bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of interfacial layers in InSb-CdTe heterostructures studied by Raman scattering
Authors: Zahn D. R. T. , Mackey K. J. , Williams R. H. , Münder H. , Geurts J. , Richter W.
Published: July 2002

The formation of a thin interfacial layer of indium telluride in InSb-CdTe heterostructures has been previously suggested by soft x-ray photoemission (SXPS) results. However, the detailed nature of this layer was difficult to interpret by SXPS. Therefore, samples being previously investigated by SXPS were studied by Raman spectroscopy. Spectra were taken of heterostructures grown by deposition of CdTe on (100) InSb substrates at room and elevated temperatures. For the room-temperature sample the vibrational modes of CdTe and crystalline Te were detected. In the case of elevated substrate temperatures it was possible to identify the interfacial layer rich in indium and tellurium as consisting largely of In2Te3 using the Raman spectrum of In2Te3 as a fingerprint. Furthermore, the segregation of antimony was confirmed by the detection of the vibrational modes of antimony at the interface.
Charge trapping in thin nitrided SiO2 films
Authors: Severi Maurizio , Impronta Maurizio
Published: July 2002

Electron avalanche injection has been used to study electron trapping and positive charge generation in thin (10–30 nm) ammonia-annealed silicon dioxide films as a function of process conditions. It is found that the electron traps induced by nitridation are characterized by a capture cross section of ∼10−16 cm2 and by a density which increases with nitridation temperature and time. These results suggest that the traps are due to the nitrogen itself at oxygen sites. Even though electron traps in conventional oxides enhance the positive charge generation rate, the turn-around effect in nitrided oxides is greatly reduced or completely eliminated under severe nitridation conditions. The process dependence of the turn-around effect suggests that the nitrogen near the interface is mainly responsible for this effect.
Low-frequency noise in silicon-gate metal-oxide-silicon capacitors before oxide breakdown
Authors: Neri B. , Olivo P. , Riccò B.
Published: July 2002

Measurements of the fluctuations in the tunneling current It through a thin SiO2 insulating layer, by means of an ultralow noise measurement set, showed that after a first time interval in which its power spectral density is stationary and proportional to I2t, an on-off modulation of It arises, just before oxide breakdown. This bistable noise seems to be related to localized phenomena controlled by trapping-detrapping processes within the oxide. Two possible mechanisms which could give rise to this bistable noise are discussed.
Crystallinity and hardness of B+-implanted Fe
Authors: Hirano Motohisa , Miyake Shojiro
Published: July 2002

Pure Fe is implanted with B+ at a target temperature range of −20 to 300 °C. The effects of the target temperature on the crystallinity and hardness in the B+ -implanted Fe are investigated. It was found that Fe2B is formed by using direct B+ implantation into pure Fe without subsequent annealing. ‘‘Cold implantation,’’ where the implantation is performed at a lower target temperature, reduces crystallinity and increases the hardness of the B+ -implanted Fe. An increase in target temperature, therefore, renders the B+ -implanted Fe with high crystallinity and relatively low hardness.
Direct overwrite in magneto-optical recording
Authors: Savage C. M. , Marquis F. , Watson M. , Meystre P.
Published: July 2002

We have used a numerical model of thermomagnetic recording to investigate direct overwrite by domain destabilization in magneto-optical thin films. Our model includes a linear dependence of domain wall velocity on the net wall pressure due to the demagnetizing field and the wall energy density. Direct overwrite by domain destabilization can be accomplished in one step, without bias fields or prior reading or erasure.
Pseudomorphic GaAs/InGaAs single quantum wells by atmospheric pressure organometallic chemical vapor deposition
Authors: Bertolet Daniel C. , Hsu Jung-Kuei , Jones Stephen H. , Lau Kei May
Published: July 2002

