bluuebunny/crossref_metadata_2025 huggingface dataset:
Fabrication of large periodic arrays of AlGaAs microdisks by laser-interference lithography and selective etching
Authors: Petter K. , Kipp T. , Heyn Ch. , Heitmann D. , Schüller C.
Published: July 2002

By laser-interference lithography, reactive-ion etching, and selective wet-chemical etching using a citric acid-based solution, we have fabricated large periodic arrays of AlGaAs microdisks with periods of 4 μm and disk diameters between 1.5 and 2 μm. The arrays are characterized by temperature-dependent photoluminescence spectroscopy. Taking into account the below-threshold absorption of the quantum wells inside the disks, we get disk quality factors close to the theoretical maximum value. We demonstrate that our technique allows one also to produce one-dimensionally or two-dimensionally coupled arrays of microdisks.
Strong wave-vector filtering and nearly 100% spin polarization through resonant tunneling antisymmetrical magnetic structure
Authors: Xu H. Z. , Shi Z.
Published: July 2002

Strong spin- and wave-vector dependent upon electron resonant tunneling through antisymmetrical double-barrier magnetic structures are revealed. The electron transmission probability and the polarization of transmitted beams as a function of incident electron energy for different structures have been calculated. It is shown that the antisymmetric magnetic structure possesses the strongest spin polarization and well-pronounced wave-vector filtering properties. Surprisingly, a polarization of nearly 100% can be achieved by spin-dependent resonant tunneling in these structures, although the average magnetic field of the structures is zero.
Influence of the growth procedure on the Cd distribution in CdSe/ZnSe heterostructures: Stranski–Krastanov versus two-dimensional islands
Authors: Litvinov D. , Rosenauer A. , Gerthsen D. , Kratzert P. , Rabe M. , Henneberger F.
Published: July 2002

Molecular beam epitaxy is used to grow different types of ZnSe/CdSe/ZnSe heterostructures. The topography of the bare CdSe surface studied with in situ atomic force microscopy is compared with high-resolution transmission electron microscopy data on overgrown structures. The growth procedure critically influences morphology and Cd distribution. Only use of thermal activation after low-temperature CdSe deposition enables the accomplishment of a distinct Stranski–Krastanov (SK) morphology with three-dimensional islands with a core of pure CdSe. Interdiffusion effects during activation of the SK transition as well as overgrowth are of minor importance.
Interband optical properties of higher manganese silicide thin films
Authors: Rebien M. , Henrion W. , Angermann H. , Teichert S.
Published: July 2002

Semiconducting MnSi1.7 has been investigated by ellipsometric as well as reflectance and transmittance measurements. The interband dielectric functions of epitaxial films on Si(001) and polycrystalline films have been determined. Below the lowest direct transition at 0.96 eV, an indirect gap of 0.40 eV is obtained. The optical properties of the native oxide of MnSi1.7 are found to closely resemble those of SiO2.
Local order of Sb and Bi dopants in hydrogenated amorphous germanium thin films studied by extended x-ray absorption fine structure
Authors: Dalba G. , Fornasini P. , Grisenti R. , Rocca F. , Chambouleyron I.
Published: July 2002

This letter reports on the investigation of the local order and coordination of Sb and Bi impurities in hydrogenated amorphous germanium thin films. The study uses the extended x-ray absorption fine structure technique in fluorescence mode at room temperature. The investigation includes doping concentrations ranging from 1.1×1019 to 5×1020 cm−3. For both impurities, the evidence is that the thermal equilibrium model is not applicable in this case. This result qualitatively follows the behavior of Ga and In impurities in hydrogenated amorphous germanium (a-Ge:H) samples except for Bi. These findings are consistent with data on the transport properties of Sb- and Bi-doped a-Ge:H films.
Model for interface defect and positive charge generation in ultrathin SiO2/ZrO2 gate dielectric stacks
Authors: Houssa M. , Autran J. L. , Stesmans A. , Heyns M. M.
Published: July 2002

The generation of interface defects and positive charge during the injection of electrons in p-Si/SiO2/ZrO2/TiN structures is investigated. The kinetics of generation of both type of defects are found to be very similar. A model is proposed to explain the interface defect generation, based on the depassivation of trivalent silicon dangling bonds (Si3≡SiH→Si3≡Si⋅) at the (100)Si/SiO2 interface by the injected electrons. A Gaussian spread for the activation energy Ed related to the dissociation of the Si–H bond is included in this model. Comparison with experimental results reveals that the mean value of the activation energy Edi decreases linearly with the electric field Eox across the SiO2 layer. This behavior is attributed to the alignment of the Si–H dipole moment with respect to Eox, which favors dissociation of the Si–H bond. The hint of a correlation between the interface defect and positive charge generation suggests that the positively charged centers might be hydrogen-induced overcoordinated oxygen centers.
Reactions of Y2O3 films with (001) Si substrates and with polycrystalline Si capping layers
Authors: Stemmer Susanne , Klenov Dmitri O. , Chen Zhiqiang , Niu Dong , Ashcraft Robert W. , Parsons Gregory N.
Published: July 2002

