 3D
pol
polymerase-catalyzed uridylylation of the protein primer for RNA synthesis, VPg. We report the solution structure of the 33-nt
cre
of human rhinovirus 14 under solution conditions optimal for uridylylation
in vitro
. The
cre
adopts a stem-loop conformation with an extended duplex stem supporting a novel 14-nt loop that derives stability from base-stacking interactions. Base-pair interactions are absent within the loop, and base substitutions within the loop that favor such interactions are detrimental to viral RNA replication. Conserved adenosines in the 5′ loop sequence that participate in a slide-back mechanism of VPg-pUpU synthesis are oriented to the inside of the loop but are available for base templating during uridylation. The structure explains why substitutions of the 3′ loop nucleotides have little impact on conformation of the critical 5′ loop bases and accounts for wide variation in the sequences of
cre
s from different enteroviruses and rhinoviruses.
Arabidopsis
micro-RNA biogenesis through Dicer-like 1 protein functions
Authors: Kurihara Yukio , Watanabe Yuichiro
Published: August 2004

Micro-RNAs (miRNAs) are small, noncoding RNAs of 18-25 nt in length that negatively regulate their complementary mRNAs at the posttranscriptional level. Previous work has shown that some RNase III-like enzymes such as Drosha and Dicer are known to be involved in miRNA biogenesis in animals. However, the mechanism of plant miRNA biogenesis still remains poorly understood. In this article, the process of
Arabidopsis
miR163 biogenesis was examined. The results revealed that two types of miR163 primary transcripts (pri-miR163s) are transcribed from a single gene by RNA polymerase II and that miR163 biogenesis requires at least three cleavage steps by RNase III-like enzymes at 21-nt-long intervals. The first step is from pri-miR163 to long miR163 precursor (premiR163), the second step is from long pre-miR163 to short premiR163, and the last step is from short pre-miR163 to mature miR163 and the remnant. It is interesting that, during the process, four small RNAs including miR163 are released. By using
dcl1
mutants, it was demonstrated that
Arabidopsis
Dicer homologue Dicer-like 1 (DCL1) catalyzes at least the first and second cleavage steps and that double-stranded RNA-binding domains of DCL1 are involved in positioning of the cleavage sites. Our result is direct evidence that DCL1 is involved in processing of pri- and pre-miRNA.
Single-molecule recognition imaging microscopy
Authors: Stroh C. , Wang H. , Bash R. , Ashcroft B. , Nelson J. , Gruber H. , Lohr D. , Lindsay S. M. , Hinterdorfer P.
Published: August 2004

Atomic force microscopy is a powerful and widely used imaging technique that can visualize single molecules and follow processes at the single-molecule level both in air and in solution. For maximum usefulness in biological applications, atomic force microscopy needs to be able to identify specific types of molecules in an image, much as fluorescent tags do for optical microscopy. The results presented here demonstrate that the highly specific antibody–antigen interaction can be used to generate single-molecule maps of specific types of molecules in a compositionally complex sample while simultaneously carrying out high-resolution topographic imaging. Because it can identify specific components, the technique can be used to map composition over an image and to detect compositional changes occurring during a process.
Cell mechanosensitivity controls the anisotropy of focal adhesions
Authors: Nicolas Alice , Geiger Benjamin , Safran Samuel A.
Published: August 2004

Cellular adhesions are modulated by cytoskeletal forces or external stresses and adapt to the mechanical properties of the extracellular matrix. We propose that this mechanosensitivity can be driven at least in part by the elastic, cell-contractility-induced deformations of protein molecules that form the adhesion. The model accounts for observations of anisotropic growth and shrinkage of focal adhesions in the direction of the force and predicts that focal adhesions only grow within a range of force that is determined by the composition and matrix properties. This prediction is consistent with the observations of a force threshold for the appearance of elongated focal adhesions and the disruption of adhesions into fibrils on a mobile extracellular matrix. The growth dynamics is calculated and the predicted sliding of focal adhesions is consistent with several experiments.
Random-coil behavior and the dimensions of chemically unfolded proteins
Authors: Kohn Jonathan E. , Millett Ian S. , Jacob Jaby , Zagrovic Bojan , Dillon Thomas M. , Cingel Nikolina , Dothager Robin S. , Seifert Soenke , Thiyagarajan P. , Sosnick Tobin R. , Hasan M. Zahid , Pande Vijay S. , Ruczinski Ingo , Doniach Sebastian , Plaxco Kevin W.
Published: August 2004

