bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural activity of a cloned potassium channel (ROMK1) monitored with the atomic force microscope: The “molecular-sandwich” technique
Authors: Oberleithner H. , Schneider S. W. , Henderson R. M.
Published: July 2002

The atomic force microscope (AFM) was used to continuously follow height changes of individual protein molecules exposed to physiological stimuli. A AFM tip was coated with ROMK1 (a cloned renal epithelial potassium channel known to be highly pH sensitive) and lowered onto atomically flat mica surface until the protein was sandwiched between AFM tip and mica. Because the AFM tip was an integral part of a highly flexible cantilever, any structural alterations of the sandwiched molecule were transmitted to the cantilever. This resulted in a distortion of the cantilever that was monitored by means of a laser beam. With this system it was possible to resolve vertical height changes in the ROMK1 protein of ≥0.2 nm (approximately 5% of the molecule’s height) with a time resolution of ≥1 msec. When bathed in electrolyte solution that contained the catalytic subunit of protein kinase A and 0.1 mM ATP (conditions that activate the native ion channel), we found stochastically occurring height fluctuations in the ROMK1 molecule. These changes in height were pH-dependent, being greatest at pH 7.6, and lowering the pH (either by titration or by the application of CO
2
) reduced their magnitude. The data show that overall changes in shape of proteins occur stochastically and increase in size and frequency when the proteins are active. This AFM “molecular-sandwich” technique, called MOST, measures structural activity of proteins in real time and could prove useful for studies on the relationship between structure and function of proteins at the molecular level.
Using a parity-sensitive sieve to count prime values of a polynomial
Authors: Friedlander John , Iwaniec Henryk
Published: July 2002

It is expected that any irreducible polynomial with integer
coefficients assumes infinitely many prime values provided that it
satisfies some obvious local conditions. Moreover, it is expected that
the frequency of these primes obeys a simple asymptotic law. This has
however been proven for only a few special classes of polynomials. In
the most famous unsolved cases the sequence of values is “thin”
in the sense that it contains fewer than N
θ
integers up
to
N
for some constant θ < 1. Quite generally it
seems to be difficult to show the infinitude of primes in a given thin
integer sequence and there is no polynomial for which this has hitherto
been done. The polynomial
x
2
+
y
4
is an example of such a thin sequence;
here, specifically, θ = 3/4. We report here the development of new
methods that rigorously demonstrate the asymptotic formula in the case
of this polynomial and that are applicable to an infinite class of
polynomials to which this one belongs. The proof is based partly on a
new sieve method that breaks the well-known parity problem of sieve
theory and partly on a careful harmonic analysis of the special
properties of biquadratic polynomial sequences.
Mutational studies of yeast transcription factor IIB
in vivo
reveal a functional surface important for gene activation
Authors: Shaw Shruti P. , Carson Daniel J. , Dorsey Michael J. , Ma Jun
Published: July 2002

Recent experiments in yeast (
Saccharomyces cerevisiae
) cells have identified a species-specific region of yeast transcription factor IIB (TFIIB) located at residues 144–157. According to the human TFIIB structure, this region is part of a solvent-exposed helix in the first repeat of the carboxyl-terminal core domain. In this report, we systematically analyze four positions in this region (Lys-147, Cys-149, Lys-151, and Glu-152) that together have been shown previously to be important for yeast TFIIB’s function
in vivo
. Our experiments suggest that all of these four positions, and in particular positions 151, 149, and 152, are critical for yeast TFIIB’s ability to support cell growth. In addition, we describe an intragenic suppressor screening experiment to identify mutations that reverse, or partially reverse, the temperature-sensitive phenotype of a yeast TFIIB derivative bearing amino acid changes at these four positions to human residues. The suppressor mutations reveal changes at positions 115, 117, and 182 that are located outside the species-specific region of yeast TFIIB, suggesting an extended surface available to interact with other proteins. Finally, we demonstrate that the suppressor mutations restore gene activation
in vivo
, further supporting the idea that one important function of yeast TFIIB in living cells is to mediate gene activation.
Hrp pilus: An
hrp
-dependent bacterial surface appendage produced by
Pseudomonas syringae
pv.
tomato
DC3000
Authors: Roine Elina , Wei Wensheng , Yuan Jing , Nurmiaho-Lassila Eeva-Liisa , Kalkkinen Nisse , Romantschuk Martin , He Sheng Yang
Published: July 2002

Hypersensitive response and pathogenicity (
hrp
) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and flagellar assembly apparatus in animal pathogenic bacteria. Here we report that
Pseudomonas syringae
pv.
tomato
strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6–8 nm in diameter in a solid minimal medium that induces
hrp
genes. Formation of the Hrp pilus is dependent on at least two
hrp
genes,
hrpS
and
hrpH
(recently renamed
hrcC
), which are involved in gene regulation and protein secretion, respectively. Our finding of the Hrp pilus, together with recent reports of
Salmonella typhimurium
surface appendages that are involved in bacterial invasion into the animal cell and of the
Agrobacterium tumefaciens virB
-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells. Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus. Finally, we show that a nonpolar
hrpA
mutant of
P. syringae
pv.
tomato
DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants.
Cosmic velocity fields and their interpretation
Authors: Davis Marc
Published: July 2002

