bluuebunny/crossref_metadata_2025 huggingface dataset:
Signaling via β1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate
in vitro
Authors: Zhu Alan Jian , Haase Ingo , Watt Fiona M.
Published: July 2002

Human epidermal stem cells express higher levels of β1 integrins and are more adhesive than keratinocytes that are destined to differentiate. To investigate whether high β1 integrin expression and adhesiveness are essential for maintaining keratinocytes in the stem cell compartment, we introduced a dominant-negative β1 integrin mutant, CD8β1, into cultured human keratinocytes, thereby interfering with β1 integrin function. Surface β1 integrin levels, adhesiveness, and mitogen-activated protein (MAP) kinase activation on fibronectin were reduced, and exit from the stem cell compartment was stimulated. Adhesiveness and proliferative potential were restored by overexpressing wild-type β1 integrin or by constitutive MAP kinase activation. Conversely, a dominant-negative MAP kinase kinase 1 mutant decreased adhesiveness and stem cell number in the absence of CD8β1. MAP kinase activation by α6β4-mediated adhesion and mitogens was normal in CD8β1 cells, and constitutive MAP kinase activation did not affect adhesion and proliferation of control keratinocytes. We conclude that β1 integrins and MAP kinase cooperate to maintain the epidermal stem cell compartment
in vitro
.
Imaging of thermal activation of actomyosin motors
Authors: Kato Hirokazu , Nishizaka Takayuki , Iga Takashi , Kinosita Kazuhiko , Ishiwata Shin’ichi
Published: July 2002

We have developed temperature-pulse microscopy in which the temperature of a microscopic sample is raised reversibly in a square-wave fashion with rise and fall times of several ms, and locally in a region of approximately 10 μm in diameter with a temperature gradient up to 2°C/μm. Temperature distribution was imaged pixel by pixel by image processing of the fluorescence intensity of rhodamine phalloidin attached to (single) actin filaments. With short pulses, actomyosin motors could be activated above physiological temperatures (higher than 60°C at the peak) before thermally induced protein damage began to occur. When a sliding actin filament was heated to 40–45°C, the sliding velocity reached 30 μm/s at 25 mM KCl and 50 μm/s at 50 mM KCl, the highest velocities reported for skeletal myosin in usual
in vitro
assay systems. Both the sliding velocity and force increased by an order of magnitude when heated from 18°C to 40–45°C. Temperature-pulse microscopy is expected to be useful for studies of biomolecules and cells requiring temporal and/or spatial thermal modulation.
Ecological inference
Authors: Schuessler Alexander A.
Published: July 2002

Ecological inference is the process of
drawing conclusions about individual-level behavior from
aggregate-level data. Recent advances involve the combination of
statistical and deterministic means to produce such inferences.
Transition state heterogeneity in GCN4 coiled coil folding studied by using multisite mutations and crosslinking
Authors: Moran Liam B. , Schneider Joel P. , Kentsis Alex , Reddy Giridher A. , Sosnick Tobin R.
Published: July 2002

We have investigated the folding behavior of dimeric and covalently crosslinked versions of the 33-residue α-helical GCN4-p1 coiled coil derived from the leucine zipper region of the transcriptional activator GCN4. The effects of multisite substitutions indicate that folding occurs along multiple routes with nucleation sites located throughout the protein. The similarity in activation energies of the different routes together with an analysis of intrinsic helical propensities indicate that minimal helix is present before a productive collision of the two chains. However, approximately one-third to one-half of the total helical structure is formed in the postcollision transition state ensemble. For the crosslinked, monomeric version, folding occurs along a single robust pathway. Here, the region nearest the crosslink, with the least helical propensity, is structured in the transition state whereas the region farthest from the tether, with the most propensity, is completely unstructured. Hence, the existence of transition state heterogeneity and the selection of folding routes critically depend on chain topology.
A role for RNA helicase A in post-transcriptional regulation of HIV type 1
Authors: Li Junzhou , Tang Hengli , Mullen Tina-Marie , Westberg Christopher , Reddy Thipparthi R. , Rose David W. , Wong-Staal Flossie
Published: July 2002

Retroviruses must bypass the tight coupling of splicing and nuclear export of mRNA in their replication cycle because unspliced genomic RNA and incompletely spliced mRNA must be exported to the cytoplasm for packaging or translation. This process is mediated by a cis-acting constitutive transport element (CTE) for simple retroviruses and by the trans-acting viral protein Rev in concert with its response element (RRE) for complex retroviruses (e.g., HIV). Recently, we identified RNA helicase A (RHA) as a potential cellular cofactor for CTE. Here, we report that RHA also plays a role in Rev/RRE-mediated gene expression and HIV replication. RHA binds weakly to HIV-1 RRE independently of Rev. Overexpression of RHA, but not of an RHA mutant lacking helicase activity, increased both Rev/RRE- and CTE-dependent gene expression and the levels of unspliced HIV mRNA. Microinjection of antibodies to RHA into nuclei dramatically inhibited both CTE- and Rev-dependent gene expression in human cells. Exogenous RHA cDNA, but not the mutant RHA, rescued this inhibition. We propose that RHA is required to release both CTE- and RRE-containing mRNA from spliceosomes before completion of splicing, thus freeing them for nuclear export.
Underdeveloped uterus and reduced estrogen responsiveness in mice with disruption of the estrogen-responsive finger protein gene, which is a direct target of estrogen receptor α
Authors: Orimo Akira , Inoue Satoshi , Minowa Osamu , Tominaga Nobuko , Tomioka Yasuhiro , Sato Miyuki , Kuno Junko , Hiroi Hisahiko , Shimizu Yoshihiko , Suzuki Masami , Noda Tetsuo , Muramatsu Masami
Published: July 2002

