bluuebunny/crossref_metadata_2025 huggingface dataset:
Impurity gettering by misfit dislocations in Si (2% Ge) epitaxy: nickel
Authors: Lee D. M. , Posthill J. B. , Shimura F. , Rozgonyi G. A.
Published: July 2002

The microstructure associated with nickel gettering by an epitaxial misfit dislocation network generated in Si(2% Ge) chemical vapor deposition films has been determined using transmission electron microscopy. The configuration of misfit dislocations is a well-characterized network of b=a/2〈011〉 60° dislocations that all lie in the (001) interfacial plane. Nickel was deliberately introduced by deposition onto the front surface of the wafer followed by diffusion at temperatures from 600 to 1000 °C. Nickel segregated on or in the immediate vicinity of the misfit dislocations to form individual or agglomerates of fine precipitates. The strong temperature dependence of Ni solubility in Si and the nucleation enhancement by the localized strain effects in the vicinity of dislocations are considered to be the dominant factors controlling gettering by the misfit dislocations. An annealing temperature of 600 °C is suggested which appears to uniformly decorate the misfit dislocations with Ni for possible buried microwire applications.
Winners don't take all: Characterizing the competition for links on the web
Authors: Pennock David M. , Flake Gary W. , Lawrence Steve , Glover Eric J. , Giles C. Lee
Published: July 2002

As a whole, the World Wide Web displays a striking “rich get richer” behavior, with a relatively small number of sites receiving a disproportionately large share of hyperlink references and traffic. However, hidden in this skewed global distribution, we discover a qualitatively different and considerably less biased link distribution among subcategories of pages—for example, among all university homepages or all newspaper homepages. Although the connectivity distribution over the entire web is close to a pure power law, we find that the distribution within specific categories is typically unimodal on a log scale, with the location of the mode, and thus the extent of the rich get richer phenomenon, varying across different categories. Similar distributions occur in many other naturally occurring networks, including research paper citations, movie actor collaborations, and United States power grid connections. A simple generative model, incorporating a mixture of preferential and uniform attachment, quantifies the degree to which the rich nodes grow richer, and how new (and poorly connected) nodes can compete. The model accurately accounts for the true connectivity distributions of category-specific web pages, the web as a whole, and other social networks.
Evidence of a mate-finding cue in the hermaphrodite nematode
Caenorhabditis elegans
Authors: Simon Jasper M. , Sternberg Paul W.
Published: July 2002

When males of the roundworm
Caenorhabditis elegans
come into association with their hermaphroditic counterparts they cease foraging behavior and begin to mate. Here we detail several assays used to demonstrate that a diffusible cue is correlated with this process. This cue is sexually dimorphic, given off only by the hermaphrodite and eliciting a response only in the male. Males are attracted to, reverse direction of movement frequently, and remain in regions of agar conditioned with hermaphrodites. From our studies we suggest a form of kinesis that works by attracting males to their mating partners from a distance and functions, once males arrive, in holding attracted males in close proximity. The hermaphrodite vulva is not required for the cue. Males from general sensory mutants
osm-5
and
osm-6
fail to respond to the cue, whereas male-specific mutants
lov-1
and
pkd-2
respond. Finally, that males from multiple isolates of
C. elegans
also respond similarly to this cue indicates that this cue is robust and has been maintained during recent evolution.
Adenovirus-directed expression of Q227L-Gα
s
inhibits growth of established tumors of later-stage human breast cancer cells in athymic mice
Authors: Santore Tara Ann , Chen Yibang , Smit Martine J. , Iyengar Ravi
Published: July 2002