We review the current status of our knowledge of cosmic velocity
fields, on both small and large scales. A new statistic is described
that characterizes the incoherent, thermal component of the velocity
field on scales less than 2
h
−1
Mpc
(
h
is
H
0
/100
km·s
−1
·Mpc
−1
, where
H
0
is the Hubble constant and 1 Mpc =
3.09 × 10
22
m) and smaller. The derived velocity is
found to be quite stable across different catalogs and is of remarkably
low amplitude, consistent with an effective Ω ∼ 0.15 on this scale.
We advocate the use of this statistic as a standard diagnostic of the
small-scale kinetic energy of the galaxy distribution. The analysis of
large-scale flows probes the velocity field on scales of 10–60
h
−1
Mpc and should be adequately described
by linear perturbation theory. Recent work has focused on the
comparison of gravity or density fields derived from whole-sky redshift
surveys of galaxies [e.g., the Infrared Astronomical Satellite
(IRAS)] with velocity fields derived from a variety of sources. All
the algorithms that directly compare the gravity and velocity fields
suggest low values of the density parameter, while the POTENT analysis,
using the same data but comparing the derived IRAS galaxy
density
field with the Mark-III derived matter
density
field, leads to much higher estimates of the
inferred density. Since the IRAS and Mark-III fields are not fully
consistent with each other, the present discrepancies might result from
the very different weighting applied to the data in the competing
methods.
Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53
Authors: Zeng Xiaoya , Levine Arnold J. , Lu Hua
Published: July 2002

The transactivation activity of the p53 tumor suppressor protein is critical for regulating cell growth and apoptosis. We describe the identification of a transcription factor that is functionally similar to p53 and contains the same DNA binding and transcription activities specific for the p53 responsive DNA element (p53RE). This protein was highly purified through chromatography from HeLa cell extracts. The purified protein was able to bind specifically to the p53RE derived from a p21
waf1
promoter and to stimulate p53RE-dependent transcription but not basal transcription
in vitro
. Its DNA-binding activity was inhibited by the wild type but not mutant p53RE-containing DNA oligomers. Also, this p53RE-binding activity was found in human p53 null Saos-2 osteosarcoma and H1299 small cell lung carcinoma cells. Interestingly, this activity exhibited a p53RE sequence preference that was distinct from the p53 protein. The activity is neither p53 nor p73, because anti-p53 or anti-73 antibodies were unable to detect this purified protein nor were the antibodies able to alter the p53-like activity, the p53RE-protein complex. These results demonstrate that, besides p73, an additional p53-like protein exists in cells, which is named NBP for
n
on-p53, p53RE
b
inding
p
rotein.
Blocking ligand occupancy of the αVβ3 integrin inhibits insulin-like growth factor I signaling in vascular smooth muscle cells
Authors: Zheng Bo , Clemmons David R.
Published: July 2002

Blocking αVβ3 integrin occupancy results in attenuation of the cellular migration response to insulin-like growth factor I (IGF-I). To determine whether integrin antagonists alter other IGF-I-stimulated biologic actions, quiescent smooth muscle cells (SMCs) were exposed to echistatin and their ability to respond to IGF-I was determined. Echistatin (10
−7
M) inhibited IGF-I-stimulated DNA synthesis by 80%, and the protein synthesis response also was inhibited. Therefore blocking occupancy of αVβ3 inhibited multiple target cell actions of IGF-I. To determine whether blocking αVβ3 occupancy could alter IGF-I receptor-mediated signal transduction, the ability of IGF-I to stimulate phosphorylation of insulin receptor substrate-1 (IRS-1) was analyzed. A 10-min exposure to 100 ng/ml of IGF-I resulted in a substantial increase in phosphorylated IRS-1, and echistatin (10
−7
M) blocked the IGF-I-induced IRS-1 phosphorylation response. Echistatin also attenuated downstream signaling because the capacity of the p85 subunit of phosphatidylinositol-3 kinase (PI-3 kinase) to bind to IRS-1 was blocked. In contrast, exposure of SMCs to vitronectin (1.0 μg/cm
2
) or thrombospondin (0.25 μg/cm
2
), two known ligands for αVβ3, resulted in enhancement of the IGF-I-stimulated IRS-1 response. To determine whether these effects were caused by alterations in receptor kinase activity, the IGF-I receptor was immunoprecipitated and then analyzed for phosphotyrosine. Echistatin (10
−7
M) significantly reduced IGF-I-stimulated tyrosine phosphorylation of the IGF-I receptor β subunit. We conclude that occupancy of the αVβ3 integrin is necessary for IGF-I to fully activate the kinase activity of the IGF-I receptor and phosphorylate IRS-1. Activation of the αVβ3 receptor results in an interaction with the IGF-I signal transduction pathway, which modulates SMCs responsiveness to IGF-I.
Thermodynamics of electron transfer in
Escherichia coli
cytochrome
bo
3
Authors: Schultz Brian E. , Chan Sunney I.
Published: July 2002