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (
R
G
) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The
R
G
of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (
r
2
= 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble.
Y1 receptors regulate aggressive behavior by modulating serotonin pathways
Authors: Karl Tim , Lin Shu , Schwarzer Christoph , Sainsbury Amanda , Couzens Michelle , Wittmann Walter , Boey Dana , von Hörsten Stephan , Herzog Herbert
Published: August 2004

Neuropeptide Y (NPY) is pivotal in the coordinated regulation of food intake, growth, and reproduction, ensuring that procreation and growth occur only when food is abundant and allowing for energy conservation when food is scant. Although emotional and behavioral responses from the higher brain are known to be involved in all of these functions, understanding of the coordinated regulation of emotion/behavior and physiological functions is lacking. Here, we show that the NPY system plays a central role in this process because ablation of the Y1 receptor gene leads to a strong increase in territorial aggressive behavior. After exposure to the resident-intruder test, expression of c-fos mRNA in Y1-knockout mice is significantly increased in the medial amygdala, consistent with the activation of centers known to be important in regulating aggressive behavior. Expression of the serotonin [5-hydroxytryptamine (5-HT)] synthesis enzyme tryptophan hydroxylase is significantly reduced in Y1-deficient mice. Importantly, treatment with a 5-HT-1A agonist, (±)-8-hydroxy-2-(di-
n
-propylamino)tetralin hydrobromide, abolished the aggressive behavior in Y1-knockout mice. These results suggest that NPY acting through Y1 receptors regulates the 5-HT system, thereby coordinately linking physiological survival mechanisms such as food intake with enabling territorial aggressive behavior.
Reassessing random-coil statistics in unfolded proteins
Authors: Fitzkee Nicholas C. , Rose George D.
Published: August 2004

The Gaussian-distributed random coil has been the dominant model for denatured proteins since the 1950s, and it has long been interpreted to mean that proteins are featureless, statistical coils in 6 M guanidinium chloride. Here, we demonstrate that random-coil statistics are not a unique signature of featureless polymers. The random-coil model does predict the experimentally determined coil dimensions of denatured proteins successfully. Yet, other equally convincing experiments have shown that denatured proteins are biased toward specific conformations, in apparent conflict with the random-coil model. We seek to resolve this paradox by introducing a contrived counterexample in which largely native protein ensembles nevertheless exhibit random-coil characteristics. Specifically, proteins of known structure were used to generate disordered conformers by varying backbone torsion angles at random for ≈8% of the residues; the remaining ≈92% of the residues remained fixed in their native conformation. Ensembles of these disordered structures were generated for 33 proteins by using a torsion-angle Monte Carlo algorithm with hard-sphere sterics; bulk statistics were then calculated for each ensemble. Despite this extreme degree of imposed internal structure, these ensembles have end-to-end distances and mean radii of gyration that agree well with random-coil expectations in all but two cases.
Mycobacterium tuberculosis
(
Mtb
) isocitrate dehydrogenases show strong B cell response and distinguish vaccinated controls from TB patients
Authors: Banerjee Sharmistha , Nandyala Ashok , Podili Raviprasad , Katoch V. M. , Murthy K. J. R. , Hasnain Seyed E.
Published: August 2004

Proteins released from
Mycobacterium tuberculosis
(
Mtb
) during late logarithmic growth phase are often considered candidate components of immunogenic or autolysis markers. One such protein is isocitrate dehydrogenase (ICD), a key regulatory enzyme in the citric acid cycle. We have evaluated the immunogenic properties of two isoforms of
Mtb
ICD and compared them with the control antigens heat-shock protein 60 and purified protein derivative (PPD). PPD lacks the sensitivity to distinguish between bacillus Calmette–Guérin (BCG)-vaccinated and tuberculosis (TB)-infected populations, and, therefore, epidemiological relevance of PPD in BCG-vaccinated regions is debatable. We show that
Mtb
ICDs elicit a strong B cell response in TB-infected populations and can differentiate between healthy BCG-vaccinated populations and those with TB. The study population (
n
= 215) was categorized into different groups, namely, patients with fresh infection (
n
= 42), relapsed TB cases (
n
= 32), patients with extrapulmonary TB (
n
= 35), clinically healthy donors (
n
= 44), nontuberculous mycobacteria patients (
n
= 30), and non-TB patients (culture negative for acid-fast bacteria but carrying other infections,
n
= 32). The
Mtb
ICDs showed statistically significant antigenic distinction between healthy BCG-vaccinated controls and TB patients (
P
< 0.0001) and those with other infections. Although extrapulmonary infections could not be discriminated from healthy controls by heat-shock protein 60 (
P
= 0.2177), interestingly, the
Mtb
ICDs could significantly (
P
< 0.0001) do so. Our results highlight the immunodominant, immunosensitive, and immunospecific nature of
Mtb
ICDs and point to an unusual property of this tricarboxylic acid energy cycle enzyme.
Dynamic control of liquid-core/liquid-cladding optical waveguides
Authors: Wolfe Daniel B. , Conroy Richard S. , Garstecki Piotr , Mayers Brian T. , Fischbach Michael A. , Paul Kateri E. , Prentiss Mara , Whitesides George M.
Published: August 2004

