bluuebunny/crossref_metadata_2025 huggingface dataset:
Surface processes in CF4/O2 reactive etching of silicon
Authors: Oehrlein Gottlieb S. , Robey Steve W. , Lindström J. Lennart
Published: July 2002

Silicon surfaces etched in CF4/O2 plasma have been characterized with the use of in situ x-ray photoemisson spectroscopy. A SiFxOy layer on elemental silicon is formed under all conditions. For oxygen percentages greater than 5% in the feed gas, the oxygen content of the film and the film thickness increase, whereas the fluorine content of the film decreases. The Si etch rate decreases also and appears to be controlled primarily by the thickness of the SiFxOy layer, rather than by the F atom concentration in the gas phase, which increases up to 15% O2 addition.
Epitaxial Al2O3 films on Si by low-pressure chemical vapor deposition
Authors: Ishida Makoto , Katakabe Ichiro , Nakamura Tetsuro , Ohtake Norio
Published: July 2002

Heteroepitaxial Al2O3 films were grown successfully on (100)Si substrates at substrate temperatures above 1000 °C by low-pressure chemical vapor deposition with the use of N2 bubbled Al(CH3)3 and N2O. From reflection high-energy electron diffraction analysis, the epitaxial films were found to be γ-Al2O3 with an orientation relation of (100)Al2O3//(100)Si. Capacitors with 700-Å-thick Al2O3 films as gate insulators were fabricated and showed high-frequency capacitance-voltage characteristics without hysteresis. An interface state density of 1.7×1011 cm−2 eV−1 was observed by using quasistatic capacitance-voltage measurements. The leakage current was 9.7×10−11 A/cm2 at a gate voltage of 3.0 V.
Ca
2+
marks: Miniature calcium signals in single mitochondria driven by ryanodine receptors
Authors: Pacher Pál , Thomas Andrew P. , Hajnóczky György
Published: July 2002

Propagation of cytosolic [Ca
2+
] ([Ca
2+
]
c
) signals to the mitochondria is believed to be supported by a local communication between Ca
2+
release channels and adjacent mitochondrial Ca
2+
uptake sites, but the signaling machinery has not been explored at the level of elementary Ca
2+
release events. Here, we demonstrate that [Ca
2+
]
c
sparks mediated by ryanodine receptors are competent to elicit miniature mitochondrial matrix [Ca
2+
] signals that we call “Ca
2+
marks.” Ca
2+
marks are restricted to single mitochondria and typically last less than 500 ms. The decay of Ca
2+
marks relies on extrusion of Ca
2+
from the mitochondria through the Ca
2+
exchanger, whereas [Ca
2+
]
c
sparks decline primarily by diffusion. Mitochondria also appear to have a direct effect on the properties of [Ca
2+
]
c
sparks, because inhibition of mitochondrial Ca
2+
uptake results in an increase in the frequency and duration of [Ca
2+
]
c
sparks. Thus, a short-lasting opening of a cluster of Ca
2+
release channels can yield activation of mitochondrial Ca
2+
uptake, and the competency of mitochondrial Ca
2+
handling may be an important determinant of cardiac excitability through local feedback control of elementary [Ca
2+
]
c
signals.
Isolation of a vascular cell wall-specific monoclonal antibody recognizing a cell polarity by using a phage display subtraction method
Authors: Shinohara Naoki , Demura Taku , Fukuda Hiroo
Published: July 2002

Using a strategy consisting of (
i
) the isolation of
cell walls from synchronously differentiating cells of
Zinnia
, (
ii
) the generation of mAbs with
an antibody phage display method, and (
iii
) screening
with a subtraction method, we isolated mAbs recognizing vascular
development-specific cell wall components without prior antigen
identification. One of the isolated mAbs, designated CN 8, recognized a
cell wall component contained in the hemicellulosic fraction.
Immunohistochemical analyses showed that the CN 8 epitope was localized
to the cell wall of immature tracheary elements and xylem parenchyma
cells. In immature tracheary elements, the CN 8 epitope had a polarized
localization pattern regardless of whether the cells are formed as
parts of vessels
in situ
or as single tracheary elements
in vitro
, suggesting that cell polarity autonomously
formed on the cell wall may function in tracheary element
differentiation.
An orchestrated gene expression component of neuronal programmed cell death revealed by cDNA array analysis
Authors: Chiang Lillian W. , Grenier Jill M. , Ettwiller Laurence , Jenkins Lorayne P. , Ficenec Dave , Martin John , Jin Fenyu , DiStefano Peter S. , Wood Andrew
Published: July 2002