The proton translocation mechanism of the
Escherichia
coli
cytochrome
bo
3
complex is
intimately tied to the electron transfers within the enzyme. Herein we
evaluate two models of proton translocation in this enzyme, a
cytochrome
c
oxidase-type ion-pump and a Q-cycle
mechanism, on the basis of the thermodynamics of electron transfer. We
conclude that from a thermodynamic standpoint, a Q-cycle is the more
favorable mechanism for proton translocation and is likely occurring in
the enzyme.
Impaired liver regeneration in inducible nitric oxide synthasedeficient mice
Authors: Rai Rudra M. , Lee Fung Yee J. , Rosen Anthony , Yang Shi Qi , Lin Hui Zhi , Koteish Ayman , Liew Foo Y. , Zaragoza Carlos , Lowenstein Charles , Diehl Anna Mae
Published: July 2002

The mechanisms that permit adult tissues to regenerate when injured are not well understood. Initiation of liver regeneration requires the injury-related cytokines, tumor necrosis factor (TNF) α and interleukin (IL) 6, and involves the activation of cytokine-regulated transcription factors such as NF-κβ and STAT3. During regeneration, TNFα and IL-6 promote hepatocyte viability, as well as proliferation, because interventions that inhibit either cytokine not only block hepatocyte DNA synthesis, but also increase liver cell death. These observations suggest that the cytokines induce hepatoprotective factors in the regenerating liver. Given evidence that nitric oxide can prevent TNF-mediated activation of the pro-apoptotic protease caspase 3 and protect hepatocytes from cytokine-mediated death, cytokine-inducible nitric oxide synthase (iNOS) may be an important hepatoprotective factor in the regenerating liver. In support of this hypothesis we report that the hepatocyte proliferative response to partial liver resection is severely inhibited in transgenic mice with targeted disruption of the iNOS gene. Instead, partial hepatectomy is followed by increased caspase 3 activity, hepatocyte death, and liver failure, despite preserved induction of TNFα, IL-6, NF-κβ, and STAT3. These results suggest that during successful tissue regeneration, injury-related cytokines induce factors, such as iNOS and its product, NO, that protect surviving cells from cytokine-mediated death.
Host factor I, Hfq, binds to
Escherichia coli ompA
mRNA in a growth rate-dependent fashion and regulates its stability
Authors: Vytvytska Oresta , Jakobsen Jimmy S. , Balcunaite Gina , Andersen Jens S. , Baccarini Manuela , von Gabain Alexander
Published: July 2002

The stability of the
ompA
mRNA depends on the bacterial growth rate. The 5′ untranslated region is the stability determinant of this transcript and the target of the endoribonuclease, RNase E, the key player of mRNA degradation. An RNA-binding protein with affinity for the 5′ untranslated region
ompA
was purified and identified as Hfq, a host factor initially recognized for its function in phage Qβ replication. The
ompA
RNA-binding activity parallels the amount of Hfq, which is elevated in bacteria cultured at slow growth rate, a condition leading to facilitated degradation of the
ompA
mRNA. In
hfq
mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the
ompA
mRNA showed that the growth-rate dependence of degradation is lost. Furthermore, the half-life of the
ompA
mRNA is prolonged in the mutant cells, irrespective of growth rate. Hfq has no affinity for the
lpp
transcript whose degradation, like that of bulk mRNA, is not affected by bacterial growth rate. Compatible with our results, we found that the intracellular concentration of RNase E and its associated degradosome components is independent of bacterial growth rate. Thus our results suggest a regulatory role for Hfq that specifically facilitates the
ompA
mRNA degradation in a growth rate-dependent manner.
A transient expansion of the native state precedes aggregation of recombinant human interferon-γ
Authors: Kendrick Brent S. , Carpenter John F. , Cleland Jeffrey L. , Randolph Theodore W.
Published: July 2002

Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-γ (rhIFN-γ) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-γ aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-γ and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-γ native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-γ against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.
Mammalian DNA topoisomerase IIIα is essential in early embryogenesis
Authors: Li Wei , Wang James C.
Published: July 2002

Targeted disruption of the mouse
TOP3α
gene encoding DNA topoisomerase IIIα was carried out to study the physiological functions of the mammalian type IA DNA topoisomerase. Whereas heterozygous
top3α
+/−
mutant mice were found to resemble phenotypically their
TOP3α
+/+
litermates, no viable
top3α
−/−
homozygotes were found among over 100 progeny of
top3α
+/−
intercrosses. Examination of embryos dissected from decidual swellings and
in vitro
culturing of blastocysts from
top3α
+/−
intercrosses showed that implantation of
top3α
−/−
embryos and the induction of decidualization could occur, but viability of these embryos was severely compromised at an early stage of development. The requirement of mouse DNA topoisomerase IIIα during early embryogenesis is discussed in terms of its plausible role in chromosome replication and its interaction with the RecQ/SGS1 family of DNA helicases, whose members include the Bloom’s syndrome and the Werner’s syndrome gene products.
Peptide nucleic acid-targeted mutagenesis of a chromosomal gene in mouse cells
Authors: Faruqi A. Fawad , Egholm Michael , Glazer Peter M.
Published: July 2002