The biological roles of estrogen-responsive finger protein (efp)in vivowere evaluated in mice carrying a loss-of-function mutation in efp by gene-targeted mutagenesis. Although efp homozygous mice were viable and fertile in both sexes, the uterus that expressed abundant estrogen receptor α exhibited significant underdevelopment. When the ovariectomized homozygotes were subjected to 17β-estradiol treatment, they showed remarkably attenuated responses to estrogen, as exemplified by decreased interstitial water imbibition and retarded endometrial cell increase, at least, attributable to the lower ratio of G1 to S-phase progression in epithelial cells. These results suggest that efp is essential for the normal estrogen-induced cell proliferation and uterine swelling as one of the direct targets of estrogen receptor α.
Lentiviral delivery of HIV-1 Vpr protein induces apoptosis in transformed cells
Authors: Stewart Sheila A. , Poon Betty , Jowett Jeremy B. M. , Xie Yiming , Chen Irvin S. Y.
Published: July 2002

Most current anticancer therapies act by inducing tumor cell stasis followed by apoptosis. HIV-1 Vpr effectively induces apoptosis of T cells after arrest of cells at a G
2
/M checkpoint. Here, we investigated whether this property of Vpr could be exploited for use as a potential anticancer agent. As a potentially safer alternative to transfer of genes encoding Vpr, we developed a method to efficiently introduce Vpr protein directly into cells. Vpr packaged into HIV-1 virions lacking a genome induced efficient cell cycle arrest and apoptosis. Introduction of Vpr into tumor cell lines of various tissue origin, including those bearing predisposing mutations in p53, XPA, and hMLH1, induced cell cycle arrest and apoptosis with high efficiency. Significantly, apoptosis mediated by virion-associated Vpr was more effective on rapidly dividing cells compared with slow-growing cells, thus, in concept, providing a potential differential effect between some types of tumor cells and surrounding normal cells. This model system provides a rationale and proof of concept for the development of potential cancer therapeutic agents based on the growth-arresting and apoptotic properties of Vpr.
Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity
Authors: Carmen Andrew A. , Griffin Patrick R. , Calaycay Jimmy R. , Rundlett Stephen E. , Suka Yuko , Grunstein Michael
Published: July 2002

Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and
Escherichia coli
, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity
in vitro
for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.
LFG
: An anti-apoptotic gene that provides protection from Fas-mediated cell death
Authors: Somia Nikunj V. , Schmitt Mark J. , Vetter Douglas E. , Van Antwerp Daniel , Heinemann Stephen F. , Verma Inder M.
Published: July 2002

Programmed cell death regulates a number of biological phenomena, and the apoptotic signal must itself be tightly controlled to avoid inappropriate cell death. We established a genetic screen to search for molecules that inhibit the apoptotic signal from the Fas receptor. Here we report the isolation of a gene,
LFG,
that protects cells uniquely from Fas but not from the mechanistically related tumor necrosis factor α death signal. LFG is widely distributed, but remarkably is highly expressed in the hippocampus. LFG can bind to the Fas receptor, but does not regulate Fas expression or interfere with binding of an agonist antibody. Furthermore LFG does not inhibit binding of FADD to Fas.
A mechanism of paraquat toxicity involving nitric oxide synthase
Authors: Day Brian J. , Patel Manisha , Calavetta Lisa , Chang Ling-Yi , Stamler Jonathan S.
Published: July 2002

Paraquat (PQ) is a well described pneumotoxicant that produces toxicity by redox cycling with cellular diaphorases, thereby elevating intracellular levels of superoxide (O
2⨪
). NO synthase (NOS) has been shown to participate in PQ-induced lung injury. Current theory holds that NO reacts with O
2⨪
generated by PQ to produce the toxin peroxynitrite. We asked whether NOS might alternatively function as a PQ diaphorase and reexamined the question of whether NO/O
2⨪
reactions were toxic or protective. Here, we show that: (
i
) neuronal NOS has PQ diaphorase activity that inversely correlates with NO formation; (
ii
) PQ-induced endothelial cell toxicity is attenuated by inhibitors of NOS that prevent NADPH oxidation, but is not attenuated by those that do not; (
iii
) PQ inhibits endothelium-derived, but not NO-induced, relaxations of aortic rings; and (
iv
) PQ-induced cytotoxicity is potentiated in cytokine-activated macrophages in a manner that correlates with its ability to block NO formation. These data indicate that NOS is a PQ diaphorase and that toxicity of such redox-active compounds involves a loss of NO-related activity.
A physical basis for protein secondary structure
Authors: Srinivasan Rajgopal , Rose George D.
Published: July 2002