Programmed cell death (PCD) during neuronal development and disease
has been shown to require
de novo
RNA synthesis.
However, the time course and regulation of target genes is poorly
understood. By using a brain-biased array of over 7,500 cDNAs, we
profiled this gene expression component of PCD in cerebellar granule
neurons challenged separately by potassium withdrawal, combined
potassium and serum withdrawal, and kainic acid administration. We
found that hundreds of genes were significantly regulated in discreet
waves including known genes whose protein products are involved in PCD.
A restricted set of genes was regulated by all models, providing
evidence that signals inducing PCD can regulate large assemblages of
genes (of which a restricted subset may be shared in multiple
pathways).
Drug selection with paclitaxel restores expression of linked IL-2 receptor γ-chain and multidrug resistance (
MDR
1) transgenes in canine bone marrow
Authors: Licht Thomas , Haskins Mark , Henthorn Paula , Kleiman Sandra E. , Bodine David M. , Whitwam Todd , Puck Jennifer M. , Gottesman Michael M. , Melniczek John R.
Published: July 2002

Unstable expression of transferred genes is a major obstacle to successful gene therapy of hematopoietic diseases. We have investigated in a canine large-animal model whether expression of transduced genes can be recovered
in vivo
. Mixed-breed dogs had undergone autologous bone marrow transplantation (BMT) with stem cell factor and granulocyte–colony-stimulating factor-mobilized retrovirally marked hematopoietic cells. The bicistronic retroviral vector construct allowed for coexpression of
MDR
1 and human IL-2 receptor common γ-chain cDNAs. The latter gene is deficient in X-linked severe combined immunodeficiency. After initial high-level expression, P-glycoprotein and the γ-chain were undetectable in blood and bone marrow 17 months post-BMT. Six months later, one dog was treated i.v. with 125 mg/m
2
paclitaxel. Three administrations restored expression of the two linked genes to high levels in blood and bone marrow. Two dogs treated with higher paclitaxel doses died from myelosuppression after the first administration. As determined by flow cytometry, both genes were expressed in granulocytes, monocytes, and lymphocytes of the surviving animal. PCR analysis of DNA from peripheral blood confirmed that the retroviral cDNA was increased after paclitaxel treatment, suggesting enrichment of transduced cells. P-glycoprotein was detectable for more than 1 year after cessation of paclitaxel. Repeated analyses of blood and bone marrow aspirates gave no indication of hematopoietic disturbance after BMT with transduced cells and paclitaxel treatment. In summary, we have shown that with the use of a drug-selectable marker gene, chemotherapy can select for cells that express an otherwise nonselected therapeutic gene in blood and bone marrow.
An impairment in sniffing contributes to the olfactory impairment in Parkinson's disease
Authors: Sobel Noam , Thomason Moriah E. , Stappen Iris , Tanner Caroline M. , Tetrud James W. , Bower James M. , Sullivan Edith V. , Gabrieli John D. E.
Published: July 2002

Although the presence of an olfactory impairment in Parkinson's
disease (PD) has been recognized for 25 years, its cause remains
unclear. Here we suggest a contributing factor to this impairment,
namely, that PD impairs active sniffing of odorants. We tested 10 men
and 10 women with clinically typical PD, and 20 age- and gender-matched
healthy controls, in four olfactory tasks: (
i
) the
University of Pennsylvania smell identification test;
(
ii
and
iii
) detection threshold tests
for the odorants vanillin and propionic acid; and (
iv
) a
two-alternative forced-choice detection paradigm during which sniff
parameters (airflow peak rate, mean rate, volume, and duration) were
recorded with a pneomatotachograph-coupled spirometer. An additional
experiment tested the effect of intentionally increasing sniff vigor on
olfactory performance in 20 additional patients. PD patients were
significantly impaired in olfactory identification
(
P
< 0.0001) and detection (
P
< 0.007). As predicted, PD patients were also significantly impaired
at sniffing, demonstrating significantly reduced sniff airflow rate
(
P
< 0.01) and volume (
P
<
0.002). Furthermore, a patient's ability to sniff predicted his or her
performance on olfactory tasks, i.e., the more poorly patients sniffed,
the worse their performance on olfaction tests (
P
< 0.009). Finally, increasing sniff vigor improved olfactory
performance in those patients whose baseline performance had been
poorest (
P
< 0.05). These findings implicate a
sniffing impairment as a component of the olfactory impairment in PD
and further depict sniffing as an important component of human
olfaction.
Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex
Authors: Cheng Steven Yan , Bishop J. Michael
Published: July 2002