Peptide nucleic acids (PNAs) can bind to single-stranded DNA by Watson–Crick base pairing and can form triple helices via Hoogsteen bonding to DNA/PNA duplexes. A single dimeric PNA molecule can form a clamp via both double- and triple-helix formation. We designed PNAs to bind as clamps to a site in the
supFG1
mutation reporter gene carried within a chromosomally integrated, recoverable λ phage shuttle vector in mouse fibroblasts. The PNAs were introduced into the cells via permeabilization with streptolysin-O, and cellular uptake was confirmed by fluorescein labeling and fluorescent microscopy. PNAs specific for either an 8- or a 10-bp site in the
supFG1
gene were found to induce mutations at frequencies in the range of 0.1%, 10-fold above the background. PNAs with three or four mismatches showed poor
in vitro
target site binding and were ineffective in the mutagenesis assay. No increased mutagenesis was detected with any of the PNAs in the nontargeted
cII
gene, also carried within the λ vector, further indicating the specificity of the PNA-induced mutagenesis. DNA sequence analysis revealed that the majority of the mutations were located within the PNA-binding site and consisted mostly of single base pair insertions and deletions within the poly G:C run there, suggesting that a high affinity PNA clamp constitutes a mutagenic lesion that may provoke replication slippage errors. The ability to direct mutations to a target site in chromosomal DNA by using PNAs may provide a useful tool for research and therapeutic applications.
A gravitational diffusion model without dark matter
Authors: Britten Roy J.
Published: July 2002

In this model, without dark matter, the flat rotation curves of galaxies and the mass-to-light ratios of clusters of galaxies are described quantitatively. The hypothesis is that the agent of gravitational force is propagated as if it were scattered with a mean free path of ≈5 kiloparsecs. As a result, the force between moderately distant masses, separated by more than the mean free path, diminishes as the inverse first power of the distance, following diffusion equations, and describes the flat rotation curves of galaxies. The force between masses separated by <1 kiloparsec diminishes as the inverse square of distance. The excess gravitational force (ratio of 1/r:1/r
2
) increases with the scale of structures from galaxies to clusters of galaxies. However, there is reduced force at great distances because of the ≈12 billion years that has been available for diffusion to occur. This model with a mean free path of ≈5 kiloparsecs predicts a maximum excess force of a few hundredfold for objects the size of galactic clusters a few megaparsecs in size. With only a single free parameter, the predicted curve for excess gravitational force vs. size of structures fits reasonably well with observations from those for dwarf galaxies through galactic clusters. Under the diffusion model, no matter is proposed in addition to the observed baryons plus radiation and thus the proposed density of the universe is only a few percent of that required for closure.
Cosmological implications of a large complete quasar sample
Authors: Segal I. E. , Nicoll J. F.
Published: July 2002

Objective and reproducible determinations of the probabilistic significance levels of the deviations between theoretical cosmological prediction and direct model-independent observation are made for the Large Bright Quasar Sample [Foltz, C., Chaffee, F. H., Hewett, P. C., MacAlpine, G. M., Turnshek, D. A.,
et al.
(1987)
Astron. J.
94, 1423–1460]. The Expanding Universe model as represented by the Friedman–Lemaitre cosmology with parameters q
o
= 0, Λ = 0 denoted as C1 and chronometric cosmology (no relevant adjustable parameters) denoted as C2 are the cosmologies considered. The mean and the dispersion of the apparent magnitudes and the slope of the apparent magnitude–redshift relation are the directly observed statistics predicted. The C1 predictions of these cosmology-independent quantities are deviant by as much as 11σ from direct observation; none of the C2 predictions deviate by >2σ. The C1 deviations may be reconciled with theory by the hypothesis of quasar “evolution,” which, however, appears incapable of being substantiated through direct observation. The excellent quantitative agreement of the C1 deviations with those predicted by C2 without adjustable parameters for the results of analysis predicated on C1 indicates that the evolution hypothesis may well be a theoretical artifact.
Two distinct classes of Ran-binding sites on the nucleoporin Nup-358
Authors: Yaseen Nabeel R. , Blobel Günter
Published: July 2002