This report describes the manipulation of light in waveguides that comprise a liquid core and a liquid cladding (liq/liq waveguide). These waveguides are dynamic: Their structure and function depend on a continuous, laminar flow of the core and cladding liquids. Because they are dynamic, they can be reconfigured and adapted continuously in ways that are not possible with solid-state waveguides. The liquids are introduced into the channels of a microfluidic network designed to sandwich the flowing core liquid between flowing slabs of the cladding fluid. At low and moderate Reynolds numbers, flow is laminar, and the liq/liq interfaces are optically smooth. Small irregularities in the solid walls of the channels do not propagate into these interfaces, and liq/liq waveguides therefore exhibit low optical loss because of scattering. Manipulating the rate of flow and the composition of the liquids tunes the characteristics of these optical systems.
Emissions pathways, climate change, and impacts on California
Authors: Hayhoe Katharine , Cayan Daniel , Field Christopher B. , Frumhoff Peter C. , Maurer Edwin P. , Miller Norman L. , Moser Susanne C. , Schneider Stephen H. , Cahill Kimberly Nicholas , Cleland Elsa E. , Dale Larry , Drapek Ray , Hanemann R. Michael , Kalkstein Laurence S. , Lenihan James , Lunch Claire K. , Neilson Ronald P. , Sheridan Scott C. , Verville Julia H.
Published: August 2004

The magnitude of future climate change depends substantially on the greenhouse gas emission pathways we choose. Here we explore the implications of the highest and lowest Intergovernmental Panel on Climate Change emissions pathways for climate change and associated impacts in California. Based on climate projections from two state-of-the-art climate models with low and medium sensitivity (Parallel Climate Model and Hadley Centre Climate Model, version 3, respectively), we find that annual temperature increases nearly double from the lower B1 to the higher A1fi emissions scenario before 2100. Three of four simulations also show greater increases in summer temperatures as compared with winter. Extreme heat and the associated impacts on a range of temperature-sensitive sectors are substantially greater under the higher emissions scenario, with some interscenario differences apparent before midcentury. By the end of the century under the B1 scenario, heatwaves and extreme heat in Los Angeles quadruple in frequency while heat-related mortality increases two to three times; alpine/subalpine forests are reduced by 50–75%; and Sierra snowpack is reduced 30–70%. Under A1fi, heatwaves in Los Angeles are six to eight times more frequent, with heat-related excess mortality increasing five to seven times; alpine/subalpine forests are reduced by 75–90%; and snowpack declines 73–90%, with cascading impacts on runoff and streamflow that, combined with projected modest declines in winter precipitation, could fundamentally disrupt California's water rights system. Although interscenario differences in climate impacts and costs of adaptation emerge mainly in the second half of the century, they are strongly dependent on emissions from preceding decades.
Discovery of an allosteric site in the caspases
Authors: Hardy Jeanne A. , Lam Joni , Nguyen Jack T. , O'Brien Tom , Wells James A.
Published: August 2004

Allosteric regulation of proteins by conformational change is a primary means of biological control. Traditionally it has been difficult to identify and characterize novel allosteric sites and ligands that freeze these conformational states. We present a site-directed approach using Tethering for trapping inhibitory small molecules at sites away from the active site by reversible disulfide bond formation. We screened a library of 10,000 thiol-containing compounds against accessible cysteines of two members of the caspase family of proteases, caspase-3 and -7. We discovered a previously unreported and conserved allosteric site in a deep cavity at the dimer interface 14 Å from the active site. This site contains a natural cysteine that, when disulfide-bonded with either of two specific compounds, inactivates these proteases. The allosteric site is functionally coupled to the active site, such that binding of the compounds at the allosteric site prevents peptide binding at the active site. The x-ray crystal structures of caspase-7 bound by either compound demonstrates that they inhibit caspase-7 by trapping a zymogen-like conformation. This approach may be useful to identify new allosteric sites from natural or engineered cysteines, to study allosteric transitions in proteins, and to nucleate drug discovery efforts.
The RD1 virulence locus ofMycobacterium tuberculosisregulates DNA transfer inMycobacterium smegmatis
Authors: Flint Jessica L. , Kowalski Joseph C. , Karnati Pavan K. , Derbyshire Keith M.
Published: August 2004