The Suppressor of Fused [Su(fu)] protein plays a conserved role in the regulation of Gli transcription factors of the hedgehog (Hh) signaling pathway that controls cell fate and tissue patterning during development. In both
Drosophila
and mammals, Su(fu) represses Gli-mediated transcription, but the mode of its action is not completely understood. Recent evidence suggests that Su(fu) physically interacts with the Gli proteins and, when overexpressed, sequesters Gli in the cytoplasm. However, Su(fu) also traverses into the nucleus under the influence of a serine-threonine kinase, Fused (Fu), and has the ability to form a DNA-binding complex with Gli, suggesting that it has a nuclear function. Here we report that the mouse homolog of Su(fu) [mSu(fu)] specifically interacts with SAP18, a component of the mSin3 and histone deacetylase complex. In addition, we demonstrate that mSu(fu) functionally cooperates with SAP18 to repress transcription by recruiting the SAP18-mSin3 complex to promoters containing the Gli-binding element. These results provide biochemical evidence that Su(fu) directly participates in modulating the transcriptional activity of Gli.
Epidermolysis bullosa and embryonic lethality in mice lacking the multi-PDZ domain protein GRIP1
Authors: Bladt Friedhelm , Tafuri Anna , Gelkop Sigal , Langille Lowell , Pawson Tony
Published: July 2002

Glutamate receptor-interacting protein 1 (GRIP1) is an adaptor protein composed of seven PDZ (postsynaptic density-95/Discs large/zona occludens-1) domains, capable of mediating diverse protein–protein interactions. GRIP1 has been implicated in the regulation of neuronal synaptic function, but its physiologic roles have not been defined
in vivo
. We find that elimination of murine GRIP1 results in embryonic lethality.
GRIP1
−/−
embryos develop abnormalities of the dermo-epidermal junction, resulting in extensive skin blistering around day 12 of embryonic life. Ultra-structural characterization of the blisters (or bullae) revealed cleavage of the dermo-epidermal junction below the lamina densa, an alteration reminiscent of the dystrophic form of human epidermolysis bullosa. Blisters were also observed in the lateral ventricle of the brain and in the meninges covering the cerebral cortex. These genetic data suggest that the GRIP1 scaffolding protein is required for the formation and integrity of the dermo-epidermal junction and reveal the importance of PDZ domains in the organization of supramolecular structures essential for mammalian embryonic development.
Transcription factor RF2a alters expression of the rice tungro bacilliform virus promoter in transgenic tobacco plants
Authors: Petruccelli Silvana , Dai Shunhong , Carcamo Rosa , Yin Yanhai , Chen Shouyi , Beachy Roger N.
Published: July 2002

The promoter from rice tungro bacilliform badnavirus (RTBV) is expressed only in phloem tissues in transgenic rice plants. RF2a, a b-Zip protein from rice, is known to bind to the Box II
cis
element near the TATA box of the promoter. Here, we report that the full-length RTBV promoter and a truncated fragment E of the promoter, comprising nucleotides −164 to +45, result in phloem-specific expression of β-glucuronidase (GUS) reporter genes in transgenic tobacco plants. When a fusion gene comprising the cauliflower mosaic virus 35S promoter and RF2a cDNA was coexpressed with the GUS reporter genes, GUS activity was increased by 2–20-fold. The increase in GUS activity was positively correlated with the amount of RF2a, and the expression pattern of the RTBV promoter was altered from phloem-specific to constitutive. Constitutive expression of RF2a did not induce morphological changes in the transgenic plants. In contrast, constitutive overexpression of the b-ZIP domain of RF2a had a strong effect on the development of transgenic plants. These studies suggest that expression of the b-Zip domain can interfere with the function of homologues of RF2a that regulate development of tobacco plants.
Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome
Authors: Arrighi Isabelle , Bloch-Faure May , Grahammer Florian , Bleich Markus , Warth Richard , Mengual Raymond , Drici Milou-Daniel , Barhanin Jacques , Meneton Pierre
Published: July 2002

The voltage-dependent K
+
channel responsible for the slowly activating delayed K
+
current
I
Ks
is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because
KCNQ1
and
KCNE1
genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K
+
and Na
+
intakes. On a normal K
+
diet, homozygous
kcne1
−/−
mice exhibit signs of chronic volume depletion associated with fecal Na
+
and K
+
wasting and have lower plasma K
+
concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K
+
diets or stimulated by low Na
+
diets, a high K
+
diet provokes a tremendous increase of plasma aldosterone levels in
kcne1
−/−
mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K
+
in
kcne1
−/−
mice. This exacerbated aldosterone production in
kcne1
−/−
mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where
I
Ks
may directly participate in the control of aldosterone production by plasma K
+
. These results, which show that KCNE1 and
I
Ks
are involved in K
+
homeostasis, might have important implications for patients with
I
Ks
-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.
Bat predation on nocturnally migrating birds
Authors: Ibáñez Carlos , Juste Javier , García-Mudarra Juan L. , Agirre-Mendi Pablo T.
Published: July 2002