Nup-358 is a giant nucleoporin located at the tips of the cytoplasmic fibrils of the nuclear pore complex (NPC). Its contains four RBH (RanBP1-homologous) domains and a zinc finger domain with eight zinc finger motifs. Using three recombinant fragments of Nup-358 that comprise two of the RBH domains and the zinc finger domain, we show that both RanGDP and RanGTP bind to Nup-358
in vitro
. The RBH domains bound either RanGDP or RanGTP. Interestingly, the zinc finger domain was found to bind RanGDP exclusively. Zinc chelation by EDTA treatment abolished the binding of RanGDP to the zinc finger domain without affecting the binding of Ran to the RBH domain. Ultrastructural studies with RanGDP-conjugated colloidal gold in digitonin-permeabilized cells showed a large number of Ran-binding sites on the cytoplasmic fibrils of the NPC. Of those, only a portion that is closer to the central axis of the NPC was sensitive to RanBP1 competition, suggesting that most of the RBH domains of Nup-358 are situated closer to the central axis of the NPC than the zinc finger domain. Thus, the RBH and the zinc finger domains of Nup-358 were identified as two different classes of Ran-binding sites with distinct, ultrastructural locations at the NPC.
Inhibition of RNase P RNA cleavage by aminoglycosides
Authors: Mikkelsen Nils E. , Brännvall Mathias , Virtanen Anders , Kirsebom Leif A.
Published: July 2002

A number of aminoglycosides have been reported to interact and interfere with the function of various RNA molecules. Among these are 16S rRNA, the group I intron, and the hammerhead ribozymes. In this report we show that cleavage by RNase P RNA in the absence as well as in the presence of the RNase P protein is inhibited by several aminoglycosides. Among the ones we tested, neomycin B was found to be the strongest inhibitor with a
K
i
value in the micromolar range (35 μM). Studies of lead(II)-induced cleavage of RNase P RNA suggested that binding of neomycin B interfered with the binding of divalent metal ions to the RNA. Taken together, our findings suggest that aminoglycosides compete with Mg
2+
ions for functionally important divalent metal ion binding sites. Thus, RNase P, which is an essential enzyme, is indeed a potential drug target that can be used to develop new drugs by using various aminoglycosides as lead compounds.
Location, location, location: The many addresses of memory formation
Authors: Yin Jerry Chi-Ping
Published: July 2002

Memory formation, like real estate, can be summarized
succinctly—location, location, location. It is an emergent property
involving different anatomical regions in the brain, sets of neuronal
circuits, and cellular and molecular interactions between and within
those neurons. At each of these levels of description, location
continues to be a major organizing principle guiding researchers. The
difficulty in the field is the integration of information between the
various levels of analyses, and it is proposed that molecular reporters
may help to fill that void.
Determination of the Hubble constant
Authors: Freedman Wendy L. , Feng Long Long
Published: July 2002

Establishing accurate extragalactic distances has provided an
immense challenge to astronomers since the 1920s. The situation has
improved dramatically as better detectors have become available, and as
several new, promising techniques have been developed. For the first
time in the history of this difficult field, relative distances to
galaxies are being compared on a case-by-case basis, and their
quantitative agreement is being established. New instrumentation, the
development of new techniques for measuring distances, and recent
measurements with the Hubble Space telescope all have resulted in new
distances to galaxies with precision at the ±5–20% level. The
current statistical uncertainty in some methods for
measuring H
0
is now only a few percent; with systematic
errors, the total uncertainty is approaching ±10%. Hence, the
historical factor-of-two uncertainty in the value of the H
0
is now behind us.
Continuous detection of extracellular ATP on living cells by using atomic force microscopy
Authors: Schneider Stefan W. , Egan Marie E. , Jena Bhanu P. , Guggino William B. , Oberleithner H. , Geibel John P.
Published: July 2002

Atomic force microscopy is a powerful technique used to investigate the surface of living cells under physiological conditions. The resolution of the instrument is mainly limited by the softness of living cells and the interactions with the scanning tip (cantilever). Atomic force microscopy, in combination with myosin-functionalized cantilevers, was used in the detection of ATP concentrations in solution and on living cells. Functionally active tips were used to scan the surface of cells in culture and to show that the CFTR+ cell line (S9) had a basal surface ATP concentration that could be detected with atomic force microscopy (
n
= 10). ATP-dependent signals were not detectable in cells scanned with noncoated or heat-inactivated enzyme-coated tips (
n
= 9). Enzymatically active tips may serve as a model for future development of atomic force microscopy biosensors that can simultaneously detect topographical and biologically important compounds at the surface microenvironment of living cells.
Neutrinos
Authors: Besson Dave , Cowen Doug , Selen Mats , Wiebusch Christopher
Published: July 2002

Neutrinos represent a new “window” to the Universe, spanning a large range of energy. We discuss the science of neutrino astrophysics and focus on two energy regimes. At “lower” energies (≈1 MeV), studies of neutrinos born inside the sun, or produced in interactions of cosmic rays with the atmosphere, have allowed the first incontrovertible evidence that neutrinos have mass. At energies typically one thousand to one million times higher, sources further than the sun (both within the Milky Way and beyond) are expected to produce a flux of particles that can be detected only through neutrinos.
Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products
Authors: McDaniel Robert , Thamchaipenet Arinthip , Gustafsson Claes , Fu Hong , Betlach Melanie , Betlach Mary , Ashley Gary
Published: July 2002