High-quality pseudomorphic GaAs/In0.12Ga0.88As single quantum wells (QW’s) were prepared by atmospheric-pressure organometallic chemical vapor deposition. Photoluminesence spectra measured at 2.5 and 78 K exhibit intense, sharp peaks [full width at half-maximum (FWHM)=2.6 meV for a 17-Å well at 78 K] from the quantized energy transitions of the QW’s. Peak positions agree well with a square well calculation that includes the strain-induced band-gap shift in the In0.12Ga0.88As. Quite unlike previous work with QW’s in which the FWHM was found to exponentially increase with decreasing well width, we observed a narrowing of the QW signals as the well width went below ∼30 Å. In larger well samples (300 Å), the onset of surface crosshatch patterns was observed, which is expected from critical thickness theory.
Magnetic hardening of Nd2Co14B
Authors: Fuerst C. D. , Herbst J. F.
Published: July 2002

We have investigated the room-temperature magnetic properties of a series of neodymium-cobalt-boron alloys rapidly solidified by melt spinning. Depending on the starting composition, intrinsic coercivities and energy products as large as 10.2 kOe and 5.2 MGOe, respectively, have been achieved in ribbons whose principal constituent is crystalline Nd2Co14B. Our work represents the first successful effort to magnetically harden that compound.
Independent control of spin density and hydrogen-bonding configuration in glow-discharge-hydrogenated Si-Ge alloys using a cathode-heating method
Authors: Matsuda A. , Yokoyama S. , Tanaka K.
Published: July 2002

The number density of dangling bonds (spin density) and the hydrogen content as well as the hydrogen-bonding configuration were independently controlled in the glow-discharge hydrogenated amorphous Si-Ge alloys using a cathode-heating technique. A tentative mechanism is presented in terms of the top surface process as well as the growth zone.
Rate heterogeneity among lineages of tracheophytes: Integration of molecular and fossil data and evidence for molecular living fossils
Authors: Soltis Pamela S. , Soltis Douglas E. , Savolainen Vincent , Crane Peter R. , Barraclough Timothy G.
Published: July 2002

Many efforts to date evolutionary divergences by using a molecular clock have yielded age estimates that are grossly inconsistent with the paleontological evidence. Such discrepancies often are attributed to the inadequacy of the fossil record, but many potential sources of error can affect molecular-based estimates. In this study, we minimize the potential error caused by inaccurate topology and uncertain calibration times by using a well-supported tree, multiple genes, and multiple well-substantiated dates to explore the correspondence between the fossil record and molecular-based age estimates for major clades of tracheophytes. Age estimates varied because of gene effects, codon position, lineage effects, method of inferring branch lengths, and whether or not rate constancy was assumed. However, even methods designed to ameliorate the effects of rate heterogeneity among lineages could not accommodate the substantially slower rates observed in
Marattia
+
Angiopteris
and in the tree ferns. Both of these clades of ferns have undergone dramatic decelerations in their rates of molecular evolution and are “molecular living fossils,” consistent with their relative morphological stasis for the past 165–200 million years. Similar discrepancies between the fossil record and molecular-based age estimates noted in other studies may also be explained in part by violations of rate constancy among lineages.
The organization of
Physcomitrella patens
RAD51
genes is unique among eukaryotic organisms
Authors: Markmann-Mulisch Ulrich , Hadi Masood Z. , Koepchen Kerstin , Alonso Juan C. , Russo Vincenzo E. A. , Schell Jeff , Reiss Bernd
Published: July 2002

Genetic recombination pathways and genes are well studied, but relatively little is known in plants, especially in lower plants. To study the recombination apparatus of a lower land plant, a recombination gene well characterized particularly in yeast, mouse, and man, the
RAD51
gene, was isolated from the moss
Physcomitrella patens
and characterized. Two highly homologous
RAD51
genes were found to be present. Duplicated
RAD51
genes have been found thus far exclusively in eukaryotes with duplicated genomes. Therefore the presence of two highly homologous genes suggests a recent genome duplication event in the ancestry of
Physcomitrella
. Comparison of the protein sequences to Rad51 proteins from other organisms showed that both
RAD51
genes originated within the group of plant Rad51 proteins. However, the two proteins form a separate clade in a phylogenetic tree of plant Rad51 proteins. In contrast to
RAD51
genes from other multicellular eukaryotes, the
Physcomitrella
genes are not interrupted by introns. Because introns are a common feature of
Physcomitrella
genes, the lack of introns in the
RAD51
genes is unusual and may indicate the presence of an unusual recombination apparatus in this organism. The presence of duplicated intronless
RAD51
genes is unique among eukaryotes. Studies of further members of this lineage are needed to determine whether this feature may be typical of lower plants.
Primate postcrania from the late middle Eocene of Myanmar
Authors: Ciochon Russell L. , Gingerich Philip D. , Gunnell Gregg F. , Simons Elwyn L.
Published: July 2002