Bat predation on birds is a very rare phenomenon in nature. Most
documented reports of bird-eating bats refer to tropical bats that
occasionally capture resting birds. Millions of small birds concentrate
and cross over the world's temperate regions during migration, mainly
at night, but no nocturnal predators are known to benefit from this
enormous food resource. An analysis of 14,000 fecal pellets of the
greater noctule bat (
Nyctalus lasiopterus
) reveals that
this species captures and eats large numbers of migrating passerines,
making it the only bat species so far known that regularly preys on
birds. The echolocation characteristics and wing morphology of this
species strongly suggest that it captures birds in flight.
Overexpression of
MYC
causes
p53
-dependent G
2
arrest of normal fibroblasts
Authors: Felsher Dean W. , Zetterberg Anders , Zhu Jiyue , Tlsty Thea , Bishop J. Michael
Published: July 2002

Overexpression of the proto-oncogene
MYC
has been implicated in the genesis of diverse human cancers. One explanation for the role of
MYC
in tumorigenesis has been that this gene might drive cells inappropriately through the division cycle, leading to the relentless proliferation characteristic of the neoplastic phenotype. Herein, we report that the overexpression of
MYC
alone cannot sustain the division cycle of normal cells but instead leads to their arrest in G
2
. We used an inducible form of the
MYC
protein to stimulate normal human and rodent fibroblasts. The stimulated cells passed through G
1
and S but arrested in G
2
and frequently became aneuploid, presumably as a result of inappropriate reinitiation of DNA synthesis. Absence of the tumor suppressor gene
p53
or its downstream effector
p21
reduced the frequency of both G
2
arrest and aneuploidy, apparently by compromising the G
2
checkpoint control. Thus, relaxation of the G
2
checkpoint may be an essential early event in tumorigenesis by
MYC
. The loss of
p53
function seems to be one mechanism by which this relaxation commonly occurs. These findings dramatize how multiple genetic events can collaborate to produce neoplastic cells.
Arg-735 of the 100-kDa subunit a of the yeast V-ATPase is essential for proton translocation
Authors: Kawasaki-Nishi Shoko , Nishi Tsuyoshi , Forgac Michael
Published: July 2002

The vacuolar (H
+
)-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and pump protons across specialized plasma membranes. Proton translocation occurs through the integral V
0
domain, which contains five different subunits (a, d, c, c′, and c"). Proton transport is critically dependent on buried acidic residues present in three different proteolipid subunits (c, c′, and c"). Mutations in the 100-kDa subunit a have also influenced activity, but none of these residues has proven to be required absolutely for proton transport. On the basis of previous observations on the F-ATPases, we have investigated the role of two highly conserved arginine residues present in the last two putative transmembrane segments of the yeast V-ATPase a subunit (Vph1p). Substitution of Asn, Glu, or Gln for Arg-735 in TM8 gives a V-ATPase that is fully assembled but is totally devoid of proton transport and ATPase activity. Replacement of Arg-735 by Lys gives a V-ATPase that, although completely inactive for proton transport, retains 24% of wild-type ATPase activity, suggesting a partial uncoupling of proton transport and ATP hydrolysis in this mutant. By contrast, nonconservative mutations of Arg-799 in TM9 lead to both defective assembly of the V-ATPase complex and decreases in activity of the assembled V-ATPase. These results suggest that Arg-735 is absolutely required for proton transport by the V-ATPases and is discussed in the context of a revised model of the topology of the 100-kDa subunit a.
Measuring the forces involved in polyvalent adhesion of uropathogenic
Escherichia coli
to mannose-presenting surfaces
Authors: Liang Michael N. , Smith Stephen P. , Metallo Steven J. , Choi Insung S. , Prentiss Mara , Whitesides George M.
Published: July 2002

Mechanisms of bacterial pathogenesis have become an increasingly
important subject as pathogens have become increasingly resistant to
current antibiotics. The adhesion of microorganisms to the surface of
host tissue is often a first step in pathogenesis and is a plausible
target for new antiinfective agents. Examination of bacterial adhesion
has been difficult both because it is polyvalent and because bacterial
adhesins often recognize more than one type of cell-surface molecule.
This paper describes an experimental procedure that measures the forces
of adhesion resulting from the interaction of uropathogenic
Escherichia coli
to molecularly well defined models of
cellular surfaces. This procedure uses self-assembled monolayers (SAMs)
to model the surface of epithelial cells and optical tweezers to
manipulate the bacteria. Optical tweezers orient the bacteria relative
to the surface and, thus, limit the number of points of attachment
(that is, the valency of attachment). Using this combination, it was
possible to quantify the force required to break a single interaction
between pilus and mannose groups linked to the SAM. These results
demonstrate the deconvolution and characterization of complicated
events in microbial adhesion in terms of specific molecular
interactions. They also suggest that the combination of optical
tweezers and appropriately functionalized SAMs is a uniquely
synergistic system with which to study polyvalent adhesion of bacteria
to biologically relevant surfaces and with which to screen for
inhibitors of this adhesion.
A general additive distance with time-reversibility and rate variation among nucleotide sites.
Authors: Gu X , Li W H
Published: July 2002