The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds—“unnatural” natural products—by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.
Production of infectious bovine papillomavirus from cloned viral DNA by using an organotypic raft/xenograft technique
Authors: McBride Alison A. , Dlugosz Andrzej , Baker Carl C.
Published: July 2002

Bovine papillomavirus type 1 (BPV-1) induces fibropapillomas in its natural host and can transform fibroblasts in culture. The viral genome is maintained as an episome within fibroblasts, which has allowed extensive genetic analyses of the viral functions required for DNA replication, gene expression, and transformation. Much less is known about BPV-1 gene expression and replication in bovine epithelial cells because the study of the complete viral life cycle requires an experimental system capable of generating a fully differentiated stratified bovine epithelium. Using a combination of organotypic raft cultures and xenografts on nude mice, we have developed a system in which BPV-1 can replicate and produce infectious viral particles. Organotypic cultures were established with bovine keratinocytes plated on a collagen raft containing BPV-1-transformed fibroblasts. These keratinocytes were infected with virus particles isolated from a bovine wart or were transfected with cloned BPV-1 DNA. Several days after the rafts were lifted to the air interface, they were grafted on nude mice. After 6–8 weeks, large xenografts were produced that exhibited a hyperplastic and hyperkeratotic epithelium overlying a large dermal fibroma. These lesions were strikingly similar to a fibropapilloma caused by BPV-1 in the natural host. Amplified viral DNA and capsid antigens were detected in the suprabasal cells of the epithelium. Moreover, infectious virus particles could be isolated from these lesions and quantitated by a focus formation assay on mouse cells in culture. Interestingly, analysis of grafts produced with infected and uninfected fibroblasts indicated that the fibroma component was not required for productive infection or morphological changes characteristic of papillomavirus-infected epithelium. This system will be a powerful tool for the genetic analysis of the roles of the viral gene products in the complete viral life cycle.
ABCG1 (ABC8), the human homolog of theDrosophila whitegene, is a regulator of macrophage cholesterol and phospholipid transport
Authors: Klucken Jochen , Büchler Christa , Orsó Evelyn , Kaminski Wolfgang E. , Porsch-Özcürümez Mustafa , Liebisch Gerhard , Kapinsky Michael , Diederich Wendy , Drobnik Wolfgang , Dean Michael , Allikmets Rando , Schmitz Gerd
Published: July 2002

Excessive uptake of atherogenic lipoproteins such as modified low-density lipoprotein complexes by vascular macrophages leads to foam cell formation, a critical step in atherogenesis. Cholesterol efflux mediated by high-density lipoproteins (HDL) constitutes a protective mechanism against macrophage lipid overloading. The molecular mechanisms underlying this reverse cholesterol transport process are currently not fully understood. To identify effector proteins that are involved in macrophage lipid uptake and release, we searched for genes that are regulated during lipid influx and efflux in human macrophages using a differential display approach. We report here that the ATP-binding cassette (ABC) transporter ABCG1 (ABC8) is induced in monocyte-derived macrophages during cholesterol influx mediated by acetylated low-density lipoprotein. Conversely, lipid efflux in cholesterol-laden macrophages, mediated by the cholesterol acceptor HDL3, suppresses the expression of ABCG1. Immunocytochemical and flow cytometric analyses revealed that ABCG1 is expressed on the cell surface and in intracellular compartments of cholesterol-laden macrophages. Inhibition of ABCG1 protein expression using an antisense strategy resulted in reduced HDL3-dependent efflux of cholesterol and choline-phospholipids. In a comprehensive analysis of the expression and regulation of all currently known human ABC transporters, we identified an additional set of ABC genes whose expression is regulated by cholesterol uptake or HDL3-mediated lipid release, suggesting a potential function for these transporters in macrophage lipid homeostasis. Our results demonstrating a regulator function for ABCG1 in cholesterol and phospholipid transport define a biologic activity for ABC transporters in macrophages.
Remodeling of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia
Authors: Optiz Thoralf , Grooms Sonja Y. , Bennett Michael V. L. , Zukin R. Suzanne
Published: July 2002