We use electron energy-loss spectroscopy in scanning transmission electron microscopy to investigate interfacial reactions of chemical vapor deposited Y2O3 films with the Si substrate and with in situ polycrystalline Si (“poly-Si”) capping layers after postdeposition annealing. We find that in situ capping layers significantly reduce the formation of SiO2 at the interface with the substrate, but silicates form at the substrate and the capping layer interfaces. Predeposition nitridation of the Si surface can impede the reaction at the substrate interface, resulting in crystallization of Y2O3 in the film interior. Possible mechanisms of the silicate formation are discussed.
Punctuated evolution of mitochondrial gene content: High and variable rates of mitochondrial gene loss and transfer to the nucleus during angiosperm evolution
Authors: Adams Keith L. , Qiu Yin-Long , Stoutemyer Mark , Palmer Jeffrey D.
Published: July 2002

To study the tempo and pattern of mitochondrial gene loss in plants, DNAs from 280 genera of flowering plants were surveyed for the presence or absence of 40 mitochondrial protein genes by Southern blot hybridization. All 14 ribosomal protein genes and both
sdh
genes have been lost from the mitochondrial genome many times (6 to 42) during angiosperm evolution, whereas only two losses were detected among the other 24 genes. The gene losses have a very patchy phylogenetic distribution, with periods of stasis followed by bursts of loss in certain lineages. Most of the oldest groups of angiosperms are still mired in a prolonged stasis in mitochondrial gene content, containing nearly the same set of genes as their algal ancestors more than a billion years ago. In sharp contrast, other plants have rapidly lost many or all of their 16 mitochondrial ribosomal protein and
sdh
genes, thereby converging on a reduced gene content more like that of an animal or fungus than a typical plant. In these and many lineages with more modest numbers of losses, the rate of ribosomal protein and
sdh
gene loss exceeds, sometimes greatly, the rate of mitochondrial synonymous substitutions. Most of these mitochondrial gene losses are probably the consequence of gene transfer to the nucleus; thus, rates of functional gene transfer also may vary dramatically in angiosperms.
Blockade of vascular endothelial growth factor receptor-3 signaling inhibits fibroblast growth factor-2-induced lymphangiogenesis in mouse cornea
Authors: Kubo Hajime , Cao Renhai , Bräkenhielm Ebba , Mäkinen Taija , Cao Yihai , Alitalo Kari
Published: July 2002

Vascular endothelial growth factor receptor-3 (VEGFR-3) is a major mediator of lymphangiogenesis. Recently, VEGFR-3 ligands, VEGF-C, and VEGF-D were reported to promote tumor lymphangiogenesis and lymphatic metastasis, and these processes were inhibited by blocking of the VEGFR-3-signaling pathway. Here, we have adapted the mouse corneal angiogenesis assay to study potential lymphangiogenic factors and inhibitors. Immunohistochemical analysis with lymphatic endothelial markers showed that VEGF-C induces lymphatic as well as blood vessel growth in the cornea. By contrast, VEGF induced angiogenesis but not lymphangiogenesis. Fibroblast growth factor-2 (FGF-2) stimulated both lymphangiogenesis and angiogenesis. FGF-2 up-regulated VEGF-C expression in vascular endothelial and perivascular cells. Furthermore, administration of blocking anti-VEGFR-3 antibodies inhibited the FGF-2-induced lymphangiogenesis. These findings show that VEGFR-3 can mediate lymphangiogenesis induced by other growth factors. Because increased expression of FGF-2 and VEGF-C has been associated with lymphatic metastasis, our results provide a potential strategy for the inhibition of lymphatic metastasis in cancer therapy.
Global analysis of mRNA decay and abundance in
Escherichia coli
at single-gene resolution using two-color fluorescent DNA microarrays
Authors: Bernstein Jonathan A. , Khodursky Arkady B. , Lin Pei-Hsun , Lin-Chao Sue , Cohen Stanley N.
Published: July 2002

Much of the information available about factors that affect mRNA decay in
Escherichia coli
, and by inference in other bacteria, has been gleaned from study of less than 25 of the ≈4,300 predicted
E. coli
messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted
E. coli
mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of
E. coli
, although ≈80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in
E. coli
, nor the free energy of folding of 5′ or 3′ untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.
Cooperation of two-domain Ca
2+
channel fragments in triad targeting and restoration of excitation– contraction coupling in skeletal muscle
Authors: Flucher Bernhard E. , Weiss Regina G. , Grabner Manfred
Published: July 2002

The specific incorporation of the skeletal muscle voltage-dependent Ca
2+
channel in the triad is a prerequisite of normal excitation–contraction (EC) coupling. Sequences involved in membrane expression and in targeting of Ca
2+
channels into skeletal muscle triads have been described in different regions of the α
1S
subunit. Here we studied the targeting properties of two-domain α
1S
fragments, green fluorescent protein (GFP)-I⋅II (1–670) and III⋅IV (691–1873) expressed alone or in combination in dysgenic (α
1S
-null) myotubes. Immunofluorescence analysis showed that GFP-I⋅II or III⋅IV expressed separately were not targeted into triads. In contrast, on coexpression the two α
1S
fragments were colocalized with one another and with the ryanodine receptor in the triads. Coexpression of GFP-I⋅II and III⋅IV also fully restored Ca
2+
currents and depolarization-induced Ca
2+
transients, despite the severed connection between the two channel halves and the absence of amino acids 671–690 from either α
1S
fragment. Thus, triad targeting, like the rescue of function, requires the cooperation and coassembly of the two complementary channel fragments. Transferring the C terminus of α
1S
to the N-terminal two-domain fragment (GFP-I⋅II⋅tail), or transferring the I–II connecting loop containing the β interaction domain to the C-terminal fragment (III⋅IV⋅βin) did not improve the targeting properties of the individually expressed two-domain channel fragments. Thus, the cooperation of GFP-I⋅II and III⋅IV in targeting cannot be explained solely by a sequential action of the β subunit by means of the I–II loop in releasing the channel from the sarcoplasmic reticulum and of the C terminus in triad targeting.
High-resolution structures of a chitinase complexed with natural product cyclopentapeptide inhibitors: Mimicry of carbohydrate substrate
Authors: Houston Douglas R. , Shiomi Kazuro , Arai Noriko , Ōmura Satoshi , Peter Martin G. , Turberg Andreas , Synstad Bjørnar , Eijsink Vincent G. H. , van Aalten Daan M. F.
Published: July 2002