As additivity is a very useful property for a distance measure, a general additive distance is proposed under the stationary time-reversible (SR) model of nucleotide substitution or, more generally, under the stationary, time-reversible, and rate variable (SRV) model, which allows rate variation among nucleotide sites. A method for estimating the mean distance and the sampling variance is developed. In addition, a method is developed for estimating the variance-covariance matrix of distances, which is useful for the statistical test of phylogenies and molecular clocks. Computer simulation shows (i) if the sequences are longer than, say, 1000 bp, the SR method is preferable to simpler methods; (ii) the SR method is robust against deviations from time-reversibility; (iii) when the rate varies among sites, the SRV method is much better than the SR method because the distance is seriously underestimated by the SR method; and (iv) our method for estimating the sampling variance is accurate for sequences longer than 500 bp. Finally, a test is constructed for testing whether DNA evolution follows a general Markovian model.
Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain.
Authors: Joung I , Strominger J L , Shin J
Published: July 2002

A novel human cDNA encoding a cytosolic 62-kDa protein (p62) that binds to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner has been cloned. The cDNA is composed of 2074 nucleotides with an open reading frame encoding 440 amino acids. Northern analysis suggests that p62 is expressed ubiquitously in all tissues examined. p62 is not homologous to any known protein in the data base. However, it contains a cysteine-rich region resembling a zinc finger motif, a potential G-protein-binding region, a PEST motif, and several potential phosphorylation sites. Using T7-epitope tagged p62 expression in HeLa cells, the expressed protein was shown to bind to the lck SH2 domain. Deletion of the N-terminal 50 amino acids abolished binding, but mutagenesis of the single tyrosine residue in this region had no effect on binding. Thus, the cloned cDNA indeed encodes the p62 protein, which is a phosphotyrosine-independent ligand for the lck SH2 domain. Its binding mechanism is unique with respect to binding modes of other known ligands for SH2 domains.
Defective STAT signaling by the leptin receptor in diabetic mice.
Authors: Ghilardi N , Ziegler S , Wiestner A , Stoffel R , Heim M H , Skoda R C
Published: July 2002

Leptin and its receptor, obese receptor (OB-R), comprise an important signaling system for the regulation of body weight. Splice variants of OB-R mRNA encode proteins that differ in the length of their cytoplasmic domains. We cloned a long isoform of the wild-type leptin receptor that is preferentially expressed in the hypothalamus and show that it can activate signal transducers and activators of transcription (STAT)-3, STAT-5, and STAT-6. A point mutation within the OB-R gene of diabetic (db) mice generates a new splice donor site that dramatically reduces expression of this long isoform in homozygous db/db mice. In contrast, an OB-R protein with a shorter cytoplasmic domain is present in both db/db and wild-type mice. We show that this short isoform is unable to activate the STAT pathway. These data provide further evidence that the mutation in OB-R causes the db/db phenotype and identify three STAT proteins as potential mediators of the anti-obesity effects of leptin.
RNA binding by the Wilms tumor suppressor zinc finger proteins.
Authors: Caricasole A , Duarte A , Larsson S H , Hastie N D , Little M , Holmes G , Todorov I , Ward A
Published: July 2002

The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms.
Methylation of coding region alone inhibits gene expression in plant protoplasts.
Authors: Hohn T , Corsten S , Rieke S , Müller M , Rothnie H
Published: July 2002

Derivatives of the cauliflower mosaic virus 35S promoter lacking CG and CNG methylation targets were constructed and used to direct transcription of reporter gene constructs in transiently transformed protoplasts. Such methylation-target-free (MTF) promoters, although weaker than the 35S promoter, retain significant activity despite mutation of the as-1 element. The effect of methylation on gene expression in MTF- and 35S-promoter driven constructs was examined. Even when the promoter region was free of methylation targets, reporter gene expression was markedly reduced when cytosine residues in CG dinucleotides were methylated in vitro prior to transformation. Mosaic methylation experiments, in which only specific parts of the plasmids were methylated, revealed that methylation of the coding region alone has a negative effect on reporter gene expression. Methylation nearer the 5' end of the coding region was more inhibitory, consistent with inhibition of transcription elongation.
Opposite actions of nitric oxide on cholinergic synapses: which pathways?
Authors: Mothet J P , Fossier P , Tauc L , Baux G
Published: July 2002