Elevation of cAMP inhibits proliferation and expression of the transformed phenotype in several cell types. We studied the effects of elevation of cAMP and expression of mutant (Q227L) activated Gα
s
on the proliferation and tumorigenic capability of the later stage, metastatic estrogen-independent human breast cancer cell lines MDA-231 and MDA-435. Our studies show that 8Br-cAMP inhibits proliferation of these cells in culture and their ability to form colonies in soft agar. This inhibition may occur by different mechanisms in the two cell types. In MDA-231 cells, cAMP elevation results in sustained expression of the cell cycle inhibitor p27kip1 and inhibition of CDK2 activity, whereas in MDA-435 cells inhibition of mitogen-activated protein (MAP) kinase 1,2 activity is observed. We tested whether these effects in culture could be translated into inhibition of tumor growth
in vivo
. Tumors were developed in athymic (Nu/Nu) mice by injections of MDA-231 or MDA-435 cells. Injection of Q227L-Gα
s
expressing adenoviral vector into these established tumors inhibited further tumor growth under conditions where tumors injected with either saline or adenoviral vector containing β-galactosidase grew up to four to five times their original size. These results raise the possibility that sustained elevation of cAMP may have therapeutic value in the treatment of estrogen-resistant later stage breast cancers.
Mechanical force generation by G proteins
Authors: Kosztin Ioan , Bruinsma Robijn , O'Lague Paul , Schulten Klaus
Published: July 2002

GTP-hydrolyzing G proteins are molecular switches that play a critical role in cell signaling processes. Here we use molecular dynamics simulations to show that Ras, a monomeric G protein, can generate mechanical force upon hydrolysis. The generated force levels are comparable to those produced by ATP-hydrolyzing motor proteins, consistent with the structural similarities of the catalytic region of motor proteins and G proteins. The force transduction mechanism is based on an irreversible structural change, produced by the hydrolysis, which triggers thermal switching between force-generating substates through changes in the configurational space of the protein.
An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus
Authors: Maione Domenico , Rocca Carlo Della , Giannetti Patrizia , D'Arrigo Roberta , Liberatoscioli Laura , Franlin Laura L. , Sandig Volker , Ciliberto Gennaro , La Monica Nicola , Savino Rocco
Published: July 2002

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance—i.e., 100% of treated animals showing sustained expression after 4 months—was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.
Cerebral energetics and the glycogen shunt: Neurochemical basis of functional imaging
Authors: Shulman Robert G. , Hyder Fahmeed , Rothman Douglas L.
Published: July 2002

Positron-emission tomography and functional MRS imaging signals can
be analyzed to derive neurophysiological values of cerebral blood flow
or volume and cerebral metabolic consumption rates of glucose
(CMR
Glc
) or oxygen (CMR

O
2

). Under basal
physiological conditions in the adult mammalian brain, glucose
oxidation is nearly complete so that the oxygen-to-glucose index (OGI),
given by the ratio of CMR

O
2

/CMR
Glc
, is
close to the stoichiometric value of 6. However, a survey of functional
imaging data suggests that the OGI is activity dependent, moving
further below the oxidative value of 6 as activity is increased. Brain
lactate concentrations also increase with stimulation. These results
had led to the concept that brain activation is supported by anaerobic
glucose metabolism, which was inconsistent with basal glucose
oxidation. These differences are resolved here by a proposed model of
glucose energetics, in which a fraction of glucose is cycled through
the cerebral glycogen pool, a fraction that increases with degree of
brain activation. The “glycogen shunt,” although energetically
less efficient than glycolysis, is followed because of its ability to
supply glial energy in milliseconds for rapid neurotransmitter
clearance, as a consequence of which OGI is lowered and lactate is
increased. The value of OGI observed is consistent with passive lactate
efflux, driven by the observed lactate concentration, for the few
experiments with complete data. Although the OGI changes during
activation, the energies required per neurotransmitter release
(neuronal) and clearance (glial) are constant over a wide range of
brain activity.
The complete optical spectrum of liquid water measured by inelastic x-ray scattering
Authors: Hayashi Hisashi , Watanabe Noboru , Udagawa Yasuo , Kao C.-C.
Published: July 2002

Interaction of light with matter is of paramount importance in nature. The most fundamental property of a material in relation to light is its oscillator strength distribution, i.e., how strongly it absorbs light as a function of wavelength. Once the oscillator strength distribution is determined precisely for a wide enough energy range, the optical constants such as absorbance and reflectance as well as a number of other properties of the material, some of which are seemingly unrelated to photoabsorption, can be deduced. Most important of all is the fact that the interaction of matter with fast charged particles can be described by its complete optical spectra [Inokuti, M. (1986)
Photochem. Photobiol.
44, 279–285]. Despite their importance, however, the complete optical spectra of volatile liquids including water have never been obtained accurately because of experimental difficulties inherent in vacuum UV spectroscopy. Inelastic x-ray scattering spectroscopy can provide quantitative data equivalent to those from vacuum UV absorption spectra. Herein, we show the complete optical spectrum of liquid water determined by making use of intense monochromatic x-rays supplied by the wiggler line X21 of the National Synchrotron Light Source.
RhoB is required to mediate apoptosis in neoplastically transformed cells after DNA damage
Authors: Liu Ai-xue , Cerniglia George J. , Bernhard Eric J. , Prendergast George C.
Published: July 2002