Over the past years, family 18 chitinases have been validated as potential targets for the design of drugs against human pathogens that contain or interact with chitin during their normal life cycles. Thus far, only one potent chitinase inhibitor has been described in detail, the pseudotrisaccharide allosamidin. Recently, however, two potent natural-product cyclopentapeptide chitinase inhibitors, argifin and argadin, were reported. Here, we describe high-resolution crystal structures that reveal the details of the interactions of these cyclopeptides with a family 18 chitinase. The structures are examples of complexes of a carbohydrate-processing enzyme with high-affinity peptide-based inhibitors and show in detail how the peptide backbone and side chains mimic the interactions of the enzyme with chitooligosaccharides. Together with enzymological characterization, the structures explain why argadin shows an order of magnitude stronger inhibition than allosamidin, whereas argifin shows weaker inhibition. The peptides bind to the chitinase in remarkably different ways, which may explain the differences in inhibition constants. The two complexes provide a basis for structure-based design of potent chitinase inhibitors, accessible by standard peptide chemistry.
In vivo
properties of an anti-GnRH Spiegelmer: An example of an oligonucleotide-based therapeutic substance class
Authors: Wlotzka Britta , Leva Susanne , Eschgfäller Bernd , Burmeister Jens , Kleinjung Frank , Kaduk Christine , Muhn Peter , Hess-Stumpp Holger , Klussmann Sven
Published: July 2002

Spiegelmers are high-affinity
l
-enantiomeric oligonucleotide ligands that display high resistance to enzymatic degradation compared with
d
-oligonucleotides. The target binding properties of Spiegelmers can be designed by an
in vitro
-selection process starting from a random pool of oligonucleotides. Applying this method, a Spiegelmer with high affinity (
K
D
= 20 nM) for the peptide hormone, gonadotropin-releasing hormone (GnRH) was isolated. The Spiegelmer acts as an antagonist to GnRH in Chinese hamster ovary cells stably expressing the human GnRH receptor, and its activity is unchanged by linking to 40-kDa polyethylene glycol. In a castrated rat model the Spiegelmer further demonstrated strong GnRH antagonist activity, which is more pronounced and persists longer with the polyethylene glycol-linked derivative. Furthermore, in rabbits the anti-GnRH Spiegelmer was shown to have a very low, possibly negligible immunogenic potential. These studies suggest that Spiegelmers could be of substantial interest in the development of new pharmaceutical approaches against GnRH and other targets.
Specific zinc-finger architecture required for HIV-1 nucleocapsid protein's nucleic acid chaperone function
Authors: Williams Mark C. , Gorelick Robert J. , Musier-Forsyth Karin
Published: July 2002

The nucleocapsid protein (NC) of HIV type 1 (HIV-1) is a nucleic acid chaperone that facilitates the rearrangement of nucleic acid secondary structure during reverse transcription. HIV-1 NC contains two CCHC-type zinc binding domains. Here, we use optical tweezers to stretch single λ-DNA molecules through the helix-to-coil transition in the presence of wild-type and several mutant forms of HIV-1 NC with altered zinc-finger domains. Although all forms of NC lowered the cooperativity of the DNA helix–coil transition, subtle changes in the zinc-finger structures reduced NC's effect on the transition. The change in cooperativity of the DNA helix–coil transition correlates strongly with
in vitro
nucleic acid chaperone activity measurements and
in vivo
HIV-1 replication studies using the same NC mutants. Moreover, Moloney murine leukemia virus NC, which contains a single zinc finger, had little effect on transition cooperativity. These results suggest that a specific two-zinc-finger architecture is required to destabilize nucleic acids for optimal chaperone activity during reverse transcription in complex retroviruses such as HIV-1.
Using deeply trapped intermediates to map the cytochrome
c
folding landscape
Authors: Tezcan F. Akif , Findley William M. , Crane Brian R. , Ross Scott A. , Lyubovitsky Julia G. , Gray Harry B. , Winkler Jay R.
Published: July 2002