Nitric oxide (NO) produced opposite effects on acetylcholine (ACh) release in identified neuroneuronal Aplysia synapses depending on the excitatory or the inhibitory nature of the synapse. Extracellular application of the NO donor, SIN-1, depressed the inhibitory postsynaptic currents (IPSCs) and enhanced the excitatory postsynaptic currents (EPSCs) evoked by presynaptic action potentials (1/60 Hz). Application of a membrane-permeant cGMP analog mimicked the effect of SIN-1 suggesting the participation of guanylate cyclase in the NO pathway. The guanylate cyclase inhibitor, methylene blue, blocked the NO-induced enhancement of EPSCs but only reduced the inhibition of IPSCs indicating that an additional mechanism participates to the depression of synaptic transmission by NO. Using nicotinamide, an inhibitor of ADP-ribosylation, we found that the NO-induced depression of ACh release on the inhibitory synapse also involves ADP-ribosylation mechanism(s). Furthermore, application of SIN-1 paired with cGMP-dependent protein kinase (cGMP-PK) inhibitors showed that cGMP-PK could play a role in the potentiating but not in the depressing effect of NO on ACh release. Increasing the frequency of stimulation of the presynaptic neuron from 1/60 Hz to 0.25 or 1 Hz potentiated the EPSCs and reduced the IPSCs. In these conditions, the potentiating effect of NO on the excitatory synapse was reduced, whereas its depressing effect on the inhibitory synapse was unaffected. Moreover the frequency-dependent enhancement of ACh release in the excitatory synapse was greatly reduced by the inhibition of NO synthase. Our results indicate that NO may be involved in different ways of modulation of synaptic transmission depending on the type of the synapse including synaptic plasticity.
Long-range repression in the Drosophila embryo.
Authors: Cai H N , Arnosti D N , Levine M
Published: July 2002

Transcriptional repressors can be characterized by their range of action on promoters and enhancers. Short-range repressors interact over distances of 50-150 bp to inhibit, or quench, either upstream activators or the basal transcription complex. In contrast, long-range repressors act over several kilobases to silence basal promoters. We describe recent progress in characterizing the functional properties of one such long-range element in the Drosophila embryo and discuss the contrasting types of gene regulation that are made possible by short- and long-range repressors.
Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.
Authors: Sekiguchi J , Seeman N C , Shuman S
Published: July 2002

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.
A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco.
Authors: He S , Abad A R , Gelvin S B , Mackenzie S A
Published: July 2002

In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239 gene constructs, with or without a mitochondrial targeting sequence, exhibited a semi sterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the ORF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that ORF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.
Organization and expression of the double-stranded RNA genome of Helminthosporium victoriae 190S virus, a totivirus infecting a plant pathogenic filamentous fungus.
Authors: Huang S , Ghabrial S A
Published: July 2002

The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.
The mouse
Pax2
1Neu
mutation is identical to a human
PAX2
mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney
Authors: Favor Jack , Sandulache Rodica , Neuhäuser-Klaus Angelika , Pretsch Walter , Chatterjee Bimal , Senft Elfriede , Wurst Wolfgang , Blanquet Véronique , Grimes Patricia , Spörle Ralf , Schughart Klaus
Published: July 2002

We describe a new mouse frameshift mutation
(
Pax2
1Neu
) with a 1-bp insertion in the
Pax2
gene. This mutation is identical to a previously
described mutation in a human family with renal-coloboma syndrome
[Sanyanusin, P., McNoe, L. A., Sullivan, M. J., Weaver, R. G. &
Eccles, M. R. (1995)
Hum. Mol. Genet.
4, 2183–2184].
Heterozygous mutant mice exhibit defects in the kidney, the optic
nerve, and retinal layer of the eye, and in homozygous mutant embryos,
development of the optic nerve, metanephric kidney, and ventral regions
of the inner ear is severely affected. In addition, we observe a
deletion of the cerebellum and the posterior mesencephalon in
homozygous mutant embryos demonstrating that, in contrast to mutations
in
Pax5
, which is also expressed early in the
mid-hindbrain region, loss of
Pax2
gene function alone
results in the early loss of the mid-hindbrain region. The
mid-hindbrain phenotype is similar to
Wnt1
and
En1
mutant phenotypes, suggesting the conservation of
gene regulatory networks between vertebrates and
Drosophila
.
Lack of chromosome territoriality in yeast: Promiscuous rejoining of broken chromosome ends
Authors: Haber James E. , Leung Wai-Ying
Published: July 2002

Various studies suggest that eukaryotic chromosomes may occupy
distinct territories within the nucleus and that chromosomes are
tethered to a nuclear matrix. These constraints might limit
interchromosomal interactions. We have used a molecular genetic test to
investigate whether the chromosomes of
Saccharomyces
cerevisiae
exhibit such territoriality. A chromosomal
double-strand break (DSB) can be efficiently repaired by recombination
between flanking homologous repeated sequences. We have constructed a
strain in which DSBs are delivered simultaneously to both chromosome
III and chromosome V by induction of the HO endonuclease. The
arrangement of partially duplicated
HIS4
and
URA3
sequences around each HO recognition site allows
the repair of the two DSBs in two alternative ways: (
i
)
the creation of two intrachromosomal deletions or (
ii
)
the formation of a pair of reciprocal translocations. We show that
reciprocal translocations are formed approximately as often as the pair
of intrachromosomal deletions. Similar results were obtained when one
of the target regions was moved from chromosome V to any of three
different locations on chromosome XI. These results argue that the
broken ends of mitotic chromosomes are free to search the entire genome
for appropriate partners; thus, mitotic chromosomes are not
functionally confined to isolated domains of the nucleus, at least when
chromosomes are broken.
Role of p300-family proteins in E1A oncogene induction of cytolytic susceptibility and tumor cell rejection
Authors: Cook James L. , Krantz Carol K. , Routes Barbara A.
Published: July 2002