Conjugal DNA transfer occurs by an atypical mechanism inMycobacterium smegmatis. The transfer system is chromosomally encoded and requires recipient recombination functions for both chromosome and plasmid transfer. Cis-acting sequences have been identified that confer mobility on nontransferable plasmids, but these are larger and have different properties to canonicaloriTsites found in bacterial plasmids. To identify trans-acting factors required for mediating DNA transfer, a library of transposon insertion mutants was generated in the donor strain, and individual mutants were screened for their effect on transfer. From this screen, a collection of insertion mutants was isolated that increased conjugation frequencies relative to wild type. Remarkably, the mutations map to a 25-kb region of theM. smegmatischromosome that is syntenous with the RD1 region ofMycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strainMycobacterium bovisbacillus Calmette–Guérin. The genes of the RD1 region encode a secretory apparatus responsible for exporting Cfp10- and Esat-6, both potent antigens and virulence factors. In crosses using twoM. smegmatisdonors, we show that wild-type cells can suppress the elevated transfer phenotype of mutant donors, which is consistent with the secretion of a factor that suppresses conjugation. Most importantly, the RD1 region ofM. tuberculosiscomplements the conjugation phenotype of the RD1 mutants inM. smegmatis.Our results indicate that theM. tuberculosisandM. smegmatisRD1 regions are functionally equivalent and provide a unique perspective on the role of this critical secretion apparatus.
Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain
Authors: Nassar Mohammed A. , Stirling L. Caroline , Forlani Greta , Baker Mark D. , Matthews Elizabeth A. , Dickenson Anthony H. , Wood John N.
Published: August 2004

Nine voltage-gated sodium channels are expressed in complex patterns in mammalian nerve and muscle. Three channels, Nav1.7, Nav1.8, and Nav1.9, are expressed selectively in peripheral damage-sensing neurons. Because there are no selective blockers of these channels, we used gene ablation in mice to examine the function of Nav1.7 (PN1) in pain pathways. A global Nav1.7-null mutant was found to die shortly after birth. We therefore used the Cre-loxP system to generate nociceptor-specific knockouts. Nav1.8 is only expressed in peripheral, mainly nociceptive, sensory neurons. We knocked Cre recombinase into the Nav1.8 locus to generate heterozygous mice expressing Cre recombinase in Nav1.8-positive sensory neurons. Crossing these animals with mice where Nav1.7 exons 14 and 15 were flanked by loxP sites produced nociceptor-specific knockout mice that were viable and apparently normal. These animals showed increased mechanical and thermal pain thresholds. Remarkably, all inflammatory pain responses evoked by a range of stimuli, such as formalin, carrageenan, complete Freund's adjuvant, or nerve growth factor, were reduced or abolished. A congenital pain syndrome in humans recently has been mapped to the Nav1.7 gene,SCN9A. Dominant Nav1.7 mutations lead to edema, redness, warmth, and bilateral pain in human erythermalgia patients, confirming an important role for Nav1.7 in inflammatory pain. Nociceptor-specific gene ablation should prove useful in understanding the role of other broadly expressed genes in pain pathways.
Tissue-specific codon usage and the expression of human genes
Authors: Plotkin Joshua B. , Robins Harlan , Levine Arnold J.
Published: August 2004

A diverse array of mechanisms regulate tissue-specific protein levels. Most research, however, has focused on the role of transcriptional regulation. Here we report systematic differences in synonymous codon usage between genes selectively expressed in six adult human tissues. Furthermore, we show that the codon usage of brain-specific genes has been selectively preserved throughout the evolution of human and mouse from their common ancestor. Our findings suggest that codon-mediated translational control may play an important role in the differentiation and regulation of tissue-specific gene products in humans.
A myocardin-related transcription factor regulates activity of serum response factor in
Drosophila
Authors: Han Zhe , Li Xiumin , Wu Jiang , Olson Eric N.
Published: August 2004