Fossil primates have been known from the late middle to late Eocene Pondaung Formation of Myanmar since the description ofPondaungia cotteriin 1927. Three additional primate taxa,Amphipithecus mogaungensis,Bahinia pondaungensisandMyanmarpithecus yarshensis,were subsequently described. These primates are represented mostly by fragmentary dental and cranial remains. Here we describe the first primate postcrania from Myanmar, including a complete left humerus, a fragmentary right humerus, parts of left and right ulnae, and the distal half of a left calcaneum, all representing one individual. We assign this specimen to a large species ofPondaungiabased on body size and the known geographic distribution and diversity of Myanmar primates. Body weight estimates ofPondaungiarange from 4,000 to 9,000 g, based on humeral length, humeral midshaft diameter, and tooth area by using extant primate regressions. The humerus and ulna indicate thatPondaungiawas capable of a wide variety of forelimb movements, with great mobility at the shoulder joint. Morphology of the distal calcaneus indicates that the hind feet were mobile at the transverse tarsal joint. Postcrania ofPondaungiapresent a mosaic of features, some shared in common with notharctine and adapine adapiforms, some shared with extant lorises and cebids, some shared with fossil anthropoids, and some unique. Overall,Pondaungiahumeral and calcaneal morphology is most consistent with that of other known adapiforms. It does not support the inclusion ofPondaungiain Anthropoidea.
Unnatural base pairs for specific transcription
Authors: Ohtsuki Takashi , Kimoto Michiko , Ishikawa Masahide , Mitsui Tsuneo , Hirao Ichiro , Yokoyama Shigeyuki
Published: July 2002

An unnatural base pair of
2-amino-6-(
N
,
N
-dimethylamino)purine
(designated as x) and pyridin-2-one (designated as y) has been
developed for specific transcription. The ribonucleoside triphosphates
of y and a modified y, 5-methylpyridin-2-one, are selectively
incorporated into RNA opposite x in the templates by T7 RNA polymerase.
In addition, the sequences of the DNA templates containing x can be
confirmed by a dideoxynucleotide chain-terminator method supplemented
with the deoxynucleoside triphosphate of y. The bulky dimethylamino
group of x in the templates effectively eliminates noncognate pairing
with the natural bases. These results enable RNA biosynthesis for the
specific incorporation of unnatural nucleotides at the desired
positions.
Estimates of lateral and longitudinal bond energies within the microtubule lattice
Authors: VanBuren Vincent , Odde David J. , Cassimeris Lynne
Published: July 2002

We developed a stochastic model of microtubule (MT) assembly dynamics that estimates tubulin–tubulin bond energies, mechanical energy stored in the lattice dimers, and the size of the tubulin-GTP cap at MT tips. First, a simple assembly/disassembly state model was used to screen possible combinations of lateral bond energy (Δ
G
Lat
) and longitudinal bond energy (Δ
G
Long
) plus the free energy of immobilizing a dimer in the MT lattice (Δ
G
S
) for rates of MT growth and shortening measured experimentally. This analysis predicts Δ
G
Lat
in the range of −3.2 to −5.7
k
B
T
and Δ
G
Long
plus Δ
G
S
in the range of −6.8 to −9.4
k
B
T
. Based on these estimates, the energy of conformational stress for a single tubulin-GDP dimer in the lattice is 2.1–2.5
k
B
T
. Second, we studied how tubulin-GTP cap size fluctuates with different hydrolysis rules and show that a mechanism of directly coupling subunit addition to hydrolysis fails to support MT growth, whereas a finite hydrolysis rate allows growth. By adding rules to mimic the mechanical constraints present at the MT tip, the model generates tubulin-GTP caps similar in size to experimental estimates. Finally, by combining assembly/disassembly and cap dynamics, we generate MT dynamic instability with rates and transition frequencies similar to those measured experimentally. Our model serves as a platform to examine GTP-cap dynamics and allows predictions of how MT-associated proteins and other effectors alter the energetics of MT assembly.
Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors
Authors: Kuruvilla Finny G. , Shamji Alykhan F. , Schreiber Stuart L.
Published: July 2002