A physical theory of protein secondary structure is proposed and tested by performing exceedingly simple Monte Carlo simulations. In essence, secondary structure propensities are predominantly a consequence of two competing local effects, one favoring hydrogen bond formation in helices and turns, the other opposing the attendant reduction in sidechain conformational entropy on helix and turn formation. These sequence specific biases are densely dispersed throughout the unfolded polypeptide chain, where they serve to preorganize the folding process and largely, but imperfectly, anticipate the native secondary structure.
Network formation of lipid membranes: Triggering structural transitions by chain melting
Authors: Schneider Matthias F. , Marsh Derek , Jahn Wolfgang , Kloesgen Beate , Heimburg Thomas
Published: July 2002

Phospholipids when dispersed in excess water generally form vesicular membrane structures. Cryo-transmission and freeze-fracture electron microscopy are combined here with calorimetry and viscometry to demonstrate the reversible conversion of phosphatidylglycerol aqueous vesicle suspensions to a three-dimensional structure that consists of extended bilayer networks. Thermodynamic analysis indicates that the structural transitions arise from two effects: (
i
) the enhanced membrane elasticity accompanying the lipid state fluctuations on chain melting and (
ii
) solvent-associated interactions (including electrostatics) that favor a change in membrane curvature. The material properties of the hydrogels and their reversible formation offer the possibility of future applications, for example in drug delivery, the design of structural switches, or for understanding vesicle fusion or fission processes.
Pressure-induced protein-folding/unfolding kinetics
Authors: Hillson Nathan , Onuchic José Nelson , García Angel E.
Published: July 2002

We use an off-lattice minimalist model to describe the effects of pressure in slowing down the folding/unfolding kinetics of proteins when subjected to increasingly larger pressures. The potential energy function used to describe the interactions between beads in the model includes the effects of pressure on the pairwise interaction of hydrophobic groups in water. We show that pressure affects the participation of contacts in the transition state. More significantly, pressure exponentially decreases the chain reconfigurational diffusion coefficient. These results are consistent with experimental results on the kinetics of pressure-denaturation of staphylococcal nuclease.
Splicing is required for rapid and efficient mRNA export in metazoans
Authors: Luo Ming-juan , Reed Robin
Published: July 2002

Pre-mRNA splicing is among the last known nuclear events before export of mature mRNA to the cytoplasm. At present, it is not known whether splicing and mRNA export are biochemically coupled processes. In this study, we have injected pre-mRNAs containing a single intron or the same mRNAs lacking an intron (Δi-mRNAs) intoXenopusoocyte nuclei. We find that the spliced mRNAs are exported much more rapidly and efficiently than the identical Δi-mRNAs. Moreover, competition studies using excess Δi-mRNA indicate that different factor(s) are involved in the inefficient export of Δi-mRNA vs. the efficient export of spliced mRNA. Consistent with this conclusion, spliced mRNA and Δi-mRNA, though identical in sequence, are assembled into different messenger ribonucleoprotein particles (mRNP)in vitro. Strikingly, the mRNA in the spliced mRNP, but not in the Δi-mRNP, is exported rapidly and efficiently. We conclude that splicing generates a specific nucleoprotein complex that targets mRNA for export. Our results, revealing a link between splicing and efficient mRNA export, may explain the reports that an intron is required for efficient expression of many protein-coding genes in metazoans.
The mating-type and pathogenicity locus of the fungus
Ustilago hordei
spans a 500-kb region
Authors: Lee Nancy , Bakkeren Guus , Wong Katherine , Sherwood John E. , Kronstad James W.
Published: July 2002

The fungal pathogen
Ustilago hordei
causes the covered smut disease of barley and oats. Mating and pathogenicity in this fungus are controlled by the
MAT
locus, which contains two distinct gene complexes,
a
and
b
. In this study, we tagged the
a
and
b
regions with the recognition sequence for the restriction enzyme I-
Sce
I and determined that the distance between the complexes is 500 kb in a
MAT-1
strain and 430 kb in a
MAT-2
strain. Characterization of the organization of the known genes within the
a
and
b
gene complexes provided evidence for nonhomology and sequence inversion between
MAT-1
and
MAT-2
. Antibiotic-resistance markers also were used to tag the
a
gene complex in
MAT-1
strains (phleomycin) and the
b
gene complex in
MAT-2
strains (hygromycin). Crosses were performed with these strains and progeny resistant to both antibiotics were recovered at a very low frequency, suggesting that recombination is suppressed within the
MAT
region. Overall, the chromosome homologues carrying the
MAT
locus of
U. hordei
share features with primitive sex chromosomes, with the added twist that the
MAT
locus also controls pathogenicity.
Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy
Authors: Rotsch Christian , Jacobson Ken , Radmacher Manfred
Published: July 2002