Serum response factor (SRF) regulates genes involved in cell proliferation, migration, cytoskeletal organization, and myogenesis. Myocardin and myocardin-related transcription factors (MRTFs) act as powerful transcriptional coactivators of SRF in mammalian cells. We describe an MRTF from
Drosophila
, called DMRTF, which shares high homology with the functional domains of mammalian myocardin and MRTFs. DMRTF forms a ternary complex with and stimulates the activity of
Drosophila
SRF, which has been implicated in branching of the tracheal (respiratory) system and formation of wing interveins. A loss-of-function mutation introduced into the
DMRTF
locus by homologous recombination results in abnormalities in tracheal branching similar to those in embryos lacking SRF. Misexpression in wing imaginal discs of a dominant negative DMRTF mutant also causes a diminution of wing interveins, whereas overexpression of DMRTF results in excess intervein tissue, abnormalities reminiscent of SRF loss- and gain-of-function phenotypes, respectively. Overexpression of these DMRTF mutants in mesoderm and in the tracheal system also perturbs mesoderm cell migration and tracheal branching, respectively. We conclude that the interaction of MRTFs with SRF represents an ancient protein partnership involved in cytoplasmic outgrowth and cell migration during development.
De novo
protein synthesis is required for activation-induced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination
Authors: Begum Nasim A. , Kinoshita Kazuo , Muramatsu Masamichi , Nagaoka Hitoshi , Shinkura Reiko , Honjo Tasuku
Published: August 2004

Activation-induced cytidine deaminase is required for the DNA cleavage step of Ig class switch recombination (CSR). However, its molecular mechanism is controversial. RNA-editing hypothesis postulates that activation-induced cytidine deaminase deaminates cytosine in an unknown mRNA to generate a new mRNA encoding an endonuclease for CSR and thus predicts that DNA cleavage depends on
de novo
protein synthesis. On the other hand, DNA deamination hypothesis proposes that DNA cleavage is initiated by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase. By using the chromatin immunoprecipitation assay to detect γ-H2AX focus formation as a marker for DNA cleavage, we found that cycloheximide inhibited DNA cleavage in the Ig heavy-chain locus during CSR. Requirement of protein synthesis in the DNA cleavage step of CSR strengthens the RNA-editing hypothesis.
Emi1-mediated M-phase arrest in
Xenopus
eggs is distinct from cytostatic factor arrest
Authors: Ohsumi Keita , Koyanagi Ayako , Yamamoto Tomomi M. , Gotoh Tetsuya , Kishimoto Takeo
Published: August 2004

Oocytes of most vertebrates arrest at metaphase of the second meiosis (meta-II) to await fertilization, thus preventing parthenogenetic activation. This arrest is caused by a cytoplasmic activity called cytostatic factor (CSF), which was first identified in the frog
Rana pipiens
oocyte >30 years ago. CSF arrest is executed by maintaining the activity of cyclin B-Cdc2 at elevated levels largely through prevention of cyclin B destruction. Although CSF arrest is established by the Mos-mitogen-activated protein kinase pathway and is released by the Ca-calmodulin kinase II pathway, it remains unclear precisely how cyclin B destruction is regulated. Recently, an early mitotic inhibitor, Emi1, was reported to be a critical component of CSF. This report has been expected to provide a final resolution to the CSF problem because Emi1 inhibits the anaphase-promoting complex/cyclosome, a ubiquitin ligase for cyclin B destruction, through sequestration of Cdc20, an activator for the anaphase-promoting complex/cyclosome. In mitotic cycles, however, Emi1 is destroyed in every pro-metaphase, and accordingly, it is unclear why Emi1 should be required for CSF activity, which is seen only in meta-II. Here, we show that Emi1 is absent in unfertilized mature
Xenopus
eggs and that exogenous Emi1 is destroyed in meta-II and mitotic metaphase. The expression of Emi1 in oocytes hinders meiotic progression. Although both Emi1 and Mos can inhibit progression through M phase, the Emi1-mediated arrest does not require mitogen-activated protein kinase activity and is not released by Ca. Together, our results indicate that Emi1 is unlikely to be a component of CSF.
The effect of acculturation and social support on change in mental health among young immigrants
Authors: Oppedal Brit , Røysamb Espen , Sam David Lackland
Published: August 2004

The aim of the study was to examine the mediating and moderating roles of social support in the acculturation–mental health link, and to investigate how these processes combine with self-esteem to affect mental health change. Questionnaire data were collected twice from 137 immigrant students, first at the upstart in junior high school, and then again a year later (8th and 9th grade). Acculturation was described in positive terms as a developmental process towards gaining competence within more than one sociocultural setting. Perceived discrimination and ethnic identity crisis were included as risk factors in this process. A model of structural relations was tested, which after some modifications demonstrated a close fit to the data. The results supported our suggestions of two indirect paths of effects of acculturation on mental health change: one through culture domain-specific social support and another through self-esteem. Self-esteem was also identified as a mediator of identity crisis. Significant interaction effects between social support and culture competencies were demonstrated in addition to buffer effects of class and family support in relation to identity crisis and discrimination respectively. The study adds to our understanding of positive and negative developmental pathways in multicultural societies.
Development of Hibernomas in Rats Dosed with Phentolamine Mesylate During the 24-Month Carcinogenicity Study
Authors: Poulet Frederique M. , Berardi Mark R. , Halliwell William , Hartman Barbara , Auletta Carol , Bolte Henry
Published: August 2004