The effect of neoplastic transformation on the response to
genotoxic stress is of significant clinical interest. In this study, we
offer genetic evidence that the apoptotic response of
neoplastically transformed cells to DNA damage requires RhoB, a member
of the Rho family of actin cytoskeletal regulators. Targeted deletion
of the
rhoB
gene did not affect cell cycle arrest in
either normal or transformed cells after exposure to doxorubicin or
gamma irradiation, but rendered transformed cells resistant to
apoptosis. This effect was specific insofar as
rhoB
deletion did not affect apoptotic
susceptibility to agents that do not damage DNA. However,
rhoB
deletion also affected apoptotic
susceptibility to Taxol, an agent that disrupts microtubule dynamics.
We have demonstrated that RhoB alteration mediates the
proapoptotic and antineoplastic effects of farnesyltransferase
inhibitors, and we show here that RhoB alteration is also crucial for
farnesyltransferase inhibitors to sensitize neoplastic cells to DNA
damage-induced cell death. We found RhoB to be an important determinant
of long-term survival
in vitro
and tumor response
in vivo
after gamma irradiation. Our findings identify a
pivotal role for RhoB in the apoptotic response of neoplastic
cells to DNA damage at a novel regulatory point that may involve the
actin cytoskeleton.
Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy
Authors: Huber Alexander , Padrun Vivianne , Déglon Nicole , Aebischer Patrick , Möhler Hanns , Boison Detlev
Published: July 2002

Adenosine is an inhibitor of neuronal activity in the brain. The local release of adenosine from grafted cells was evaluated as an
ex vivo
gene therapy approach to suppress synchronous discharges and epileptic seizures. Fibroblasts were engineered to release adenosine by inactivating the adenosine-metabolizing enzymes adenosine kinase and adenosine deaminase. After encapsulation into semipermeable polymers, the cells were grafted into the brain ventricles of electrically kindled rats, a model of partial epilepsy. Grafted rats provided a nearly complete protection from behavioral seizures and a near-complete suppression of afterdischarges in electroencephalogram recordings, whereas the full tonic–clonic convulsions in control rats remained unaltered. Thus, the local release of adenosine resulting in adenosine concentrations <25 nM at the site of action is sufficient to suppress seizure activity and, therefore, provides a potential therapeutic principle for the treatment of drug-resistant partial epilepsies.
Counting probability distributions: Differential geometry and model selection
Authors: Myung In Jae , Balasubramanian Vijay , Pitt Mark A.
Published: July 2002

A central problem in science is deciding among competing explanations of data containing random errors. We argue that assessing the “complexity” of explanations is essential to a theoretically well-founded model selection procedure. We formulate model complexity in terms of the geometry of the space of probability distributions. Geometric complexity provides a clear intuitive understanding of several extant notions of model complexity. This approach allows us to reconceptualize the model selection problem as one of counting explanations that lie close to the “truth.” We demonstrate the usefulness of the approach by applying it to the recovery of models in psychophysics.
Cholinergic dilation of cerebral blood vessels is abolished in M
5
muscarinic acetylcholine receptor knockout mice
Authors: Yamada Masahisa , Lamping Kathryn G. , Duttaroy Alokesh , Zhang Weilie , Cui Yinghong , Bymaster Frank P. , McKinzie David L. , Felder Christian C. , Deng Chu-Xia , Faraci Frank M. , Wess Jürgen
Published: July 2002

The M
5
muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M
1
-M
5
) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M
5
receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M
5
receptor-deficient mice (
M5R
−/−
mice).
M5R
−/−
mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However,
in vitro
neurotransmitter release experiments showed that M
5
receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M
5
receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M
5
receptors led to changes in vascular tone by using several
in vivo
and
in vitro
vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in
M5R
−/−
mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in
M5R
−/−
mice. Our findings provide direct evidence that M
5
muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimer's disease and focal cerebral ischemia, cerebrovascular M
5
receptors may represent an attractive therapeutic target.
Mitochondrial diversity and the origins of African and European cattle.
Authors: Bradley D G , MacHugh D E , Cunningham P , Loftus R T
Published: July 2002