Replacement of iron with cobalt(III) selectively introduces a deep trap in the folding-energy landscape of the heme protein cytochrome
c
. Remarkably, neither the protein structure nor the folding thermodynamics is perturbed by this metal–ion substitution, as shown by data from spectroscopic and x-ray diffraction experiments. Through kinetics measurements, we have found parallel folding pathways involving several different misligated Co(III) species, and, as these folding intermediates persist for several hours under certain conditions, we have been able to elucidate fully their spectroscopic properties. The results, along with an analysis of the fluorescence energy-transfer kinetics during refolding, show that rapidly equilibrating populations of compact and extended polypeptide conformations are present until all molecules have reached the native structure. These measurements provide direct evidence that collapsed denatured structures are not substantially more stable than extended conformations of cytochrome
c
.
Sequence determinants of the energetics of folding of a transmembrane four-helix-bundle protein
Authors: Howard Kathleen P. , Lear James D. , DeGrado William F.
Published: July 2002

Although previous studies are beginning to point to the specific types of helix–helix interactions that stabilize the folds of membrane-bound helical proteins, quantitative thermodynamic data on natural membrane proteins has been very limited. Here the database is expanded substantially by adding thermodynamic data for a series of sequence variants of M2 protein from influenza A virus. The M2 protein has a single transmembrane helix that homotetramerizes to form proton-selective channels that are essential to virus function. To determine the contributions of specific residues to the folding of this protein, a series of transmembrane peptides with single-site changes near the core of the protein were studied by using sedimentation equilibrium analytical ultracentrifugation. Remarkably, a large number of the mutations increased the stability of the protein. The free energies of tetramerization of the variants can be understood in terms of current models for the structure of the protein. In general, the energetic consequences of the mutations are smaller than those observed for similar mutations in water-soluble proteins. This observation is consistent with previous studies and hence may represent a general phenomenon.
The p53MH algorithm and its application in detecting p53-responsive genes
Authors: Hoh J. , Jin S. , Parrado T. , Edington J. , Levine A. J. , Ott J.
Published: July 2002

A computer algorithm, p53MH, was developed, which identifies putative p53 transcription factor DNA-binding sites on a genomewide scale with high power and versatility. With the sequences from the human and mouse genomes, putative p53 DNA-binding elements were identified in a scan of 2,583 human genes and 1,713 mouse orthologs based on the experimental data of el-Deiry
et al.
[el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W. & Vogelstein, B. (1992)
Nat. Genet.
1, 45–49] and Funk
et al.
[Funk, W. D., Pak, D. T., Karas, R. H., Wright, W. E. & Shay, J. W. (1992)
Mol. Cell. Biol.
12, 2866–2871] (
http://linkage.rockefeller.edu/p53
). The p53 DNA-binding motif consists of a 10-bp palindrome and most commonly a second related palindrome linked by a spacer region. By scanning from the 5′ to 3′ end of each gene with an additional 10-kb nucleotide sequence appended at each end (most regulatory DNA elements characterized in the literature are in these regions), p53MH computes the binding likelihood for each site under a discrete discriminant model and then outputs ordered scores, corresponding site positions, sequences, and related information. About 300 genes receiving scores greater than a theoretical cut-off value were identified as potential p53 targets. Semiquantitative reverse transcription–PCR experiments were performed in 2 cell lines on 16 genes that were previously unknown regarding their functional relationship to p53 and were found to have high scores in either proximal promoter or possible distal enhancer regions. Ten (∼63%) of these genes responded to the presence of p53.
Stable expression of calpain 3 from a muscle transgene
in vivo
: Immature muscle in transgenic mice suggests a role for calpain 3 in muscle maturation
Authors: Spencer M. J. , Guyon J. R. , Sorimachi H. , Potts A. , Richard I. , Herasse M. , Chamberlain J. , Dalkilic I. , Kunkel L. M. , Beckmann J. S.
Published: July 2002

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3
in vitro
raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene
in vivo
. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur
in vivo
. Unexpectedly, we found that full-length C3 can be overexpressed at high levels
in vivo
, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably
in vivo
from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development.
Identification of a gene encoding MGAT1, a monoacylglycerol acyltransferase
Authors: Yen Chi-Liang Eric , Stone Scot J. , Cases Sylvaine , Zhou Ping , Farese Robert V.
Published: July 2002

Acyl-CoA:monoacylglycerol acyltransferase (MGAT) catalyzes the synthesis of diacylglycerol, the precursor of physiologically important lipids such as triacylglycerol and phospholipids. In the intestine, MGAT plays a major role in the absorption of dietary fat because resynthesis of triacylglycerol is required for the assembly of lipoproteins that transport absorbed fat to other tissues. MGAT activity has also been reported in mammalian liver and white adipose tissue. However, MGAT has never been purified to homogeneity from mammalian tissues, and its gene has not been cloned. We identified a gene that encodes an MGAT (MGAT1) in mice. This gene has sequence homology with members of a recently identified diacylglycerol acyltransferase gene family. Expression of the MGAT1 cDNA in insect cells markedly increased MGAT activity in cell membranes. In addition, MGAT activity was proportional to the level of MGAT1 protein expressed, and the amount of diacylglycerol produced depended on the concentration of either of its substrates, oleoyl-CoA or monooleoylglycerol. In mice, MGAT1 expression and MGAT activity were detected in the stomach, kidney, white and brown adipose tissue, and liver. However, MGAT1 was not expressed in the small intestine, implying the existence of a second MGAT gene. The identification of the MGAT1 gene should greatly facilitate research on the identification of the intestinal MGAT gene and on the function of MGAT enzymes in mammalian glycerolipid metabolism.
Sialyltransferase ST3Gal-IV operates as a dominant modifier of hemostasis by concealing asialoglycoprotein receptor ligands
Authors: Ellies Lesley G. , Ditto David , Levy Gallia G. , Wahrenbrock Mark , Ginsburg David , Varki Ajit , Le Dzung T. , Marth Jamey D.
Published: July 2002