The atomic force microscope (AFM) was employed to investigate the extension and retraction dynamics of protruding and stable edges of motile 3T3 fibroblasts in culture. Such dynamics closely paralleled the results of earlier studies employing video microscopy that indicated that the AFM force-mapping technique does not appreciably perturb these dynamics. Force scans permitted height determinations of active and stable edges. Whereas the profiles of active edges are flat with average heights of 0.4–0.8 μm, stable edges smoothly ascend to 2–3 μm within about 6 μm of the edge. In the region of the leading edge, the height fluctuates up to 50% (SD) of the mean value, much more than the stable edge; this fluctuation presumably reflects differences in underlying cytoskeletal activity. In addition, force mapping yields an estimate of the local Young’s modulus or modulus of elasticity (E, the cortical stiffness). This stiffness will be related to “cortical tension,” can be accurately calculated for the stable edges, and is ≈12 kPa in this case. The thinness of the leading edge precludes accurate estimation of the E values, but within 4 μm of the margin it is considerably smaller than that for stable edges, which have an upper limit of 3–5 kPa. Although blebbing cannot absolutely be ruled out as a mechanism of extension, the data are consistent with an actin polymerization and/or myosin motor mechanism in which the average material properties of the extending margin would be nearly constant to the edge. Because the leading edge is softer than the stable edge, these data also are consistent with the notion that extension preferentially occurs in regions of lower cortical tension.
Two differently regulated nuclear factor κB activation pathways triggered by the cytoplasmic tail of CD40
Authors: Tsukamoto Nobuo , Kobayashi Norihiko , Azuma Sakura , Yamamoto Tadashi , Inoue Jun-ichiro
Published: July 2002

CD40 signaling modulates the immune response at least in part by activation of nuclear factor κB (NFκB). It has been shown that two distinct domains in the CD40 cytoplasmic tail (cyt), namely cyt-N and cyt-C, independently activate NFκB. Although four members of the tumor necrosis factor receptor-associated factor (TRAF) family, including TRAF2, TRAF3, TRAF5, and TRAF6, bind to the CD40 cyt, how each TRAF protein contributes to the NFκB activation by CD40 is not clear. Here we report that TRAF2, TRAF3, and TRAF5 bind cyt-C, whereas TRAF6 binds cyt-N. cyt-N is conserved poorly between human and mouse CD40, while cyt-C is highly conserved. However, single aa substitution of Glu-235 in cyt-N of human CD40 with Ala abolishes the binding of TRAF6 to cyt-N and NFκB activation by cyt-N. Conservation of this Glu between mouse and human CD40 strongly suggests that TRAF6 could link cyt-N to signals essential for CD40-mediated immune response. Furthermore, NFκB activation by cyt-C is inhibited by a kinase-negative form of NFκB-inducing kinase more efficiently than that by cyt-N, consistent with the result that NFκB activation by TRAF2 and TRAF5 is inhibited by a kinase-negative form of NFκB-inducing kinase more efficiently than that by TRAF6. These results indicate that NFκB activating signals emanating from cyt-N and cyt-C are mediated by the different members of the TRAF family and could be regulated in a distinct manner.
The role of immunophilins in mutant superoxide dismutase-1linked familial amyotrophic lateral sclerosis
Authors: Lee Jean-pyo , Palfrey H. Clive , Bindokas Vytautas P. , Ghadge Ghanashyam D. , Ma Li , Miller Richard J. , Roos Raymond P.
Published: July 2002

It has been reported that expression of familial amyotrophic lateral sclerosis (FALS)-associated mutant Cu/Zn superoxide dismutase-1 (SOD) induces apoptosis of neuronal cells in culture associated with an increase in reactive oxygen species. SOD recently has been shown to prevent calcineurin inactivation, initiating the present investigations examining the role of calcineurin in mutant SOD-induced cell death. Wild-type or mutant SOD was expressed in neuronal cells by infection with replication-deficient adenoviruses. PC12 cells overexpressing human wild-type SOD exhibited higher calcineurin activity than cells expressing FALS-related mutant SOD (SODV148G); however, cells expressing SODV148G had calcineurin activity equal to mock-infected cells, suggesting that cell death induced by mutant SOD was not related to a decrease in calcineurin activity. Calcineurin antagonists such as cyclosporin A and FK506, as well as nonimmunosuppressant analogs of cyclosporin A, significantly enhanced SODV148G- and SODA4V-induced cell death. Because both groups of drugs inhibit the rotamase activity of cyclophilins (CyP), but only the immunosuppressant analogs inhibit calcineurin activity, these data suggest that rotamase inhibition underlies the enhanced cell death after SODV148G expression. The importance of rotamase activity in mutant SOD-mediated apoptosis was supported by experiments showing that overexpressed wild-type cyclophilin A (CyPA), but not CyPA with a rotamase active site point mutation, protected cells from death after SODV148G expression. These data suggest that mutant SOD produces a greater need for rotamase and, also, highlights possible new therapeutic strategies in FALS.
Energy transduction in the sodium F-ATPase of
Propionigenium modestum
Authors: Dimroth Peter , Wang Hongyun , Grabe Michael , Oster George
Published: July 2002

The F-ATPase of the bacterium
Propionigenium modestum
is driven by an electrochemical sodium gradient between the cell interior and its environment. Here we present a mechanochemical model for the transduction of transmembrane sodium-motive force into rotary torque. The same mechanism is likely to operate in other F-ATPases, including the proton-driven F-ATPases of
Escherichia coli
.
Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins
Authors: Schwille Petra , Kummer Susanne , Heikal Ahmed A. , Moerner W. E. , Webb Watt W.
Published: July 2002