The nature of domestic cattle origins in Africa are unclear as archaeological data are relatively sparse. The earliest domesticates were humpless, or Bos taurus, in morphology and may have shared a common origin with the ancestors of European cattle in the Near East. Alternatively, local strains of the wild ox, the aurochs, may have been adopted by peoples in either continent either before or after cultural influence from the Levant. This study examines mitochondrial DNA displacement loop sequence variation in 90 extant bovines drawn from Africa, Europe, and India. Phylogeny estimation and analysis of molecular variance verify that sequences cluster significantly into continental groups. The Indian Bos indicus samples are most markedly distinct from the others, which is indicative of a B. taurus nature for both European and African ancestors. When a calibration of sequence divergence is performed using comparisons with bison sequences and an estimate of 1 Myr since the Bison/Bos Leptobos common ancestor, estimates of 117-275,000 B.P. and 22-26,000 B.P. are obtained for the separation between Indians and others and between African and European ancestors, respectively. As cattle domestication is thought to have occurred approximately 10,000 B.P., these estimates suggest the domestication of genetically discrete aurochsen strains as the origins of each continental population. Additionally, patterns of variation that are indicative of population expansions (probably associated with the domestication process) are discernible in Africa and Europe. Notably, the genetic signatures of these expansions are clearly younger than the corresponding signature of African/European divergence.
Cloning and expression of human deoxyguanosine kinase cDNA.
Authors: Johansson M , Karlsson A
Published: July 2002

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.
UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells.
Authors: Lu M L , Sato M , Cao B , Richie J P
Published: July 2002

UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirement for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-alpha- and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis.
Synthesis and characterization of a photoactivatable analog of corticotropin-releasing factor for specific receptor labeling.
Authors: Rühmann A , Köpke A K , Dautzenberg F M , Spiess J
Published: July 2002

A novel photoactivatable analog of ovine corticotropin-releasing factor (ovine photoCRF) has been synthesized and characterized. A diazirine group, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl residue, was covalently bound to the amino terminus of ovine CRF (oCRF), which was N-terminally extended by a tyrosyl residue for radioactive labeling with 125I. Under mild conditions, photolysis yielded highly reactive carbenes, responsible for the formation of covalent bonds to the CRF receptor. Ovine photoCRF was shown to bind to the high-affinity site of the CRF receptor with a similar Kd value as oCRF. When radioactively iodinated ovine photoCRF (ovine 125I-photoCRF) was covalently linked to rat CRF receptor, type 1 (rCRFR1), permanently transfected into human embryonic kidney (HEK) 293 cells, a highly glycosylated 75-kDa protein was identified with SDS/PAGE. The specificity of ovine 125I-photoCRF was demonstrated by the finding that this analog could be displaced from the receptor by oCRF, but not other unrelated peptides such as vasoactive intestinal peptide. The observed size of the 75-kDa cross-link was in agreement with the molecular weight reported earlier for native CRFR1 from rat brain. Deglycosylation of the 75-kDa cross-link with peptide:N-glycosidase (PNGase) yielded a 46-kDa protein, in agreement with the molecular weight estimated from cDNA coding for rat CRFR1. The developed CRF analog, photoCRF, is expected to facilitate future biochemical and physiological analysis of CRF receptors and--by analogous strategies--of other peptide receptors.
Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA.
Authors: Zabaleta E , Mouras A , Hernould M , Suharsono  , Araya A
Published: July 2002

We have previously shown that the expression of an unedited atp9 chimeric gene correlated with male-sterile phenotype in transgenic tobacco plant. To study the relationship between the expression of chimeric gene and the male-sterile trait, hemizygous and homozygous transgenic tobacco lines expressing the antisense atp9 RNA were constructed. The antisense producing plants were crossed with a homozygous male-sterile line, and the F1 progeny was analyzed. The offspring from crosses between homozygous lines produced only male-fertile plants, suggesting that the expression antisense atp9 RNA abolishes the effect of the unedited chimeric gene. In fact, the plants restored to male fertility showed a dramatic reduction of the unedited atp9 transcript levels, resulting in normal flower development and seed production. These results support our previous observation that the expression of unedited atp9 gene can induce male sterility.
Inhibition of interleukin 1β-converting enzyme-mediated apoptosis of mammalian cells by baculovirus IAP
Authors: Hawkins Christine J. , Uren Anthony G. , Häcker Georg , Medcalf Robert L. , Vaux David L.
Published: July 2002