Transient global ischemia induces selective delayed cell death,
primarily of principal neurons in the hippocampal CA1. However, the
molecular mechanisms underlying ischemia-induced cell death are as yet
unclear. The present study shows that global ischemia triggers a
pronounced and cell-specific reduction in GluR2 [the subunit that
limits Ca
2+
permeability of
α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)
receptors] in vulnerable CA1 neurons, as evidenced by
immunofluorescence of brain sections and Western blot analysis of
microdissected hippocampal subfields. At 72 h after ischemia (a
time before cell death), virtually all CA1 pyramidal neurons exhibited
greatly reduced GluR2 immunolabeling throughout their somata and
dendritic processes. GluR2 immunolabeling was unchanged in pyramidal
cells of the CA3 and granule cells of the dentate gyrus, regions
resistant to ischemia-induced damage. Immunolabeling of the AMPA
receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus.
Western analysis indicated that GluR2 subunit abundance was markedly
reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1
abundance was unchanged in all subfields at all times examined. These
findings, together with the previous observation of enhanced
AMPA-elicited Ca
2+
influx in postischemic CA1 neurons, show
that functional GluR2-lacking, Ca
2+
-permeable AMPA
receptors are expressed in vulnerable neurons before cell death. Thus,
the present study provides an important link in the postulated causal
chain between global ischemia and delayed death of CA1 pyramidal
neurons.
Britain's Regional Shopping Centres: New Urban Forms?
Authors: Lowe Michelle S.
Published: July 2002

There have been a number of recent attempts to represent Britain's regional shopping centres as potentially being the cores of new towns. Using the example of the Merry Hill Centre in the West Midlands, this paper debates that issue. The paper suggests that new town centres such as the one in formation at Merry Hill pose a number of challenges to UK planning policy. Such centres might fruitfully be regarded as developing 'edge cities' and are likely to have a considerable impact on Britain's urban futures into the 21st century and beyond. This paper serves as a plea that, whatever our prejudices, it is surely now time to take these new urban forms seriously.
Local Underemployment and the Discouraged Worker Effect
Authors: van Ham Maarten , Mulder Clara H. , Hooimeijer Pieter
Published: July 2002

The effect of poor local labour market opportunities on occupational achievement is an important aspect of the spatial mismatch hypothesis. Much of the research has concentrated on the direct link between geographical access to jobs and employment outcomes. In contrast, little attention has been given to the discouraging effect of poor chances on job search activities. The discouraged worker effect is defined as the decision to refrain from job search as a result of poor chances on the labour market. Discouragement effects can arise from a lack of individual qualifications, from discrimination in the labour market or from a high local level of underemployment. The empirical findings of this paper, based on the Netherlands Labour Force Surveys 1994-97, show that discouragement can enter the job search process both at the stage of deciding to enter the labour force and at the stage of deciding to engage actively in a job search. Gender differentials in discouragement are revealed in the process of self-selection into the labour force. Poor labour market chances lead to less activity in both off-the-job and on-the-job search, indicating a role of discouragement in the spatial mismatch. Individual qualifications and ascribed characteristics turn out to be more decisive than the local level of underemployment.
Beyond the Third World City: The New Urban Geography of South-east Asia
Authors: Dick H.W. , Rimmer P.J.
Published: July 2002

Scholars, as area specialists, have typified south-east Asian cities as Third World cities and emphasised their uniquely south-east Asian or even national characteristics. This paper will argue that the early decades of decolonisation which gave rise to this perspective were in fact a transitional phase. In the late colonial period south-east Asian cities were already becoming more like Western cities. Since the 1980s, in the era of globalisation, this process of convergence has re-emerged. Clearly, there should now be a single urban discourse. This is not to deny that south-east Asian (or Third World) cities have distinctive elements. The problem is the paradigm which shuts out First World elements.
Comments: Contesting And Constructing National Identity in Central Europe
Authors: Unowsky Daniel
Published: July 2002

With his contingent of geographers, historians, and other academic “experts” collectively known as The Inquiry in tow, Woodrow Wilson arrived in Paris in January 1919 to redraw the map of Europe. Wilson wanted to fulfill his Fourteen Points and guarantee national self-determination to the peoples of Europe. A peaceful community of ethnically homogeneous nation-states was to replace the great multinational empires (defined by central European nationalists as prisons of the peoples) that had previously dominated central and eastern Europe. During the inter-war period, the governing elites of central Europe, their new “nation-states” legitimated by the post-war settlement, created new national holidays, national anthems, and nationalist school text books lauding the history and achievements of the state-bearing nation. These simple and seemingly coherent national narratives elided the messy, confusing, and jumbled past of multiple identities, mingled ethnic groups, and alienated social orders, and legitimized political, economic, and territorial claims made in the name of the “national community” lending its name to the new state.
Educational Motivation and Engagement: Qualitative accounts from three countries
Authors: Hufton Neil R. , Elliott Julian G. , Illushin Leonid
Published: July 2002

AbstractThis article extends and develops earlier survey studies by reporting findings from detailed interviews with adolescents in Sunderland, Kentucky and St Petersburg. The interviews sought to examine a number of key factors underpinning educational motivation and engagement, in particular, attitudes about schooling, self‐evaluations of academic performance, patterns and rate of work at home and at school, reasons why education may be valuable and aspirations for the future. A number of reasons for the presence of high levels of English and American self‐satisfaction, and lower Russian self‐evaluations are presented. In line with the earlier studies, yet contrary to a widely held position, effort appeared to be emphasised more than ability in explaining differential performance. Differences in the meaning and understanding of such constructs are considered. The article then examines the important influence of peers, and teacher‐pupil relationships, upon classroom behaviour and work rates. Differing perceptions as to the intrinsic and extrinsic value of education are explored. In conclusion, the implications for educational reform and attempts to raise educational achievement are considered.
Constraining Explicit and Implicit Content by Means of a Norwegian Scalar Particle
Authors: Fretheim Thorstein
Published: July 2002