The target of rapamycin (Tor) proteins sense nutrients and control transcription and translation relevant to cell growth. Treating cells with the immunosuppressant rapamycin leads to the intracellular formation of an Fpr1p-rapamycin-Tor ternary complex that in turn leads to translational down-regulation. A more rapid effect is a rich transcriptional response resembling that when cells are shifted from high- to low-quality carbon or nitrogen sources. This transcriptional response is partly mediated by the nutrient-sensitive transcription factors
GLN3
and
NIL1
(also named
GAT1
). Here, we show that these GATA-type transcription factors control transcriptional responses that mediate translation by several means. Four observations highlight upstream roles of GATA-type transcription factors in translation. In their absence, processes caused by rapamycin or poor nutrients are diminished: translation repression, eIF4G protein loss, transcriptional down-regulation of proteins involved in translation, and RNA polymerase I/III activity repression. The Tor proteins preferentially use Gln3p or Nil1p to down-regulate translation in response to low-quality nitrogen or carbon, respectively. Functional consideration of the genes regulated by Gln3p or Nil1p reveals the logic of this differential regulation. Besides integrating control of transcription and translation, these transcription factors constitute branches downstream of the multichannel Tor proteins that can be selectively modulated in response to distinct (carbon- and nitrogen-based) nutrient signals from the environment.
Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair
Authors: Bleuit Jill S. , Xu Hang , Ma Yujie , Wang Tongsheng , Liu Jie , Morrical Scott W.
Published: July 2002

Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3′ ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 recombination/replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative “bubble migration” mode of RDR could play a role in lesion bypass by the T4 replication machinery.
Positive selection of an MHC class-I restricted TCR in the absence of classical MHC class I molecules
Authors: Maurice Madelon M. , Gould Dina S. , Carroll John , Vugmeyster Yulia , Ploegh Hidde L.
Published: July 2002

The H-2Ldalloreactive 2C T cell receptor (TCR) is commonly considered as being positively selected on the H-2Kbmolecule. Surprisingly, 2C TCR+CD8+single-positive T cells emerge in massive numbers in fetal thymic organ culture originating from 2C transgenic, H-2KbDb−/−(2C+KbDb−/−) but not in fetal thymic organ culture from β2-microglobulin−/−2C transgenic animals. Mature CD8+T cells are observed in newborn but not in adult 2C+KbDb−/−mice. These CD8+T cells express the α4β7integrin, which allows them to populate the intestine, a pattern of migration visualized by intrathymic injection of FITC and subsequent accrual of FITC-labeled lymphocytes in the gut. We conclude that the 2C TCR is reactive not only with H-2Ldand H-2Kb, but also with nonclassical MHC class I products to enable positive selection of 2C+T cells in the fetal and newborn thymus and to support their maintenance in the intestine.
Excited-state dynamics in photosystem II: Insights from the x-ray crystal structure
Authors: Vasil'ev Sergej , Orth Peter , Zouni Athina , Owens Thomas G. , Bruce Doug
Published: July 2002