Apoptosis can be a potent weapon against viral infection and consequently has selected for viruses carrying antiapoptosis genes. Two baculovirus proteins, IAP and p35, can prevent insect cells from dying in response to infection. p35, which interferes with members of the Ced-3 family of cysteine proteases, can also function in mammalian cells. We investigated the ability of IAP fromOrgyia pseudotsugatanuclear polyhedrosis virus to prevent death of mammalian cells. IAP was transiently expressed in mammalian cells and its ability to block cell death caused by expression of interleukin-1β converting enzyme (ICE), FADD, or the ICE homologues ICH-1 and ICE-Lap3, was investigated. IAP strongly inhibited ICE- and ICH-1-induced cell death but protected only partially against death by overexpression of FADD and not at all against death due to enforced ICE-Lap3 expression. These results demonstrate that a baculoviral IAP protein can functionally interact with conserved components of the apoptosis machinery in mammalian cells.
A close relative of the adrenoleukodystrophy (ALD) gene codes for a peroxisomal protein with a specific expression pattern.
Authors: Lombard-Platet G , Savary S , Sarde C O , Mandel J L , Chimini G
Published: July 2002

Adrenoleukodystrophy (ALD), a severe demyelinating disease, is caused by mutations in a gene coding for a peroxisomal membrane protein (ALDP), which belongs to the superfamily of ATP binding cassette (ABC) transporters and has the structure of a half transporter. ALDP showed 38% sequence identity with another peroxisomal membrane protein, PMP70, up to now its closest homologue. We describe here the cloning and characterization of a mouse ALD-related gene (ALDR), which codes for a protein with 66% identity with ALDP and shares the same half transporter structure. The ALDR protein was overexpressed in COS cells and was found to be associated with the peroxisomes. The ALD and ALDR genes show overlapping but clearly distinct expression patterns in mouse and may thus play similar but nonequivalent roles. The ALDR gene, which appears highly conserved in man, is a candidate for being a modifier gene that could account for some of the extreme phenotypic variability of ALD. The ALDR gene is also a candidate for being implicated in one of the complementation groups of Zellweger syndrome, a genetically heterogeneous disorder of peroxisome biogenesis, rare cases of which were found to be associated with mutations in the PMP70 (PXMP1) gene.
Organ-specific and Agamous-regulated expression and glycosylation of a pollen tube growth-promoting protein.
Authors: Cheung A Y , Zhan X Y , Wang H , Wu H M
Published: July 2002

Transmitting tissue-specific (TTS) protein is a pollen tube growth-promoting and attracting glycoprotein located in the stylar transmitting tissue extracellular matrix of the pistil of tobacco. The TTS protein backbones have a deduced molecular mass of about 28 kDa, whereas the glycosylated stylar TTS proteins have apparent molecular masses ranging between 50 and 100 kDa. TTS mRNAs and proteins are ectopically produced in transgenic tobacco plants that express either a cauliflower mosaic virus (CaMV) 35S promoter-TTS2 transgene or a CaMV 35S-promoter-NAG1 (NAG1 = Nicotiana tabacum Agamous gene) transgene. However, the patterns of TTS mRNA and protein accumulation and the quality of the TTS proteins produced are different in these two types of transgenic plants. In 35S-TTS transgenic plants, TTS mRNAs and proteins accumulate constitutively in vegetative and floral tissues. However, the ectopically expressed TTS proteins in these transgenic plants accumulate as underglycosylated protein species with apparent molecular masses between 30 and 50 kDa. This indicates that the capacity to produce highly glycosylated TTS proteins is restricted to the stylar transmitting tissue. In 35S-NAG transgenic plants, NAG1 mRNAs accumulate constitutively in vegetative and floral tissues, and TTS mRNAs are induced in the sepals of these plants. Moreover, highly glycosylated TTS proteins in the 50- to 100-kDa molecular mass range accumulate in the sepals of these transgenic, 35S-NAG plants. These results show that the tobacco NAGI gene, together with other yet unidentified regulatory factors, control the expression of TTS genes and the cellular capacity to glycosylate TTS proteins, which are normally expressed very late in the pistil developmental pathway and function in the final stage of floral development. The sepals in the transgenic 35S-NAG plants also support efficient pollen germination and tube growth, similar to what normally occurs in the pistil, and this ability correlates with the accumulation of the highest levels of the 50- to 100-kDa glycosylated TTS proteins.
Position- and orientation-independent activity of the
Schizosaccharomyces pombe
meiotic recombination hot spot
M26
Authors: Fox Mary E. , Virgin Jeffrey B. , Metzger Jens , Smith Gerald R.
Published: July 2002