Phentolamine is a reversible competitive α-adrenergic antagonist with similar affinities for α1 and α2 receptors. It has a long history of safe clinical use, and was developed as a potential therapy for male erectile dysfunction because of its capacity to increase the arteriolar blood flow to the corpora cavernosa. Phentolamine mesylate was administered to rats by oral gavage at daily doses of 10, 50, and 150 mg/kg for 24 months. A dose-related increase in mortality, ascribed to an exaggerated pharmacologic effect, was seen at high doses. Systemic exposure as measured by plasma drug concentration increased with dose and duration of dosing and slight drug accumulation occurred, particularly in high-dose males. In the treated groups, 10 males and 1 female were diagnosed with hibernomas, neoplasms of brown adipose tissue, which appeared in the thoracic cavity or retroperitoneal area as circumscribed, tan to reddish-brown lobulated masses. Histologically, the masses were well circumscribed with variably sized lobules defined by a rich capillary network and consisted of closely apposed oval to polygonal cells with large amounts of cytoplasm and a centrally located nucleus. The cytoplasm's appearance varied from multivacuolated to univacuolated to granular eosinophilic. In a few cases, neoplastic emboli were observed in capsular vessels. Ultrastructurally, the neoplastic cells contained numerous mitochondria with transverse parallel cristae that occupied over 60% of the cytoplasm and lipid droplets. This study documents the previously unreported development of hibernomas in rats treated with phentolamine mesylate.
Statins Inhibit HIV-1 Infection by Down-regulating Rho Activity
Authors: del Real Gustavo , Jiménez-Baranda Sonia , Mira Emilia , Lacalle Rosa Ana , Lucas Pilar , Gómez-Moutón Concepción , Alegret Marta , Peña Jose María , Rodríguez-Zapata Manuel , Alvarez-Mon Melchor , Martínez-A. Carlos , Mañes Santos
Published: August 2004

Human immunodeficiency virus (HIV)-1 infectivity requires actin-dependent clustering of host lipid raft–associated receptors, a process that might be linked to Rho guanosine triphosphatase (GTPase) activation. Rho GTPase activity can be negatively regulated by statins, a family of drugs used to treat hypercholesterolemia in man. Statins mediate inhibition of Rho GTPases by impeding prenylation of small G proteins through blockade of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We show that statins decreased viral load and increased CD4+ cell counts in acute infection models and in chronically HIV-1–infected patients. Viral entry and exit was reduced in statin-treated cells, and inhibition was blocked by the addition of l-mevalonate or of geranylgeranylpyrophosphate, but not by cholesterol. Cell treatment with a geranylgeranyl transferase inhibitor, but not a farnesyl transferase inhibitor, specifically inhibited entry of HIV-1–pseudotyped viruses. Statins blocked Rho-A activation induced by HIV-1 binding to target cells, and expression of the dominant negative mutant RhoN19 inhibited HIV-1 envelope fusion with target cell membranes, reducing cell infection rates. We suggest that statins have direct anti–HIV-1 effects by targeting Rho.
CD24 Controls Expansion and Persistence of Autoreactive T Cells in the Central Nervous System during Experimental Autoimmune Encephalomyelitis
Authors: Bai Xue-Feng , Li Ou , Zhou Qunmin , Zhang Huiming , Joshi Pramod S. , Zheng Xincheng , Liu Yan , Wang Yin , Zheng Pan , Liu Yang
Published: August 2004

In the development of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), autoreactive T cells must be activated and clonally expand in the lymphoid organs, and then migrate into the central nervous system (CNS) where they undergo further activation. It is unclear whether the autoreactive T cells further expand in the CNS and if so, what interactions are required for this process. We have demonstrated previously that expression by the host cells of the heat-stable antigen (CD24), which was recently identified as a genetic modifier for MS, is essential for their susceptibility to EAE. Here we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on either hematopoietic cells or nonhematopoietic antigen-presenting cells in the recipient is sufficient to confer susceptibility to EAE.
Heat Shock Protein 70 Promotes Cell Survival by Inhibiting Lysosomal Membrane Permeabilization
Authors: Nylandsted Jesper , Gyrd-Hansen Mads , Danielewicz Agnieszka , Fehrenbacher Nicole , Lademann Ulrik , Høyer-Hansen Maria , Weber Ekkehard , Multhoff Gabriele , Rohde Mikkel , Jäättelä Marja
Published: August 2004