Fast excitation-driven fluctuations in the fluorescence emission of yellow-shifted green fluorescent protein mutants T203Y and T203F, with S65G/S72A, are discovered in the 10
−6
–10
−3
-s time range, by using fluorescence correlation spectroscopy at 10
−8
M. This intensity-dependent flickering is conspicuous at high pH, with rate constants independent of pH and viscosity with a minor temperature effect. The mean flicker rate increases linearly with excitation intensity for at least three decades, but the mean dark fraction of the molecules undergoing these dynamics is independent of illumination intensity over ≈6 × 10
2
to 5 × 10
6
W/cm
2
. These results suggest that optical excitation establishes an equilibration between two molecular states of different spectroscopic properties that are coupled only via the excited state as a gateway. This reversible excitation-driven transition has a quantum efficiency of ≈10
−3
. Dynamics of external protonation, reversibly quenching the fluorescence, are also observed at low pH in the 10- to 100-μs time range. The independence of these two bright–dark flicker processes implies the existence of at least two separate dark states of these green fluorescent protein mutants. Time-resolved fluorescence measurements reveal a single exponential decay of the excited state population with 3.8-ns lifetime, after 500-nm excitation, that is pH independent. Our fluorescence correlation spectroscopy results are discussed in terms of recent theoretical studies that invoke isomerization of the chromophore as a nonradiative channel of the excited state relaxation.
The IgG Fc receptor, FcγRIIB, is a target for deregulation by chromosomal translocation in malignant lymphoma
Authors: Callanan Mary B. , Le Baccon Patricia , Mossuz Pascal , Duley Samuel , Bastard Christian , Hamoudi Rifat , Dyer Martin J. , Klobeck Gustav , Rimokh Ruth , Sotto Jean Jacques , Leroux Dominique
Published: July 2002

Rearrangement of chromosomal bands 1q21–23 is one of the most frequent chromosomal aberrations observed in hematological malignancy. The genes affected by these rearrangements remain poorly characterized. Typically, 1q21–23 rearrangements arise during tumor evolution and accompany disease-specific chromosomal rearrangements such as t(14;18) (BCL2) and t(8;14) (MYC), where they are thus thought to play an important role in tumor progression. The pathogenetic basis of this 1q21–23-associated disease progression is currently unknown. In this setting, we surveyed our series of follicular lymphoma for evidence of recurring 1q21–23 breaks and identified three cases in which a t(14;18)(q32;q21) was accompanied by a novel balanced t(1;22)(q22;q11). Molecular cloning of the t(1;22) in a cell line (B593) derived from one of these cases and detailed fluorescentin situhybridization mapping in the two remaining cases identified theFCGR2Bgene, which encodes the immunoreceptor tyrosine-based inhibition motif-bearing IgG Fc receptor, FcγRIIB, as the target gene of the t(1;22)(q22;q11). We demonstrate deregulation ofFCGR2Bleading to hyperexpression of FcγRIIb2 as the principal consequence of the t(1;22). This is evidence that IgG Fc receptors can be targets for deregulation through chromosomal translocation in lymphoma. It suggests that dysregulation ofFCGR2Bmay play a role in tumor progression in follicular lymphoma.
Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets
Authors: Bieschke J. , Giese A. , Schulz-Schaeffer W. , Zerr I. , Poser S. , Eigen M. , Kretzschmar H.
Published: July 2002

A definite diagnosis of prion diseases such as Creutzfeldt–Jakob disease (CJD) relies on the detection of pathological prion protein (PrP
Sc
). However, no test for PrP
Sc
in cerebrospinal fluid (CSF) has been available thus far. Based on a setup for confocal dual-color fluorescence correlation spectroscopy, a technique suitable for single molecule detection, we developed a highly sensitive detection method for PrP
Sc
. Pathological prion protein aggregates were labeled by specific antibody probes tagged with fluorescent dyes, resulting in intensely fluorescent targets, which were measured by dual-color fluorescence intensity distribution analysis in a confocal scanning setup. In a diagnostic model system, PrP
Sc
aggregates were detected down to a concentration of 2 pM PrP
Sc
, corresponding to an aggregate concentration of approximately 2 fM, which was more than one order of magnitude more sensitive than Western blot analysis. A PrP
Sc
-specific signal could also be detected in a number of CSF samples from patients with CJD but not in control samples, providing the basis for a rapid and specific test for CJD and other prion diseases. Furthermore, this method could be adapted to the sensitive detection of other disease-associated amyloid aggregates such as in Alzheimer's disease.
Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals
Authors: Artsimovitch Irina , Landick Robert
Published: July 2002

Transcript elongation by RNA polymerase is discontinuous and interrupted by pauses that play key regulatory roles. We show here that two different classes of pause signals punctuate elongation. Class I pauses, discovered in enteric bacteria, depend on interaction of a nascent RNA structure with RNA polymerase to displace the 3′ OH away from the catalytic center. Class II pauses, which may predominate in eukaryotes, cause RNA polymerase to slide backwards along DNA and RNA and to occlude the active site with nascent RNA. These pauses differ in their responses to antisense oligonucleotides, pyrophosphate, GreA, and general elongation factors NusA and NusG. In contrast, substitutions in RNA polymerase that increase or decrease the rate of RNA synthesis affect both pause classes similarly. We propose that both pause classes, as well as arrest and termination, arise from a common intermediate that itself binds NTP substrate weakly.
The Flory isolated-pair hypothesis is not valid for polypeptide chains: Implications for protein folding
Authors: Pappu Rohit V. , Srinivasan Rajgopal , Rose George D.
Published: July 2002