A number of poorly characterized genetic modifiers contribute to the extensive variability of von Willebrand disease, the most prevalent bleeding disorder in humans. We find that a genetic lesion inactivating the murine ST3Gal-IV sialyltransferase causes a bleeding disorder associated with an autosomal dominant reduction in plasma von Willebrand factor (VWF) and an autosomal recessive thrombocytopenia. Although both ST3Gal-IV and ST6Gal-I sialyltransferases mask galactose linkages implicated as asialoglycoprotein receptor ligands, only ST3Gal-IV deficiency promotes asialoglycoprotein clearance mechanisms with a reduction in plasma levels of VWF and platelets. Exposed galactose on VWF was also found in a subpopulation of humans with abnormally low VWF levels. Oligosaccharide branch-specific sialylation by the ST3Gal-IV sialyltransferase is required to sustain the physiologic half-life of murine hemostatic components and may be an important modifier of plasma VWF level in humans.
Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization
Authors: Hessle Lovisa , Johnson Kristen A. , Anderson H. Clarke , Narisawa Sonoko , Sali Adnan , Goding James W. , Terkeltaub Robert , Millán José Luis
Published: July 2002

Osteoblasts mineralize bone matrix by promoting hydroxyapatite crystal formation and growth in the interior of membrane-limited matrix vesicles (MVs) and by propagating the crystals onto the collagenous extracellular matrix. Two osteoblast proteins, tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-
1
(PC-1) are involved in this process. Mutations in the TNAP gene result in the inborn error of metabolism known as hypophosphatasia, characterized by poorly mineralized bones, spontaneous fractures, and elevated extracellular concentrations of inorganic pyrophosphate (PP
i
). PP
i
suppresses the formation and growth of hydroxyapatite crystals. PP
i
is produced by the nucleoside triphosphate pyrophosphohydrolase activity of a family of isozymes, with PC-1 being the only member present in MVs. Mice with spontaneous mutations in the PC-1 gene have hypermineralization abnormalities that include osteoarthritis and ossification of the posterior longitudinal ligament of the spine. Here, we show the respective correction of bone mineralization abnormalities in knockout mice null for both the TNAP (
Akp2
) and PC-1 (
Enpp1
) genes. Each allele of
Akp2
and
Enpp1
has a measurable influence on mineralization status
in vivo. Ex vivo
experiments using cultured double-knockout osteoblasts and their MVs demonstrate normalization of PP
i
content and mineral deposition. Our data provide evidence that TNAP and PC-1 are key regulators of the extracellular PP
i
concentrations required for controlled bone mineralization. Our results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia. Conversely, interfering with TNAP activity may correct pathological hyperossification because of PP
i
insufficiency.
The barley stem rust-resistance gene
Rpg1
is a novel disease-resistance gene with homology to receptor kinases
Authors: Brueggeman R. , Rostoks N. , Kudrna D. , Kilian A. , Han F. , Chen J. , Druka A. , Steffenson B. , Kleinhofs A.
Published: July 2002

Stem rust caused by
Puccinia graminis
f. sp.
tritici
was among the most devastating diseases of barley in the northern Great Plains of the U.S. and Canada before the deployment of the stem rust-resistance gene
Rpg1
in 1942. Since then,
Rpg1
has provided durable protection against stem rust losses in widely grown barley cultivars (cvs.). Extensive efforts to clone
Rpg1
by synteny with rice provided excellent flanking markers but failed to yield the gene because it does not seem to exist in rice. Here we report the map-based cloning and characterization of
Rpg1
. A high-resolution genetic map constructed with 8,518 gametes and a 330-kb bacterial artificial chromosome contig physical map positioned the gene between two crossovers ≈0.21 centimorgan and 110 kb apart. The region including
Rpg1
was searched for potential candidate genes by sequencing low-copy probes. Two receptor kinase-like genes were identified. The candidate gene alleles were sequenced from resistant and susceptible cvs. Only one of the candidate genes showed a pattern of apparently functional gene structure in the resistant cvs. and defective gene structure in the susceptible cvs. identifying it as the
Rpg1
gene.
Rpg1
encodes a receptor kinase-like protein with two tandem protein kinase domains, a novel structure for a plant disease-resistance gene. Thus, it may represent a new class of plant resistance genes.
A structural basis for the activity of retro-Diels–Alder catalytic antibodies: Evidence for a catalytic aromatic residue
Authors: Hugot Marina , Bensel Nicolas , Vogel Monique , Reymond Martine T. , Stadler Beda , Reymond Jean-Louis , Baumann Ulrich
Published: July 2002