The heart of oxygenic photosynthesis is photosystem II (PSII), a
multisubunit protein complex that uses solar energy to drive the
splitting of water and production of molecular oxygen. The
effectiveness of the photochemical reaction center of PSII depends on
the efficient transfer of excitation energy from the surrounding
antenna chlorophylls. A kinetic model for PSII, based on the x-ray
crystal structure coordinates of 37 antenna and reaction center pigment
molecules, allows us to map the major energy transfer routes from the
antenna chlorophylls to the reaction center chromophores. The model
shows that energy transfer to the reaction center is slow compared with
the rate of primary electron transport and depends on a few bridging
chlorophyll molecules. This unexpected energetic isolation of the
reaction center in PSII is similar to that found in the bacterial
photosystem, conflicts with the established view of the photophysics of
PSII, and may be a functional requirement for primary photochemistry in
photosynthesis. In addition, the model predicts a value for the
intrinsic photochemical rate constant that is 4 times that found in
bacterial reaction centers.
The Eurasian Heartland: A continental perspective on Y-chromosome diversity
Authors: Wells R. Spencer , Yuldasheva Nadira , Ruzibakiev Ruslan , Underhill Peter A. , Evseeva Irina , Blue-Smith Jason , Jin Li , Su Bing , Pitchappan Ramasamy , Shanmugalakshmi Sadagopal , Balakrishnan Karuppiah , Read Mark , Pearson Nathaniel M. , Zerjal Tatiana , Webster Matthew T. , Zholoshvili Irakli , Jamarjashvili Elena , Gambarov Spartak , Nikbin Behrouz , Dostiev Ashur , Aknazarov Ogonazar , Zalloua Pierre , Tsoy Igor , Kitaev Mikhail , Mirrakhimov Mirsaid , Chariev Ashir , Bodmer Walter F.
Published: July 2002

The nonrecombining portion of the human Y chromosome has proven to be a valuable tool for the study of population history. The maintenance of extended haplotypes characteristic of particular geographic regions, despite extensive admixture, allows complex demographic events to be deconstructed. In this study we report the frequencies of 23 Y-chromosome biallelic polymorphism haplotypes in 1,935 men from 49 Eurasian populations, with a particular focus on Central Asia. These haplotypes reveal traces of historical migrations, and provide an insight into the earliest patterns of settlement of anatomically modern humans on the Eurasian continent. Central Asia is revealed to be an important reservoir of genetic diversity, and the source of at least three major waves of migration leading into Europe, the Americas, and India. The genetic results are interpreted in the context of Eurasian linguistic patterns.
Posttranslational modification of the glycosylation inhibiting factor (GIF) gene product generates bioactive GIF
Authors: Watarai Hiroshi , Nozawa Risa , Tokunaga Ayako , Yuyama Noriko , Tomas Mayumi , Hinohara Atsushi , Ishizaka Kimishige , Ishii Yasuyuki
Published: July 2002

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF/MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF/MIF, possessed immunosuppressive effects on thein vitroIgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells.In vitrotreatment of wild-type recombinant human GIF/MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in α2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wild-type GIF/MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF/MIF molecules are responsible for the generation of GIF bioactivity.
Activated glycogen synthase-3β suppresses cardiac hypertrophy
in vivo
Authors: Antos Christopher L. , McKinsey Timothy A. , Frey Norbert , Kutschke William , McAnally John , Shelton John M. , Richardson James A. , Hill Joseph A. , Olson Eric N.
Published: July 2002

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart
in vivo
, we generated transgenic mice that express a constitutively active form of GSK-3β under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3β diminished hypertrophy in response to chronic β-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3β as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3β activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.
A pre-Columbian Y chromosome-specific transition and its implications for human evolutionary history.
Authors: Underhill P A , Jin L , Zemans R , Oefner P J , Cavalli-Sforza L L
Published: July 2002

A polymorphic C-->T transition located on the human Y chromosome was found by the systematic comparative sequencing of Y-specific sequence-tagged sites by denaturing high-performance liquid chromatography. The results of genotyping representative global indigenous populations indicate that the locus is polymorphic exclusively within the Western Hemisphere. The pre-Columbian T allele occurs at > 90% frequency within the native South and Central American populations examined, while its occurrence in North America is approximately 50%. Concomitant genotyping at the polymorphic tetranucleotide microsatellite DYS19 locus revealed that the C-->T mutation displayed significant linkage disequilibrium with the 186-bp allele. The data suggest a single origin of linguistically diverse native Americans with subsequent haplotype differentiation within radiating indigenous populations as well as post-Columbian European and African gene flow. The mutation may have originated either in North America at a very early time during the expansion or before it, in the ancestral population(s) from which all Americans may have originated. The analysis of linkage of the DYS199 and the DYS19 tetranucleotide loci suggests that the C-->T mutation may have occurred around 30,000 years ago. We estimate the nucleotide diversity over 4.2 kb of the nonrecombining portion of the Y chromosome to be 0.00014. compared to autosomes, the majority of variation is due to the smaller effective population size of the Y chromosome rather than selective sweeps. There begins to emerge a pattern of pronounced geographical localization of Y-specific nucleotide substitution polymorphisms.
The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.
Authors: Boyd N D , Kage R , Dumas J J , Krause J E , Leeman S E
Published: July 2002