Using an all-atom representation, we exhaustively enumerate all
sterically allowed conformations for short polyalanyl chains. Only
intrachain interactions are considered, including one adjustable
parameter, a favorable backbone energy (e.g., a peptide hydrogen bond).
The counting is used to reevaluate Flory's isolated-pair hypothesis,
the simplifying assumption that each φ,ψ pair is sterically
independent. This hypothesis is a conceptual linchpin in helix–coil
theories and protein folding. Contrary to the hypothesis, we find that
systematic local steric effects can extend beyond nearest-chain
neighbors and can restrict the size of accessible conformational space
significantly. As a result, the entropy price that must be paid to
adopt any specific conformation is far less than previously thought.
Charge conductivity in peptides: Dynamic simulations of a bifunctional model supporting experimental data
Authors: Schlag E. W. , Sheu Sheh-Yi , Yang Dah-Yen , Selzle H. L. , Lin S. H.
Published: July 2002

Our previous finding and the given mechanism of charge and electron transfer in polypeptides are here integrated in a bifunctional model involving electronic charge transfer coupled to special internal rotations. Present molecular dynamics simulations that describe these motions in the chain result in the mean first passage times for the hopping process of an individual step. This “rest and fire” mechanism is formulated in detail—i.e., individual amino acids are weakly coupled and must first undergo alignment to reach the special strong coupling. This bifunctional model contains the essential features demanded by our prior experiments. The molecular dynamics results yield a mean first passage time distribution peaked at about 140 fs, in close agreement with our direct femtosecond measurements. In logic gate language this is a strongly conducting ON state resulting from small firing energies, the system otherwise being a quiescent OFF state. The observed time scale of about 200 fs provides confirmation of our simulations of transport, a model of extreme transduction efficiency. It explains the high efficiency of charge transport observed in polypeptides. We contend that the moderate speed of weak coupling is required in our model by the bifunctionality of peptides. This bifunctional mechanism agrees with our data and contains valuable features for a general model of long-range conductivity, final reactivity, and binding at a long distance.
Structure of
Escherichia coli
ribosomal protein L25 complexed with a 5S rRNA fragment at 1.8-Å resolution
Authors: Lu Min , Steitz Thomas A.
Published: July 2002

The crystal structure of
Escherichia coli
ribosomal protein L25 bound to an 18-base pair portion of 5S ribosomal RNA, which contains “loop E,” has been determined at 1.8-Å resolution. The protein primarily recognizes a unique RNA shape, although five side chains make direct or water-mediated interactions with bases. Three β-strands lie in the widened minor groove of loop E formed by noncanonical base pairs and cross-strand purine stacks, and an α-helix interacts in an adjacent widened major groove. The structure of loop E is largely the same as that of uncomplexed RNA (rms deviation of 0.4 Å for 11 base pairs), and 3 Mg
2+
ions that stabilize the noncanonical base pairs lie in the same or similar locations in both structures. Perhaps surprisingly, those residues interacting with the RNA backbone are the most conserved among known L25 sequences, whereas those interacting with the bases are not.
Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes
Authors: Lange B. Markus , Wildung Mark R. , Stauber Einar J. , Sanchez Christopher , Pouchnik Derek , Croteau Rodney
Published: July 2002

Functional genomics approaches, which use combined computational and expression-based analyses of large amounts of sequence information, are emerging as powerful tools to accelerate the comprehensive understanding of cellular metabolism in specialized tissues and whole organisms. As part of an ongoing effort to identify genes of essential oil (monoterpene) biosynthesis, we have obtained sequence information from 1,316 randomly selected cDNA clones, or expressed sequence tags (ESTs), from a peppermint (
Mentha
x
piperita
) oil gland secretory cell cDNA library. After bioinformatic selection, candidate genes putatively involved in essential oil biosynthesis and secretion have been subcloned into suitable expression vectors for functional evaluation in
Escherichia coli
. On the basis of published and preliminary data on the functional properties of these clones, it is estimated that the ESTs involved in essential oil metabolism represent about 25% of the described sequences. An additional 7% of the recognized genes code for proteins involved in transport processes, and a subset of these is likely involved in the secretion of essential oil terpenes from the site of synthesis to the storage cavity of the oil glands. The integrated approaches reported here represent an essential step toward the development of a metabolic map of oil glands and provide a valuable resource for defining molecular targets for the genetic engineering of essential oil formation.
Lsh, an SNF2/helicase family member, is required for proliferation of mature T lymphocytes
Authors: Geiman T. M. , Muegge K.
Published: July 2002