The nitroxyl synthase catalytic antibodies 10F11, 9D9, and 27C5 catalyze the release of nitroxyl from a bicyclic pro-drug by accelerating a retro-Diels–Alder reaction. The Fabs (antigen-binding fragments) of these three catalytic antibodies were cloned and sequenced. Fab 9D9 was crystallized in the apo-form and in complex with one transition state analogue of the reaction. Crystal structures of Fab 10F11 in complex with ligands mimicking substrate, transition state, and product have been determined at resolutions ranging from 1.8 to 2.3 Å. Antibodies 9D9 and 10F11 show increased shape complementarity (as quantified by the program
sc
) to the hapten and to a modeled transition state as compared with substrate and product. The shape complementarity is mediated to a large extent by an aromatic residue (tyrosine or tryptophan) at the bottom of the hydrophobic active pocket, which undergoes π-stacking interactions with the aromatic rings of the ligands. Another factor contributing to the different reactivity of the regioisomers probably arises because of hydrogen-bonding interactions between the nitroxyl bridge and the backbone amide of PheH101 and possibly a conserved water molecule.
Hyporesponsiveness of SPRET/Ei mice to lethal shock induced by tumor necrosis factor and implications for a TNF-based antitumor therapy
Authors: Staelens Jan , Wielockx Ben , Puimège Leen , Van Roy Frans , Guénet Jean-Louis , Libert Claude
Published: July 2002

Tumor necrosis factor (TNF) is a central mediator in lethal shock and an interesting cytokine for anticancer therapy. To inhibit TNF-induced lethal shock, it is important to identify protective genes. Here we demonstrate that the SPRET/Ei mouse strain, derived fromMus spretus, exhibits an extremely dominant resistance to TNF-induced lethal inflammation. An interspecific backcross experiment revealed that the TNF hyporesponse is linked to loci on chromosomes 2, 6, and 11. Treatment of inoculated tumors with TNF and IFN-γ leads to regression and a highly reduced toxicity in (C57BL/6 × SPRET/Ei)F1mice.
CD28 plays a critical role in the segregation of PKCθ within the immunologic synapse
Authors: Huang Jianyong , Lo Pei-Fen , Żal Tomasz , Gascoigne Nicholas R. J. , Smith Brian A. , Levin Steven D. , Grey Howard M.
Published: July 2002

The signaling pathways that lead to the localization of cellular protein to the area of interaction between T cell and antigen-presenting cell and the mechanism by which these molecules are further sorted to the peripheral supramolecular activation cluster or central supramolecular activation cluster regions of the immunologic synapse are poorly understood. In this study, we investigated the functional involvement of CD28 costimulation in the T cell receptor (TCR)-mediated immunologic synapse formation with respect to protein kinase C (PKC)θ localization. We showed that CD3 crosslinking alone was sufficient to induce PKCθ capping in naïve CD4+T cells. Studies with pharmacologic inhibitors and knockout mice showed that the TCR-derived signaling that drives PKCθ membrane translocation requires the Src family kinase, Lck, but not Fyn. In addition, a time course study of the persistence of T cell molecules to the immunologic synapse indicated that PKCθ, unlike TCR, persisted in the synapse for at least 4 h, a time that is sufficient for commitment of a T cell to cell division. Finally, by using TCR-transgenic T cells from either wild-type or CD28-deficient mice, we showed that CD28 expression was required for the formation of the mature immunologic synapse, because antigen stimulation of CD28−T cells led to a diffuse pattern of localization of PKCθ and lymphocyte function-associated antigen-1 in the immunologic synapse, in contrast to the central supramolecular activation cluster localization of PKCθ in CD28+T cells.
Identification and characterization of CIA/ASF1 as an interactor of bromodomains associated with TFIID
Authors: Chimura Takahiko , Kuzuhara Takashi , Horikoshi Masami
Published: July 2002

General transcription initiation factor IID (TFIID) plays a central and critical role in transcription initiation from both naked and chromatin templates. Although interaction between several DNA-binding proteins and TFIID were identified and well characterized, functional linkage between TFIID and chromatin factors has remained to be elucidated. Here we show the identification and characterization of human CIA/hASF1 (identified previously as a histone chaperone) as an interactor of two tandem bromodomain modules of human (h)TAF
II
250/CCG1, the largest subunit of TFIID. Although yeast (y)TAF
II
145, a homologue of hTAF
II
250/CCG1 in
Saccharomyces cerevisiae
, lacks bromodomains, glutathione
S
-transferase pull-down and immunoprecipitation assays revealed that Asf1p (
a
nti
s
ilencing
f
unction 1), the counterpart of CIA in
S. cerevisiae
, interacts with Bdf1p (
b
romo
d
omain
f
actor 1), which is reported to serve as the missing bromodomain in yTAF
II
145. Furthermore, yeast strain lacking the
BDF1
gene shows the Spt phenotype that is shown also by the
ASF1
gene disruptant, and a double-knockout strain of both genes shows synthetic lethality, indicating that
ASF1
genetically interacts with bromodomains associated with yTFIID. We also found that Asf1p coprecipitates with yTFIID subunits from yeast whole-cell extract, and overexpression of yTFIID subunits suppress the Spt phenotype caused by gene disruption of the
ASF1
. This study describes the functional linkage between TFIID and a histone chaperone.
Bradykinin-12-lipoxygenase-VR1 signaling pathway for inflammatory hyperalgesia
Authors: Shin Jieun , Cho Hawon , Hwang Sun Wook , Jung Jooyoung , Shin Chan Young , Lee Soon-Youl , Kim So Hee , Lee Myung Gull , Choi Young Hae , Kim Jinwoong , Haber Nicole Alessandri , Reichling David B. , Khasar Sachia , Levine Jon D. , Oh Uhtaek
Published: July 2002