The activity of the
M26
meiotic recombination hot spot of
Schizosaccharomyces pombe
depends on the presence of the heptamer 5′-ATGACGT-3′. Transplacement of DNA fragments containing the
ade6-M26
gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1–4 bp in the
ade6
and
ura4
genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for
M26
hot spot activity.
Representation of sound localization cues in the auditory thalamus of the barn owl
Authors: Proctor Larry , Konishi Masakazu
Published: July 2002

Barn owls can localize a sound source using either the map of auditory space contained in the optic tectum or the auditory forebrain. The auditory thalamus, nucleus ovoidalis (N.Ov), is situated between these two auditory areas, and its inactivation precludes the use of the auditory forebrain for sound localization. We examined the sources of inputs to the N.Ov as well as their patterns of termination within the nucleus. We also examined the response of single neurons within the N.Ov to tonal stimuli and sound localization cues. Afferents to the N.Ov originated with a diffuse population of neurons located bilaterally within the lateral shell, core, and medial shell subdivisions of the central nucleus of the inferior colliculus. Additional afferent input originated from the ipsilateral ventral nucleus of the lateral lemniscus. No afferent input was provided to the N.Ov from the external nucleus of the inferior colliculus or the optic tectum. The N.Ov was tonotopically organized with high frequencies represented dorsally and low frequencies ventrally. Although neurons in the N.Ov responded to localization cues, there was no apparent topographic mapping of these cues within the nucleus, in contrast to the tectal pathway. However, nearly all possible types of binaural response to sound localization cues were represented. These findings suggest that in the thalamo-telencephalic auditory pathway, sound localization is subserved by a nontopographic representation of auditory space.
Control of alternative pre-mRNA splicing by distributed pentameric repeats
Authors: Hedjran Farah , Yeakley Joanne M. , Huh Gene S. , Hynes Richard O. , Rosenfeld Michael G.
Published: July 2002

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.
Hereditary hemochromatosis: Effects of C282Y and H63D mutations on association with β
2
-microglobulin, intracellular processing, and cell surface expression of the HFE protein in COS-7 cells
Authors: Waheed Abdul , Parkkila Seppo , Zhou Xiao Yan , Tomatsu Shunji , Tsuchihashi Zenta , Feder John N. , Schatzman Randall C. , Britton Robert S. , Bacon Bruce R. , Sly William S.
Published: July 2002

Hereditary hemochromatosis (HH) is the most common autosomal recessive disorder known in humans. A candidate gene for HH called HFE has recently been cloned that encodes a novel member of the major histocompatibility complex class I family. Most HH patients are homozygous for a Cys-282→Tyr (C282Y) mutation in HFE gene, which has been shown to disrupt interaction with β
2
-microglobulin; a second mutation, His-63→Asp (H63D), is enriched in HH patients who are heterozygous for C282Y mutation. The aims of this study were to determine the effects of the C282Y and H63D mutations on the cellular trafficking and degradation of the HFE protein in transfected COS-7 cells. The results indicate that, while the wild-type and H63D HFE proteins associate with β
2
-microglobulin and are expressed on the cell surface of COS-7 cells, these capabilities are lost by the C282Y HFE protein. We present biochemical and immunofluorescence data that indicate that the C282Y mutant protein: (
i
) is retained in the endoplasmic reticulum and middle Golgi compartment, (
ii
) fails to undergo late Golgi processing, and (
iii
) is subject to accelerated degradation. The block in intracellular transport, accelerated turnover, and failure of the C282Y protein to be presented normally on the cell surface provide a possible basis for impaired function of this mutant protein in HH.
Transgenic rescue from embryonic lethality and renal carcinogenesis in the Eker rat model by introduction of a wild-type
Tsc2
gene
Authors: Kobayashi Toshiyuki , Mitani Hiroaki , Takahashi Ri-ichi , Hirabayashi Masumi , Ueda Masatsugu , Tamura Hiroshi , Hino Okio
Published: July 2002