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.
Mutation of a conserved serine in TM4 of opioid receptors confers full agonistic properties to classical antagonists.
Authors: Claude P A , Wotta D R , Zhang X H , Prather P L , McGinn T M , Erickson L J , Loh H H , Law P Y
Published: July 2002

The involvement of a conserved serine (Ser196 at the mu-, Ser177 at the delta-, and Ser187 at the kappa-opioid receptor) in receptor activation is demonstrated by site-directed mutagenesis. It was initially observed during our functional screening of a mu/delta-opioid chimeric receptor, mu delta2, that classical opioid antagonists such as naloxone, naltrexone, naltriben, and H-Tyr-Tic[psi,CH2NH]Phe-Phe-OH (TIPPpsi; Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing the chimeric receptor. Antagonists also activated the G protein-coupled inward rectifying potassium channel (GIRK1) in Xenopus oocytes coexpressing the mu delta2 opioid receptor and the GIRK1 channel. By sequence analysis and back mutation, it was determined that the observed antagonist activity was due to the mutation of a conserved serine to leucine in the fourth transmembrane domain (S196L). The importance of this serine was further demonstrated by analogous mutations created in the mu-opioid receptor (MORS196L) and delta-opioid receptor (DORS177L), in which classical opioid antagonists could inhibit forskolin-stimulated adenylyl cyclase activity in CHO cells stably expressing either MORS196L or DORS177L. Again, antagonists could activate the GIRK1 channel coexpressed with either MORS196L or DORS177L in Xenopus oocytes. These data taken together suggest a crucial role for this serine residue in opioid receptor activation.
Structure and conformational changes of DNA topoisomerase II visualized by electron microscopy.
Authors: Schultz P , Olland S , Oudet P , Hancock R
Published: July 2002

Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy. Here we describe the structure of the molecule of human topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] as seen by scanning transmission electron microscopy. A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations. When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted. About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region. We propose that structural rearrangements lead to this displacement of an internal tunnel. The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits. These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.
Distinct desmocollin isoforms occur in the same desmosomes and show reciprocally graded distributions in bovine nasal epidermis.
Authors: North A J , Chidgey M A , Clarke J P , Bardsley W G , Garrod D R
Published: July 2002

The adhesive core of the desmosome is composed of cadherin-like glycoproteins of two families, desmocollins and desmogleins. Three isoforms of each are expressed in a tissue-specific and developmentally regulated pattern. In bovine nasal epidermis, the three desmocollin (Dsc) isoforms are expressed in overlapping domains; Dsc3 expression is strongest in the basal layer, while Dsc2 and Dsc1 are strongly expressed in the suprabasal layers. Herein we have investigated whether different isoforms are assembled into the same or distinct desmosomes by performing double immunogold labeling using isoform-specific antibodies directed against Dsc1 and Dsc3. The results show that individual desmosomes harbor both isoforms in regions where their expression territories overlap. Quantification showed that the ratio of the proteins in each desmosome altered gradually from basal to immediately suprabasal and upper suprabasal layers, labeling for Dsc1 increasing and Dsc3 decreasing. Thus desmosomes are constantly modified as cells move up the epidermis, with continuing turnover of the desmosomal glycoproteins. Statistical analysis of the quantitative data showed a possible relationship between the distributions of the two isoforms. This gradual change in desmosomal composition may constitute a vertical adhesive gradient within the epidermis, having important consequences for cell positioning and differentiation.
Transgenic expression of PML/RARalpha impairs myelopoiesis.
Authors: Early E , Moore M A , Kakizuka A , Nason-Burchenal K , Martin P , Evans R M , Dmitrovsky E
Published: July 2002

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.
Characterizations of diverse residue clusters in protein three-dimensional structures.
Authors: Karlin S , Zhu Z Y
Published: July 2002