The capsaicin-sensitive vanilloid receptor (VR1) was recently shown to play an important role in inflammatory pain (hyperalgesia), but the underlying mechanism is unknown. We hypothesized that pain-producing inflammatory mediators activate capsaicin receptors by inducing the production of fatty acid agonists of VR1. This study demonstrates that bradykinin, acting at B2 bradykinin receptors, excites sensory nerve endings by activating capsaicin receptors via production of 12-lipoxygenase metabolites of arachidonic acid
.
This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain.
Chk2 is dispensable for p53-mediated G1arrest but is required for a latent p53-mediated apoptotic response
Authors: Jack Melissa T. , Woo Richard A. , Hirao Atsushi , Cheung Alison , Mak Tak W. , Lee Patrick W. K.
Published: July 2002

In response to genotoxic stress, mammalian cells can activate cell cycle checkpoint pathways to arrest the cell for repair of DNA damage or induce apoptosis to eliminate damaged cells. The checkpoint kinase, Chk2, has been implicated in both of these responses and is believed to function in an ataxia telangiectasia (Atm)-dependent manner. We show here that Chk2−/− mouse embryo fibroblasts (MEFs), unlike Atm−/− or p53−/− MEFs, behaved like normal MEFs in manifesting p21 induction and G1arrest upon exposure to γ-irradiation. Therefore, Chk2 is not involved in p53-mediated G1arrest. To examine the role of Chk2 in p53-dependent apoptotic response, we used adenovirus E1A-expressing MEFs. We show that Chk2−/− cells, like p53−/− cells, did not undergo DNA damage-induced apoptosis, whereas Atm−/− cells behaved like normal cells in invoking an apoptotic response. Furthermore, this apoptosis could occur in the absence of protein synthesis, suggesting that it is preexisting, or “latent,” p53 that mediates this response. We conclude that Chk2 is not involved in Atm- and p53-dependent G1arrest, but is involved in the activation of latent p53, independently of Atm, in triggering DNA damage-induced apoptosis.
The Epstein–Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases
Authors: Tsai Chi-Neu , Tsai Chia-Lung , Tse Ka-Po , Chang Hwan-You , Chang Yu-Sun
Published: July 2002

The latent membrane protein (LMP1) of Epstein–Barr virus (EBV) is expressed in EBV-associated nasopharyngeal carcinoma, which is notoriously metastatic. Although it is established that LMP1 represses E-cadherin expression and enhances the invasive ability of carcinoma cells, the mechanism underlying this repression remains to be elucidated. In this study, we demonstrate that LMP1 induces the expression and activity of the DNA methyltransferases 1, 3a, and 3b, using real-time reverse transcription–PCR and enzyme activity assay. This results in hypermethylation of the E-cadherin promoter and down-regulation of E-cadherin gene expression, as revealed by methylation-specific PCR, real-time reverse transcription–PCR and Western blotting data. The DNA methyltransferase inhibitor, 5′-Aza-2′dC, restores E-cadherin promoter activity and protein expression in LMP1-expressing cells, which in turn blocks cell migration ability, as demonstrated by the Transwell cell migration assay. Our findings suggest that LMP1 down-regulates E-cadherin gene expression and induces cell migration activity by using cellular DNA methylation machinery.
Transcripts of
Vp
-
1
homeologues are misspliced in modern wheat and ancestral species
Authors: McKibbin Rowan S. , Wilkinson Mark D. , Bailey Paul C. , Flintham John E. , Andrew Lucy M. , Lazzeri Paul A. , Gale Mike D. , Lenton John R. , Holdsworth Michael J.
Published: July 2002

The maize (
Zea mays
) Viviparous 1 (Vp1) transcription factor has been shown previously to be a major regulator of seed development, simultaneously activating embryo maturation and repressing germination. Hexaploid bread wheat (
Triticum
aestivum
) caryopses are characterized by relatively weak embryo dormancy and are susceptible to preharvest sprouting (PHS), a phenomenon that is phenotypically similar to the maize
vp1
mutation. Analysis of
Vp
-
1
transcript structure in wheat embryos during grain development showed that each homeologue produces cytoplasmic mRNAs of different sizes. The majority of transcripts are spliced incorrectly, contain insertions of intron sequences or deletions of coding region, and do not have the capacity to encode full-length proteins. Several VP-1-related lower molecular weight protein species were present in wheat embryo nuclei. Embryos of a closely related tetraploid species (
Triticum
turgidum
) and ancestral diploids also contained misspliced
Vp
-
1
transcripts that were structurally similar or identical to those found in modern hexaploid wheat, which suggests that compromised structure and expression of
Vp
-
1
transcripts in modern wheat are inherited from ancestral species. Developing embryos from transgenic wheat grains expressing the
Avena fatua Vp
1
gene showed enhanced responsiveness to applied abscisic acid compared with the control. In addition, ripening ears of transgenic plants were less susceptible to PHS. Our results suggest that missplicing of wheat
Vp
-
1
genes contributes to susceptibility to PHS in modern hexaploid wheat varieties and identifies a possible route to increase resistance to this environmentally triggered disorder.
Molecular basis for the dichotomy in
Plasmodium falciparum
adhesion to CD36 and chondroitin sulfate A
Authors: Gamain Benoit , Gratepanche Sylvie , Miller Louis H. , Baruch Dror I.
Published: July 2002