Heat shock protein 70 (Hsp70) is a potent survival protein whose depletion triggers massive caspase-independent tumor cell death. Here, we show that Hsp70 exerts its prosurvival function by inhibiting lysosomal membrane permeabilization. The cell death induced by Hsp70 depletion was preceded by the release of lysosomal enzymes into the cytosol and inhibited by pharmacological inhibitors of lysosomal cysteine proteases. Accordingly, the Hsp70-mediated protection against various death stimuli in Hsp70-expressing human tumor cells as well as in immortalized Hsp70 transgenic murine fibroblasts occurred at the level of the lysosomal permeabilization. On the contrary, Hsp70 failed to inhibit the cytochrome c–induced, apoptosome-dependent caspase activation in vitro and Fas ligand–induced, caspase-dependent apoptosis in immortalized fibroblasts. Immunoelectron microscopy revealed that endosomal and lysosomal membranes of tumor cells contained Hsp70. Permeabilization of purified endo/lysosomes by digitonin failed to release Hsp70, suggesting that it is physically associated with the membranes. Finally, Hsp70 positive lysosomes displayed increased size and resistance against chemical and physical membrane destabilization. These data identify Hsp70 as the first survival protein that functions by inhibiting the death-associated permeabilization of lysosomes.
Basophils Produce IL-4 and Accumulate in Tissues after Infection with a Th2-inducing Parasite
Authors: Min Booki , Prout Melanie , Hu-Li Jane , Zhu Jinfang , Jankovic Dragana , Morgan Ellen S. , Urban Joseph F. , Dvorak Ann M. , Finkelman Fred D. , LeGros Graham , Paul William E.
Published: August 2004

Using mice in which the eGfp gene replaced the first exon of the Il4 gene (G4 mice), we examined production of interleukin (IL)-4 during infection by the intestinal nematode Nippostrongylus brasiliensis (Nb). Nb infection induced green fluorescent protein (GFP)pos cells that were FcεRIpos, CD49bbright, c-kitneg, and Gr1neg. These cells had lobulated nuclei and granules characteristic of basophils. They were found mainly in the liver and lung, to a lesser degree in the spleen, but not in the lymph nodes. Although some liver basophils from naive mice express GFP, Nb infection enhanced GFP expression and increased the number of tissue basophils. Similar basophil GFP expression was found in infected Stat6−/− mice. Basophils did not increase in number in infected Rag2−/− mice; Rag2−/− mice reconstituted with CD4 T cells allowed significant basophil accumulation, indicating that CD4 T cells can direct both tissue migration of basophils and enhanced IL-4 production. IL-4 production was immunoglobulin independent and only partially dependent on IL-3. Thus, infection with a parasite that induces a “Th2-type response” resulted in accumulation of tissue basophils, and these cells, stimulated by a non-FcR cross-linking mechanism, are a principal source of in vivo IL-4 production.
Bone Marrow Microenvironmental Changes Underlie Reduced RAG-mediated Recombination and B Cell Generation in Aged Mice
Authors: Labrie Joseph E. , Sah Alex P. , Allman David M. , Cancro Michael P. , Gerstein Rachel M.
Published: August 2004

During aging, adaptive immunity is severely compromised, due in part to decreased production of B lymphocytes and loss of immunoglobulin (Ig) diversity. However, the molecular mechanisms that underlie age-associated diminished B cell production remain unclear. Using in vivo labeling, we find that this reduction in marrow pre–B cells reflects increased attrition during passage from the pro–B to pre–B cell pool. Analyses of reciprocal bone marrow chimeras reveal that the magnitude and production rates of pre–B cells are controlled primarily by microenvironmental factors, rather than intrinsic events. To understand changes in pro–B cells that could diminish production of pre–B cells, we evaluated rag2 expression and V(D)J recombinase activity in pro–B cells at the single cell level. The percentage of pro–B cells that express rag2 is reduced in aged mice and is correlated with both a loss of V(D)J recombinase activity in pro–B cells and reduced numbers of pre–B cells. Reciprocal bone marrow chimeras revealed that the aged microenvironment also determines rag2 expression and recombinase activity in pro–B cells. Together, these observations suggest that extrinsic factors in the bone marrow that decline with age are largely responsible for less efficient V(D)J recombination in pro–B cells and diminished progression to the pre–B cell stage.
The Ins and Outs of CCR7 in the Thymus
Authors: Witt Colleen M. , Robey Ellen A.
Published: August 2004