The mechanism by which the adenoviral (Ad) E1A oncogene induces cellular susceptibility to lysis by killer lymphocytes involves interactions between its first exon and different second-exon accessory regions. Mutational analysis showed that two first-exon regions—one in the N terminus and one in the conserved region 1 (CR1) domain—are necessary for this activity. E1A complex formation with cellular p300 protein through these first-exon-encoded regions correlated with induction of the cytolytic susceptible phenotype but was only effective in the context of E1A second-exon expression. An E1A first-exon deletion that prevented p300 binding eliminated both oncoprotein-induced cytolytic susceptibility and rejection of transfected sarcoma cells by immunocompetent animals. These results suggest that the E1A oncogene induces cytolytic susceptibility and tumor rejection by interactions with cellular proteins of the p300 family that affect transcription of genes involved in the cellular response to injury inflicted by host killer cells.
Skeletal matrices, muci, and the origin of invertebrate calcification.
Authors: Marin F , Smith M , Isa Y , Muyzer G , Westbroek P
Published: July 2002

The sudden appearance of calcified skeletons among many different invertebrate taxa at the Precambrian-Cambrian transition may have required minor reorganization of preexisting secretory functions. In particular, features of the skeletal organic matrix responsible for regulating crystal growth by inhibition may be derived from mucous epithelial excretions. The latter would have prevented spontaneous calcium carbonate overcrusting of soft tissues exposed to the highly supersaturated Late Proterozoic ocean [Knoll, A. H., Fairchild, I. J. & Swett, K. (1993) Palaios 8, 512-525], a putative function for which we propose the term "anticalcification." We tested this hypothesis by comparing the serological properties of skeletal water-soluble matrices and mucous excretions of three invertebrates--the scleractinian coral Galaxea fascicularis and the bivalve molluscs Mytilus edulis and Mercenaria mercenaria. Crossreactivities recorded between muci and skeletal water-soluble matrices suggest that these different secretory products have a high degree of homology. Furthermore, freshly extracted muci of Mytilus were found to inhibit calcium carbonate precipitation in solution.
Ca(2+)-independent reduction of N-methyl-D-aspartate channel activity by protein tyrosine phosphatase.
Authors: Wang Y T , Yu X M , Salter M W
Published: July 2002

Regulation of ion channel function by intracellular processes is fundamental for controlling synaptic signaling and integration in the nervous system. Currents mediated by N-methyl-D-aspartate (NMDA) receptors decline during whole-cell recordings and this may be prevented by ATP. We show here that phosphorylation is necessary to maintain NMDA currents and that the decline is not dependent upon Ca2+. A protein tyrosine phosphatase or a peptide inhibitor of protein tyrosine kinase applied intracellularly caused a decrease in NMDA currents even when ATP was included. On the other hand, pretreating the neurons with a membrane-permeant tyrosine kinase inhibitor occluded the decline in NMDA currents when ATP was omitted. In inside-out patches, applying a protein tyrosine phosphatase to the cytoplasmic face of the patch caused a decrease in probability of opening of NMDA channels. Conversely, open probability was increased by a protein tyrosine phosphatase inhibitor. These results indicate that NMDA channel activity is reduced by a protein tyrosine phosphatase associated with the channel complex.
Temperature effects in hydrophobic interaction chromatography.
Authors: Haidacher D , Vailaya A , Horváth C
Published: July 2002

The effect of temperature from 5 degrees C to 50 degrees C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy, and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.
Reduced sympathetic innervation after alteration of target cell neurotransmitter phenotype in transgenic mice.
Authors: Cho S , Son J H , Park D H , Aoki C , Song X , Smith G P , Joh T H
Published: July 2002