Lsh (Hells) is closely related to SNF2/helicase family members that remodel chromatin and thus regulate gene transcription. In the adult mouse Lsh is expressed primarily in lymphoid tissue, showing the highest level in thymocytes. Lsh gene expression can be induced in thymic pro-T cells by pre-T cell receptor crosslinking and in mature T cells by T cell receptor crosslinking together with costimulation via CD28. The time course ofLshgene and protein expression correlated closely with the onset of S phase of the cell cycle. To explore the function of Lsh during lymphoid development or activation, we deleted theLshgene by homologous recombination in ES cells. Fetal liver cells from Lsh−/− were used as a source of hematopoietic precursors to reconstitute lymphoid development in Rag2−/− mice. Lsh−/− (compared to Lsh+/+ or ±) chimeras showed a modest reduction in thymocyte numbers due to a partial arrest at the transition from the CD4−CD8−stage to the CD4+CD8+stage of T cell development. Mature peripheral lymphocytes were reduced in number to ≈60% for T cells and 40% for B cells; however, V(D)J recombination of the immune receptor genes was normal. Although polyclonal activation of Lsh−/− T cells induced normal levels of cytokines, cell proliferation was severely suppressed and cells underwent apoptosis. Several genes involved in the regulation of apoptosis were expressed normally with the exception of Bcl-2 that was actually elevated. These findings demonstrate that Lsh is not obligatory for normal lymphoid development but is essential for normal proliferation of peripheral T lymphocytes.
Embryonic expression of the tumor-associated PAX3-FKHR fusion protein interferes with the developmental functions of Pax3
Authors: Anderson Michael J. , Shelton G. Diane , Cavenee Webster K. , Arden Karen C.
Published: July 2002

A unique chromosomal translocation involving the genes
PAX3
and
FKHR
is characteristic of most
human alveolar rhabdomyosarcomas. The resultant chimeric protein fuses
the PAX3 DNA-binding domains to the transactivation domain of FKHR,
suggesting that PAX3-FKHR exerts its role in alveolar rhabdomyosarcomas
through dysregulation of PAX3-specific target genes. Here, we have
produced transgenic mice in which PAX3-FKHR expression was driven by
mouse
Pax3
promoter/enhancer sequences. Five
independent lines expressed PAX3-FKHR in the dorsal neural tube and
lateral dermomyotome. Each line exhibited phenotypes that correlated
with PAX3-FKHR expression levels and predominantly involved pigmentary
disturbances of the abdomen, hindpaws, and tail, with additional
neurological related alterations. Phenotypic severity could be
increased by reducing Pax3 levels through matings with
Pax3
-defective
Splotch
mice, and
interference between PAX3 and PAX3-FKHR was apparent in transcription
reporter assays. These data suggest that the tumor-associated PAX3-FKHR
fusion protein interferes with normal Pax3 developmental functions as a
prelude to transformation.
Differences in antigen recognition and cytolytic activity of CD8+and CD8−T cells that express the same antigen-specific receptor
Authors: Cho Bryan K. , Lian Kuo-Chiang , Lee Peter , Brunmark Anders , McKinley Carol , Chen Jianzhu , Kranz David M. , Eisen Herman N.
Published: July 2002

CD8+and CD8−T cell lines expressing the same antigen-specific receptor [the 2C T cell receptor (TCR)] were compared for ability to bind soluble peptide-MHC and to lyse target cells. The 2C TCR on CD8−cells bound a syngeneic MHC (Kb+)-peptide complex 10–100 times less well than the same TCR on CD8+cells, and the CD8−2C cells lysed target cells presenting this complex very poorly. Surprisingly, however, the CD8−cells differed little from CD8+cells in ability to bind an allogeneic MHC (Ld+)-peptide complex and to lyse target cells presenting this complex. The CD8+/CD8−difference provided an opportunity to estimate how long TCR engagements with peptide-MHC have to persist to initiate the cytolytic T cell response.
La compréhension d'un texte procédural: Un processus à profondeur variable
Authors: Köhler Corinna , Kékenbosch Christiane , Verstiggel Jean Claude
Published: July 2002

La recherche porte sur la compréhension d'une posologie selon le but assigné avant la lecture. Les résultats des deux expériences réalisées montrent que seules les activités cognitives liées à l'exécution des instructions (verus compréhension/mémorisation) requièrent la traduction de la représentation déclarative en une représentation de nature procédurale. Les inférences produites témoignent d'une relation résultat/déroulement et d'une organisation hiérarchique de la planification des actions à mettre en oeuvre. Par ailleurs, les informations qui participent directement à la satisfaction du but reçoivent un traitement plus approfondi que les autres.
Land Readjustment, Urban Planning and Urban Sprawl in the Tokyo Metropolitan Area
Authors: Sorensen Andre
Published: July 2002

This paper examines the role of land readjustment (LR) projects in suburban planning and land development in a case-study area in the Tokyo Metropolitan Area. LR projects have been the most important planning tool in Japan, yet the results of their use to develop over 30 per cent of Japanese urban areas have been little examined. The paper challenges the conventional wisdom that LR projects are an effective means of preventing urban sprawl. Through interviews with local planners and participants in projects, and a detailed GIS mapping of land-use change and infrastructure development in the case-study area, the research presents an in-depth study of the use of LR for suburban land development, and a close look at the patterns of urbanisation on the fringe of Japan's most dynamic metropolitan region. It appears that while LR projects do clearly prevent sprawl within the project areas themselves, they tend to exacerbate problems of sprawl at the scale of the city and region.
Sex Positive: Feminism, Queer Theory, and the Politics of Transgression
Authors: Glick Elisa
Published: July 2002