Although it is widely supposed that chemokines play a role in the thymus, most existing evidence is circumstantial. In this issue, two groups provide direct evidence that the chemokine receptor CCR7 is required for normal thymocyte migration (Ueno, T., F. Saito, D. Gray, S. Kuse, K. Hieshima, H. Nakano, T. Kakiuchi, M. Lipp, R. Boyd, and Y. Takahama. 2004. J. Exp. Med. 200:493–505; Misslitz, A., O. Pabst, G. Hintzen, L. Ohl, E. Kremmer, H. T. Petrie, and R. Forster. 2004. J. Exp. Med. 200:481–491). The two papers focus on distinct and opposite migration events, an early outward migration and a later inward migration. Together these papers provide a fascinating picture of the complex role of CCR7 in orchestrating thymocyte migration.
Dynamics of Ca2+-Calmodulin–dependent Inhibition of Rod Cyclic Nucleotide-gated Channels Measured by Patch-clamp Fluorometry
Authors: Trudeau Matthew C. , Zagotta William N.
Published: August 2004

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca2+ ions. Rod CNG channels are directly inhibited by Ca2+-calmodulin (Ca2+/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca2+/CaM-dependent modulation. The molecular basis for Ca2+/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca2+/CaM to a site in the NH2-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH2-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca2+/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca2+/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH2- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca2+/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH2- and COOH-terminal regions underlies Ca2+/CaM-dependent inhibition.
Schizosaccharomyces pombe RanGAP Homolog, SpRna1, Is Required for Centromeric Silencing and Chromosome Segregation
Authors: Kusano Ayumi , Yoshioka Tomoko , Nishijima Hitoshi , Nishitani Hideo , Nishimoto Takeharu
Published: August 2004

We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP. Resulting Sprna1ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation. In addition to Sprna1+ and Spksp1+, the clr4+ (histone H3-K9 methyltransferase), the S. pombe gene, SPAC25A8.01c, designated snf2SR+ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1+ (S. pombe Ran homolog), rescued a lethality of Sprna1ts. Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres. Consistently, Sprna1ts was defective in gene-silencing at the centromeres. But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1ts strains, indicating SpRna1 in general did not function for a heterochromatin formation. snf2SR+ rescued a centromeric silencing defect and Δclr4+ was synthetic lethal with Sprna1ts. Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR. Loss of SpRna1 activity, therefore, caused chromosome missegregation.
Identification of UK fashion retailer use of Web sites
Authors: Marciniak Ruth , Bruce Margaret
Published: August 2004

This exploratory study examines fashion retailer use of Web sites of retailers operating within the UK. A survey of 990 fashion retailers was undertaken, 780 of which have a registered domain name. A content analysis of their Web sites took place in order to examine: first, retailer characteristics in terms of product focus, marketing positioning, operational structure type and Web site functionality; and second, to establish whether a relationship exists between online levels of involvement and specific fashion retailer characteristics. Results indicate that a high number of those surveyed (78.7 per cent) had registered a domain name and over half of those who had registered, had a Web site that was transactional. Fashion designer retailers predominantly use their Web sites as an informational tool. Whereas transactional retailers were predominantly made up of product specialist retailers who were multiple, independent, mail order or pure play retailers. In conclusion it is product specialists who have greater levels of involvement in e‐commerce.
Job share: can this work in management?
Authors: Tiney Claire
Published: August 2004

This article looks at the retail industry's obligation to provide late opening hours, seven days a week and, in some instances, 24/7. Assesses the affects on employees’ working patterns, childcare issues and whether these obligations have a positive or negative effect on the business. Concludes that job‐share is a good way to retain experienced individuals, giving management cover throughout the year and allows the business to capitalise on the strengths and skills of two people.
Numerical study of turbulent boundary layers with heat transfer and tangential transpiration
Authors: Çuhadaroğlu Burhan
Published: August 2004

The hydrodynamic and thermal characteristics of the turbulent boundary layer developed on a porous wall with heat transfer and various angles of transpiration are analyzed numerically with the proper boundary conditions. The wall functions of the viscous and turbulent sub‐layers for velocity and temperature are modified to allow for the effect of the angle of injection and suction through the porous wall. The finite difference method based on a control volume approach is used for solving the time averaged Navier‐Stokes equations for incompressible flow in conjunction with the standard k‐ε turbulence model equations. A non‐uniform staggered grid arrangement is used. The parameters studied include the suction and injection velocity (Vw) and the angle (α) of the injection and suction. The present numerical results of the normal injection and suction are compared with a known experimental data and a good agreement is obtained. The numerical results also indicate that the characteristics of the turbulent boundary layer such as local friction coefficient and thermal boundary layer thickness are substantially influenced by the velocity and the angle of transpiration.