Plasmodium falciparum
-infected erythrocytes adhere dichotomously to the host receptors CD36 and chondroitin sulfate A (CSA). This dichotomy is associated with parasite sequestration to microvasculature beds (CD36) or placenta (CSA), leading to site-specific pathogenesis. Both properties are mediated by members of the variant
P. falciparum
erythrocyte membrane protein 1 (PfEMP-1) family and reside on nonoverlapping domains of the molecule. To identify the molecular basis for the apparent dichotomy, we expressed various domains of PfEMP-1 individually or in combination and tested their binding properties. We found that the CD36-binding mode of the cysteine-rich interdomain region-1 (CIDR1) ablates the ability of the Duffy binding-like γ domain to bind CSA. In contrast, neither a non-CD36-binding CIDR1 nor an intercellular adhesion molecule 1 binding domain had any affect on CSA binding. Our findings point out that interactions between different domains of PfEMP-1 can alter the adhesion phenotype of infected erythrocytes and provide a molecular basis for the apparent dichotomy in adhesion. We suggest that the basis for the dichotomy is structural and that mutually exclusive conformations of PfEMP-1 are involved in binding to CD36 or CSA. Furthermore, we propose a model explaining the requirement for structural dichotomy between placental and nonplacental isolates.
Association of calnexin with mutant peripheral myelin protein-22
ex vivo
: A basis for “gain-of-function” ER diseases
Authors: Dickson K. M. , Bergeron J. J. M. , Shames I. , Colby J. , Nguyen D. T. , Chevet E. , Thomas D. Y. , Snipes G. J.
Published: July 2002

Schwann cell-derived peripheral myelin protein-22 (PMP-22) when mutated or overexpressed causes heritable neuropathies with a previously unexplained “gain-of-function” endoplasmic reticulum (ER) retention phenotype. In wild-type sciatic nerves, PMP-22 associates in a specific, transient (
t
1/2
≈ 11 min), and oligosaccharide processing-dependent manner with the lectin chaperone calnexin (CNX), but not calreticulin nor BiP. In
Trembler
-
J
(
Tr
-
J
) sciatic nerves, prolonged association of mutant PMP-22 with CNX is found (
t
1/2
> 60 min). In 293A cells overexpressing PMP-22

Tr-J

, CNX and PMP-22 colocalize in large intracellular structures identified at the electron microscopy level as myelin-like figures with CNX localization in the structures dependent on PMP-22 glucosylation. Similar intracellular myelin-like figures were also present in Schwann cells of sciatic nerves from homozygous
Trembler
-
J
mice with no detectable activation of the stress response pathway as deduced from BiP and CHOP expression. Sequestration of CNX in intracellular myelin-like figures may be relevant to the autosomal dominant Charcot–Marie–Tooth-related neuropathies.
Self-assembling biomaterials: Liquid crystal phases of cholesteryl oligo(l-lactic acid) and their interactions with cells
Authors: Hwang Julia J. , Iyer Subramani N. , Li Li-Sheng , Claussen Randal , Harrington Daniel A. , Stupp Samuel I.
Published: July 2002

We report here on the synthesis and characterization of a series of self-assembling biomaterials with molecular features designed to interact with cells and scaffolds for tissue regeneration. The molecules of these materials contain cholesteryl moieties, which have universal affinity for cell membranes, and short chains of lactic acid, a common component of biodegradable tissue engineering matrices. The materials were synthesized in good yields with low polydispersities in the range of 1.05–1.15, and their characterization was carried out by small-angle x-ray diffraction, transmission electron microscopy, electron diffraction, differential scanning calorimetry, and atomic force microscopy. These molecular materials form layered structures that can be described as smectic phases and can also order into single-crystal stacks with an orthorhombic unit cell. Their layer spacings range from 58 to 99 Å, corresponding to bilayers of oligomers with an average of 10 and 37 lactic acid residues, respectively. The self-organized layered structures were found to promote improved fibroblast adhesion and spreading, although the specific mechanism for this observed response remains unknown. The ability of self-assembling materials to present ordered and periodic bulk structures to cells could be a useful strategy in tissue engineering.
Prospects for the Progress of Heterodox Economics
Authors: Dow Sheila C.
Published: July 2002

What are the prospects for progress in heterodox economics? The question posed for this roundtable discussion raises a wide range of important issues, for heterodox economics and for economics as a whole. In order to clarify some of these issues, this contribution approaches the question from a practical point of view: what strategy would promote the progress of heterodox economics?
Reply to Hollander and Peart's “John Stuart Mill's Method”
Authors: Hirsch Abraham
Published: July 2002

I do not think that it would serve much of a purpose to answer Hollander and Peart (1999) point by point, to repeat old arguments, and to once again cite long quotations from Mill. Furthermore, I doubt that the Editor of this Journal would give me the space and time to do so. Instead I propose to ask a number of basic questions about Mill's methodology, give my answers to them and show why I find fault with what I take to be Hollander and Peart's answers. I doubt that this will convince my adversaries. But I hope to show our readers what the major issues are in the disagreement and, perhaps, even to get Hollander and Peart to agree that these are the issues that separate us. If I fail in this latter task they will have the opportunity to point this out in their Rejoinder.