We recently reported that a germ-line insertion in the rat homologue of the human tuberous sclerosis gene (
TSC2
) gives rise to dominantly inherited cancer in the Eker rat model. In this study, we constructed transgenic Eker rats with introduction of a wild-type
Tsc2
gene to ascertain whether suppression of the Eker phenotype is possible. Rescue from embryonic lethality of mutant homozygotes (Eker/Eker) and suppression of
N
-ethyl-
N
-nitrosourea-induced renal carcinogenesis in heterozygotes (Eker/+) were both observed, defining the germ-line
Tsc2
mutation in the Eker rat as embryonal lethal and tumor predisposing mutation. To the best of our knowledge, this is the first report of rescue from a naturally occurring dominantly inherited cancer. This transgenic rescue system will be useful to analyze
Tsc2
gene function, its relation to tumorigenesis
in vivo
, and genetic–environmental interactions in carcinogenesis.
Nonneuronal isoforms of STOP protein are responsible for microtubule cold stability in mammalian fibroblasts
Authors: Denarier Eric , Fourest-Lieuvin Anne , Bosc Christophe , Pirollet Fabienne , Chapel Agnès , Margolis Robert L. , Job Didier
Published: July 2002

A number of cycling mammalian cells, such as NIH 3T3, contain abundant subsets of cold-stable microtubules. The origin of such microtubule stabilization in nonneuronal cells is unknown. We have previously described a neuronal protein, stable tubule-only polypeptide (STOP), that binds to microtubules and induces cold stability. We find that NIH 3T3 fibroblasts contain a major 42-kDa isoform of STOP (fibroblastic STOP, F-STOP). F-STOP contains the central repeats characteristic of brain STOP but shows extensive deletions of N- and C-terminal protein domains that are present in brain STOP. These deletions arise from differences in STOP RNA splicing. Despite such deletions, F-STOP has full microtubule stabilizing activity. F-STOP accumulates on cold-stable microtubules of interphase arrays and is present on stable microtubules within the mitotic spindle of NIH 3T3 cells. STOP inhibition by microinjection of affinity-purified STOP central repeat antibodies into NIH 3T3 cells abolishes both interphase and spindle microtubule cold stability. Similar results were obtained with Rat2 cells. These results show that STOP proteins have nonneuronal isoforms that are responsible for the microtubule cold stability observed in mammalian fibroblasts.
Antigen binding forces of individually addressed single-chain Fv antibody molecules
Authors: Ros Robert , Schwesinger Falk , Anselmetti Dario , Kubon Martina , Schäfer Rolf , Plückthun Andreas , Tiefenauer Louis
Published: July 2002

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.
Long-distance transcriptional enhancement by the histone acetyltransferase PCAF
Authors: Krumm Anton , Madisen Linda , Yang Xiang-Jiao , Goodman Richard , Nakatani Yoshihiro , Groudine Mark
Published: July 2002

Enhancers are defined by their ability to stimulate gene activity from remote sites and their requirement for promoter-proximal upstream activators to activate transcription. Here we demonstrate that recruitment of the p300/CBP-associated factor PCAF to a reporter gene is sufficient to stimulate promoter activity. The PCAF-mediated stimulation of transcription from either a distant or promoter-proximal position depends on the presence of an upstream activator (Sp1). These data suggest that acetyltransferase activity may be a primary component of enhancer function, and that recruitment of polymerase and enhancement of transcription are separable. Transcriptional activation by PCAF requires both its acetyltransferase activity and an additional activity within its N terminus. We also show that the simian virus 40 enhancer and PCAF itself are sufficient to counteract Mad-mediated repression. These results are compatible with recent models in which gene activity is regulated by the competition between deacetylase-mediated repression and enhancer-mediated recruitment of acetyltransferases.
Impairing follicle-stimulating hormone (FSH) signalingin vivo: Targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance
Authors: Dierich Andrée , Sairam M. Ram , Monaco Lucia , Fimia Gian Maria , Gansmuller Anne , LeMeur Marianne , Sassone-Corsi Paolo
Published: July 2002