We present new methods for identifying and analyzing statistically significant residue clusters that occur in three-dimensional (3D) protein structures. Residue clusters of different kinds occur in many contexts. They often feature the active site (e.g., in substrate binding), the interface between polypeptide units of protein complexes, regions of protein-protein and protein-nucleic acid interactions, or regions of metal ion coordination. The methods are illustrated with 3D clusters centering on four themes. (i) Acidic or histidine-acidic clusters associated with metal ions. (ii) Cysteine clusters including coordination of metals such as zinc or iron-sulfur structures, cysteine knots prominent in growth factors, multiple sets of buried disulfide pairings that putatively nucleate the hydrophobic core, or cysteine clusters of mostly exposed disulfide bridges. (iii) Iron-sulfur proteins and charge clusters. (iv) 3D environments of multiple histidine residues. Study of diverse 3D residue clusters offers a new perspective on protein structure and function. The algorithms can aid in rapid identification of distinctive sites, suggest correlations among protein structures, and serve as a tool in the analysis of new structures.
Mapping rat megalin: the second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis.
Authors: Saito A , Yamazaki H , Rader K , Nakatani A , Ullrich R , Kerjaschki D , Orlando R A , Farquhar M G
Published: July 2002

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.
Mammalian dwarfins are phosphorylated in response to transforming growth factor beta and are implicated in control of cell growth.
Authors: Yingling J M , Das P , Savage C , Zhang M , Padgett R W , Wang X F
Published: July 2002

The dwarfin protein family has been genetically implicated in transforming growth factor beta (TGF-beta)-like signaling pathways in Drosophila and Caenorhabditis elegans. To investigate the role of these proteins in mammalian signaling pathways, we have isolated and studied two murine dwarfins, dwarfin-A and dwarfin-C. Using antibodies against dwarfin-A and dwarfin-C, we show that these two dwarfins and an immunogenically related protein, presumably also a dwarfin, are phosphorylated in a time- and dose-dependent manner in response to TGF-beta. Bone morphogenetic protein 2, a TGF-beta superfamily ligand, induces phosphorylation of only the related dwarfin protein. Thus, TGF-beta superfamily members may use overlapping yet distinct dwarfins to mediate their intracellular signals. Furthermore, transient overexpression of either dwarfin-A or dwarfin-C causes growth arrest, implicating the dwarfins in growth regulation. This work provides strong biochemical and preliminary functional evidence that dwarfin-A and dwarfin-C represent prototypic members of a family of mammalian proteins that may serve as mediators of signaling pathways for TGF-beta superfamily members.
An intrinsic genetic program for autonomous differentiation of muscle cells in the ascidian embryo.
Authors: Satoh N , Araki I , Satou Y
Published: July 2002

The B-line presumptive muscle cells of ascidian embryos have extensive potential for self-differentiation dependent on determinants prelocalized in the myoplasm of fertilized eggs. Ascidian larval muscle cells therefore provide an experimental system with which to explore an intrinsic genetic program for autonomous specification of embryonic cells. Experiments with egg fragments suggested that maternal mRNAs are one of the components of muscle determinants. Expression of larval muscle actin genes begins as early as the 32-cell stage, prior to the developmental fate restriction of the cells. The timing of initiation of the actin gene expression proceeds the expression of an ascidian homologue of vertebrate MyoD by a few hours. Mutations in the proximal E-box of the 5' flanking region of the actin genes did not alter the promoter activity for muscle-specific expression of reporter gene. These results, together with results of deletion constructs of fusion genes, suggest that muscle determinants regulate directly, or indirectly via regulatory factors other than MyoD, the transcription of muscle-specific structural genes leading to the terminal differentiation.
Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule.
Authors: Shimojo N , Kohno Y , Yamaguchi K , Kikuoka S , Hoshioka A , Niimi H , Hirai A , Tamura Y , Saito Y , Kohn L D , Tahara K
Published: July 2002