From the feminist ‘sex wars’ of the 1980s to the queer theory and politics of the 1990s, debates about the politics of sexuality have been at the forefront of contemporary theoretical, social, and political demands. This article seeks to intervene in these debates by challenging the terms through which they have been defined. Investigating the importance of ‘sex positivity’ and transgression as conceptual features of feminist and queer discourses, this essay calls for a new focus on the political and material effects of pro-sexuality.
Feminism and the Third Way
Authors: McRobbie Angela
Published: July 2002

This article argues that the Third Way’, as the ideological rationale for the New Labour Government in the UK, attempts to resolve the tensions around women and social policy confronted by the present Government. The Third Way addresses ‘women’ without ‘feminism’, in particular those floating women voters for whom feminism holds little attraction. But affluent, middle England, corporate women, though central to the popular imagination of the Daily Mail, and thus to Tony Blair, are in practice a tiny minority. New Labour in office thus finds itself committed to reconciling the irreconcilable. It wants to see women as a social group move more fully into employment, and on this many feminists would agree. At the same time it wants to see through further transformations of the welfare state, along the lines set in motion by Mrs Thatcher. Inevitably this involves further cuts in spending and privatization of social insurance. The former principle is made more difficult by the latter policy. Recent feminist analysis indicates the scale of the needs of women to allow full and equal participation in work and in society.
‘But Most of all mi Love me Browning’: The Emergence in Eighteenth and Nineteenth-Century Jamaica of the Mulatto Woman as the Desired
Authors: Mohammed Patricia
Published: July 2002

One of the most common threads in the Caribbean tapestry races which have populated the region over the last five centuries largely through forced or voluntary migration, is that there have emerged mixtures of the different racial groups. A large proportion of Caribbean women and men are referred to euphemistically as ‘mixed race’. The terms used to describe people of mixed race vary by territory and have been incrementally added to or changed over time. The original nomenclatures such as sambo, musteephino, mulatto, creole, etc. have been replaced at present to include terms like brown skin, mulatto, clear skin, light skin, red-nigger, dougla and browning. The title of the article comes from a contemporary dancehall song in Jamaica in which the black singer, Buju Banton, unwittingly echoes an unspoken yet shared notion of female desirability in the Caribbean: a preference for ‘brown’ as opposed to black women or unmixed women. In the ongoing constructions of femininity in the region, class and skin colour have intersected with race to produce hierarchies and stereotypes of femininity based on racial mixing. Drawing on some of the historical data available, particularly that of the pioneering research in this area produced by Lucille Mathurin in 1974, this article interrogates some aspects of miscegenation in the Jamaican past, to configure these with gender, race and class relations in the present. The article does not attempt to arrive at conclusive findings but to contribute to the ongoing process in the region, and elsewhere, of differentiating the category ‘woman’ in historiography and sociology.
Female Faith and the Politics of the Personal: Five Mission Encounters in Twentieth-Century South Africa
Authors: Gaitskell Deborah
Published: July 2002

This article explores female religious interaction in racially divided ‘colonial’ South Africa through the lives of five unmarried Anglican women missionaries who worked in and around Johannesburg between 1907 and 1960. It particularly analyses the quality of their personal relationships with African women converts, colleagues and students.

Deaconess Julia Gilpin, in the imperial, anglicizing post-Boer War years, encouraged devout, respectable wifehood on the mine compounds, contributing to the corporate solidarity of praying mothers as a deeply entrenched feature of most black churches. Dora Earthy interacted in a warmer, more egalitarian way with black churchwomen. Her transfer to Mozambique led not only to evangelistic building on African cultural traditions and pioneering anthropological research but also to the protection and validation of Christian widows’ autonomy against coerced remarriage.

Frances Chilton and Dorothy Maud, despite sharply contrasting class origins, struggled to connect with more assertive networks of married women in the segregationist 1920s and 1930s, devoting their energies rather to guiding and enhancing African girlhood and youth. Hannah Stanton, in the more politically fraught 1950s, recognized the necessary independence of African actors and the limitations of outdated white liberal advocacy. Her training of African women promised new theological and organizational maturity, a potential collegiality, cut short by her detention in the 1960 State of Emergency and subsequent expulsion.

The historical sweep of this survey suggests which personal and political conjunctures opened up or closed down meaningful interaction between colonized and colonizing women. It also offers a more positive evaluation of the intersection of race and religion as not invariably a ‘fatal combination’. The politics of the personal in women's joint educational, mission and social welfare ventures across the racial divide helped keep alive the idea of a single society and thus aided the surprisingly peaceful transition to democracy in South Africa in 1994.
The Limits of European-Ness: Immigrant Women in Fortress Europe
Authors: Lutz Helma
Published: July 2002

This article is intended to contribute to the ongoing debate on the ideological, social and political formation of a New Europe. By focusing on the position of immigrant women it examines the gendered nature of the changing configurations of cultural and social European landscapes. Two features of immigrant women's positioning are the key issues of this analysis: regulations through national and European law and ideological representation. It is argued that the debate on European citizenship should be closely linked to the question of formal and substantive and also of symbolic rights. Moreover, feminists, when using the concept of difference in this context, should be aware of the power structures underlying differentiated social positions in society. European-ness will lose its exclusive character only if it provides a solid place in the symbolic order of Europe for immigrants.