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R −/− mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R −/− mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.
Vancomycin-resistant enterococci in intensive-care hospital settings: Transmission dynamics, persistence, and the impact of infection control programs
Authors: Austin Daren J. , Bonten Marc J. M. , Weinstein Robert A. , Slaughter Sarah , Anderson Roy M.
Published: July 2002

Vancomycin-resistant enterococci (VRE) recently have emerged as a nosocomial pathogen especially in intensive-care units (ICUs) worldwide. Transmission via the hands of health-care workers is an important determinant of spread and persistence in a VRE-endemic ICU. We describe the transmission of nosocomial pathogens by using a micro-epidemiological framework based on the transmission dynamics of vector-borne diseases. By using the concept of a basic reproductive number,R0, defined as the average number of secondary cases generated by one primary case, we show quantitatively how infection control measures such as hand washing, cohorting, and antibiotic restriction affect nosocomial cross-transmission. By using detailed molecular epidemiological surveillance and compliance monitoring, we found that the estimated basic reproductive number for VRE during a study at the Cook County Hospital, Chicago, was approximately 3–4 without infection control and 0.7 when infection control measures were included. The impact of infection control was to reduce the prevalence from a predicted 79% to an observed 36%. Hand washing and staff cohorting are the most powerful control measures although their efficacy depends on the magnitude ofR0. Under the circumstances tested, endemicity of VRE was stabilized despite infection control measures, by the constant introduction of colonized patients. Multiple stochastic simulations of the model revealed excellent agreement with observed pattern. In conjunction with detailed microbiological surveillance, a mathematical framework provides a precise template to describe the colonization dynamics of VRE in ICUs and impact of infection control measures. Our analyses suggest that compliance for hand washing significantly in excess of reported levels, or the cohorting of nursing staff, are needed to prevent nosocomial transmission of VRE in endemic settings.
The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene
Authors: Guo Yiru , Jones W. Keith , Xuan Yu-Ting , Tang Xian-Liang , Bao Weike , Wu Wen-Jian , Han Hui , Laubach Victor E. , Ping Peipei , Yang Zequan , Qiu Yumin , Bolli Roberto
Published: July 2002

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC)
in vivo
. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion/4-min reperfusion exhibited a significant (
P
< 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent
l
-citrulline formation), and nitrite + nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion/4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%;
P
< 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null
iNOS
allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (
i
) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (
ii
) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC
in vivo
.
Blood-borne tissue factor: Another view of thrombosis
Authors: Giesen Peter L. A. , Rauch Ursula , Bohrmann Bernd , Kling Dorothee , Roqué Merce , Fallon John T. , Badimon Juan J. , Himber Jacques , Riederer Markus A. , Nemerson Yale
Published: July 2002

Arterial thrombosis is considered to arise from the interaction of tissue factor (TF) in the vascular wall with platelets and coagulation factors in circulating blood. According to this paradigm, coagulation is initiated after a vessel is damaged and blood is exposed to vessel-wall TF. We have examined thrombus formation on pig arterial media (which contains no stainable TF) and on collagen-coated glass slides (which are devoid of TF) exposed to flowing native human blood. In both systems the thrombi that formed during a 5-min perfusion stained intensely for TF, much of which was not associated with cells. Antibodies against TF caused ≈70% reduction in the amount of thrombus formed on the pig arterial media and also reduced thrombi on the collagen-coated glass slides. TF deposited on the slides was active, as there was abundant fibrin in the thrombi. Factor VII
ai
, a potent inhibitor of TF, essentially abolished fibrin production and markedly reduced the mass of the thrombi. Immunoelectron microscopy revealed TF-positive membrane vesicles that we frequently observed in large clusters near the surface of platelets. TF, measured by factor X
a
formation, was extracted from whole blood and plasma of healthy subjects. By using immunostaining, TF-containing neutrophils and monocytes were identified in peripheral blood; our data raise the possibility that leukocytes are the main source of blood TF. We suggest that blood-borne TF is inherently thrombogenic and may be involved in thrombus propagation at the site of vascular injury.