This study is a semantic and pragmatic analysis of the Norwegian right-detached particleså, which occurs exclusively with sentence fragments. The framework of description is relevance theory, a cognitively based theory of communication whose objective is to account for how we are able to understand utterances and to make ourselves understood, in spite of the fact that the linguistic code that we use vastly underdetermines what we mean, and even what we say in the strict sense (truth conditions). It is argued thatsåencodes a procedure for the addressee to follow in his inferential processing of the linguistic signal. The lexical entry forsåcontains an instruction to the addressee to contextually activate one or more positive propositions which resemble the one asserted by the utterance of theså-fragment, and to arrange them mentally on a scale whose lower bound is represented by theså-fragment proposition. The speaker implicitly communicates her lack of commitment to the more highly ranked propositions on the scale. The principle behind the scalar ranking is argued to be highly context-dependent.
Heroin‐Related Deaths in Sydney, Australia. How Common Are They?
Authors: Garrick Therese M. , Sheedy Donna , Abernethy John , Hodda Allan E. , Harper Clive G.
Published: July 2002

The objective of this article was to determine the number of recent deaths caused by accidental illicit drug overdoses seen at the NSW Institute of Forensic Medicine, Glebe (Sydney). All Forensic cases (3559) were reviewed during the period July 1995‐February 1997. Any that were classified as accidental illicit drug overdose were followed up, and demographic and toxicological data were collected for analysis. Our results found that one hundred and forty three accidental illicit drug overdoses were identified from 3359 autopsies during the 20 month data collection period (4%). Male to female ratio was 5:1, but females predominated in the methadone toxicity group. Most of the cases were under 40 years of age. Toxicological results showed that 80% of the deaths were associated with morphine (heroin) levels in the toxic range, although 91% had morphine present at some level. Only 35% of cases had significant levels of bile morphine, suggesting “chronic” usage. In many cases, multiple illicit substances and/or alcohol were thought to be important contributing factors. Cocaine was found in 13% of cases, and all of these had morphine (heroin) in their blood. Methadone was found in 13% of cases, and 13/19 had toxic levels—9/19 also had morphine in their blood. Only two cases had amphetamines or methamphetamines in their blood.

The authors conclude that Heroin overdose is by far the most common cause of accidental illicit drug overdose. Those at greatest risk are naive users and those who are not tolerant. There is an urgent need for increased awareness and further education concerning the dangers of heroin use, particularly of multiple drug use (including alcohol). Only about one‐third of these cases appear to be “chronic” users.
Nonuniform Microtubular Polarity Established by CHO1/MKLP1 Motor Protein Is Necessary for Process Formation of Podocytes
Authors: Kobayashi Naoto , Reiser Jochen , Kriz Wilhelm , Kuriyama Ryoko , Mundel Peter
Published: July 2002

Podocytes are unique cells that are decisively involved in glomerular filtration. They are equipped with a complex process system consisting of major processes and foot processes whose function is insufficiently understood (Mundel, P., and W. Kriz. 1995. Anat. Embryol. 192:385–397). The major processes of podocytes contain a microtubular cytoskeleton. Taking advantage of a recently established cell culture system for podocytes with preserved ability to form processes (Mundel, P., J. Reiser, A. Zúñiga Mejía Borja, H. Pavenstädt, G.R. Davidson, W. Kriz, and R. Zeller. 1997b. Exp. Cell Res. 36:248–258), we studied the functional significance of the microtubular system in major processes. The following data were obtained: (a) Microtubules (MTs) in podocytes show a nonuniform polarity as revealed by hook-decoration. (b) CHO1/ MKLP1, a kinesin-like motor protein, is associated with MTs in podocytes. (c) Treatment of differentiating podocytes with CHO1/MKLP1 antisense oligonucleotides abolished the formation of processes and the nonuniform polarity of MTs. (d) During the recovery from taxol treatment, taxol-stabilized (nocodazole- resistant) MT fragments were distributed in the cell periphery along newly assembled nocodazole-sensitive MTs. A similar distribution pattern of CHO1/MKLP1 was found under these circumstances, indicating its association with MTs. (e) In the recovery phase after complete depolymerization, MTs reassembled exclusively at centrosomes. Taken together, these findings lead to the conclusion that the nonuniform MT polarity in podocytes established by CHO1/MKLP1 is necessary for process formation.
Hyphal Death during Colony Development in Streptomyces antibioticus: Morphological Evidence for the Existence of a Process of Cell Deletion in a Multicellular Prokaryote
Authors: Miguélez Elisa M. , Hardisson Carlos , Manzanal Manuel B.
Published: July 2002

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.