Graves disease is an autoimmune thyroid disease characterized by the presence of antibodies against the thyrotropin receptor (TSHR), which stimulate the thyroid to cause hyperthyroidism and/or goiter. By immunizing mice with fibroblasts transfected with both the human TSHR and a major histocompatibility complex class II molecule, but not by either alone, we have induced immune hyperthyroidism that has the major humoral and histological features of Graves disease: stimulating TSHR antibodies, thyrotropin binding inhibiting immunoglobulins, which are different from the stimulating TSHR antibodies, increased thyroid hormone levels, thyroid enlargement, thyrocyte hypercellularity, and thyrocyte intrusion into the follicular lumen. The results suggest that the aberrant expression of major histocompatibility complex class II molecules on cells that express a native form of the TSHR can result in the induction of functional anti-TSHR antibodies that stimulate the thyroid. They additionally suggest that the acquisition of antigen-presenting ability on a target cell containing the TSHR can activate T and B cells normally present in an animal and induce a disease with the major features of autoimmune Graves.
The design of an agent to bend DNA.
Authors: Akiyama T , Hogan M E
Published: July 2002

An artificial DNA bending agent has been designed to assess helix flexibility over regions as small as a protein binding site. Bending was obtained by linking a pair of 15-base-long triple helix forming oligonucleotides (TFOs) by an adjustable polymeric linker. By design, DNA bending was introduced into the double helix within a 10-bp spacer region positioned between the two sites of 15-base triple helix formation. The existence of this bend has been confirmed by circular permutation and phase-sensitive electrophoresis, and the directionality of the bend has been determined as a compression of the minor helix groove. The magnitude of the resulting duplex bend was found to be dependent on the length of the polymeric linker in a fashion consistent with a simple geometric model. Data suggested that a 50-70 degrees bend was achieved by binding of the TFO chimera with the shortest linker span (18 rotatable bonds). Equilibrium analysis showed that, relative to a chimera which did not bend the duplex, the stability of the triple helix possessing a 50-70 degrees bend was reduced by less than 1 kcal/mol of that of the unbent complex. Based upon this similarity, it is proposed that duplex DNA may be much more flexible with respect to minor groove compression than previously assumed. It is shown that this unusual flexibility is consistent with recent quantitation of protein-induced minor groove bending.
A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis
Authors: Dong Feng , Weitzel Steven E. , von Hippel Peter H.
Published: July 2002

We have developed a coupled helicase–polymerase DNA unwinding
assay and have used it to monitor the rate of double-stranded DNA
unwinding catalyzed by the phage T4 DNA replication helicase (gp41).
This procedure can be used to follow helicase activity in
subpopulations in systems in which the unwinding-synthesis reaction is
not synchronized on all the substrate-template molecules. We show that
T4 replication helicase (gp41) and polymerase (gp43) can be assembled
onto a loading site located near the end of a long double-stranded DNA
template in the presence of a macromolecular crowding agent, and that
this coupled “two-protein” system can carry out ATP-dependent
strand displacement DNA synthesis at physiological rates (400 to 500 bp
per sec) and with high processivity in the absence of other T4 DNA
replication proteins. These results suggest that a direct
helicase–polymerase interaction may be central to fast and processive
double-stranded DNA replication, and lead us to reconsider the roles of
the other replication proteins in processivity control.
Compressibility as a means to detect and characterize globular protein states.
Authors: Chalikian T V , Breslauer K J
Published: July 2002

We report compressibility data on single-domain, globular proteins which suggest a general relationship between protein conformational transitions and delta kzeroS, the change in the partial specific adiabatic compressibility which accompanies the transition. Specifically, we find transitions between native and compact intermediate states to be accompanied by small increases in kzeroS of +(1-4) x 10(-6) cm3.g-1.bar-1 (1 bar = 100 kPa). By contrast, transitions between native and partially unfolded states are accompanied by small decreases in kzeroS of -(3-7) x 10(-6) cm3.g-1.bar-1, while native-to-fully unfolded transitions result in large decreases in kzeroS of -(18-20) x 10(-6) cm3.g-1.bar-1. Thus, for the single-domain, globular proteins studied here, changes in kzeroS correlate with the type of transition being monitored, independent of the specific protein. Consequently, kzeroS measurements may provide a convenient approach for detecting the existence of and for defining the nature of protein transitions, while also characterizing the hydration properties of individual protein states.