Neurotransmitters play a variety of important roles during nervous system development. In the present study, we hypothesized that neurotransmitter phenotype of both projecting and target cells is an important factor for the final synaptic linkage and its specificity. To test this hypothesis, we used transgenic techniques to convert serotonin/melatonin-producing cells of the pineal gland into cells that also produce dopamine and investigated the innervation of the phenotypically altered target cells. This phenotypic alteration markedly reduced the noradrenergic innervation originating from the superior cervical ganglia. Although the mechanism by which the reduction occurs is presently unknown, quantitative enzyme-linked immunoassay showed the presence of the equivalent amounts of nerve growth factor (NGF) in the control and transgenic pineal glands, suggesting that it occurred in a NGF-independent manner. The results suggest that target neurotransmitter phenotype influences the formation of afferent connections during development.
Increased competitiveness of nematode sperm bearing the male X chromosome
Authors: LaMunyon Craig W. , Ward Samuel
Published: July 2002

Male offspring, which cannot reproduce independently, represent a cost of sexual reproduction. This cost is eliminated by the production of hermaphroditic offspring in the self-fertilizing nematode
Caenorhabditis briggsae
. However, these hermaphrodites can outcross by mating with males. Half the sperm received from males contain no sex chromosome and therefore give rise to male progeny. Mating with males should thus impose the cost of making male offspring. We found that male sperm took immediate precedence over hermaphrodite sperm, resulting in maximized outcrossing, but the appearance of male progeny was delayed after mating. This delay is caused by the male X-bearing sperm outcompeting their nullo-X counterparts. The competitive advantage of X-bearing sperm over nullo-X sperm is limited to sperm from males; it did not occur in a mutant hermaphrodite that produces both types of sperm. The chromosomal effect on sperm competitiveness in
C. briggsae
, which has not been observed in other species, suggests that the X chromosome has evolved a form of meiotic drive, selfishly increasing the competitiveness of sperm that bear it over those that do not. Thus, the multiple levels of sperm competitiveness found in
C. briggsae
maximize outcrossing after mating while delaying the cost of making male offspring.
Interleukin (IL) 1β, IL-1 receptor antagonist, IL-10, and IL-13 gene expression in the central nervous system and anterior pituitary during systemic inflammation: Pathophysiological implications
Authors: Wong Ma-Li , Bongiorno Peter B. , Rettori Valeria , McCann Samuel M. , Licinio Julio
Published: July 2002

The pathophysiology of systemic inflammation and sepsis involves peripheral organs, causing gastrointestinal, renal, and cardiovascular alterations, as well as the central nervous system (CNS), affecting sleep, temperature regulation, behavior, and neuroendocrine function. The molecular basis of the CNS effects of systemic inflammation are not fully elucidated. Here we show that the CNS responds to systemic inflammation with pronounced IL-1β gene expression and limited IL-1 receptor antagonist (IL-1ra), IL-10, and IL-13 gene expression. This pattern occurs throughout the CNS, including areas such as the subfornical organ, pineal gland, neurohypophysis, and hypothalamus. In contrast, in the anterior pituitary, we found limited IL-1β gene expression but marked induction of the mRNA encoding for the secreted isoform of IL-1ra, secreted IL-1ra. We conclude that the central manifestations of peripheral inflammation are mediated by endogenous brain IL-1β synthesized during systemic inflammation in the context of limited central cytokine counter regulation of IL-1. As IL-1β is a potent stimulus for inducible nitric oxide synthase expression and activity, these findings explain our previous observation that systemic inflammation promotes inducible nitric oxide synthase gene expression in the brain and the spillover of NO metabolites into cerebrospinal fluid. The CNS transcription of the HIV-1 replication factor IL-1β in the context of limited transcription of the IL-1 replication inhibitors IL-1ra, IL-10, and IL-13 might help explain the negative impact of systemic inflammation on the clinical course of AIDS. In addition, we propose that IL-1ra may be secreted by the anterior pituitary as a systemic anti-inflammatory hormone that is released in response to IL-1β originated from multiple sources.
Reactivation of silenced, virally transduced genes by inhibitors of histone deacetylase
Authors: Chen Wen Yong , Bailey Evans C. , McCune Steven L. , Dong Jian-Yun , Townes Tim M.
Published: July 2002

Retroviral and adeno-associated viral sequences can dramatically silence transgene expression in mice. We now report that this repression also occurs in stably infected HeLa cells when the cells are grown without selection. Expression of a transduced
lac
Z gene (rAAV/CMV
lac
Z) is silenced in greater than 90% of cells after 60 days in culture. Surprisingly, high-level expression can be reactivated by treating the cells with sodium butyrate or trichostatin A but not with 5-azacytidine. When cell clones with integrated copies of rAAV/CMV
lac
Z were isolated,
lac
Z expression was silenced in 80% of the clones; however,
lac
Z expression was reactivated in all of the silenced clones by treatment with butyrate or trichostatin A. The two drugs also reactivated a silenced globin gene construct (rAAV/HS2αβ
AS3
) in stably infected K562 cells. Trichostatin A is a specific inhibitor of histone deacetylase; therefore, we propose that hyperacetylation of histones after drug treatment changes the structure of chromatin on integrated viral sequences and relieves repression of transduced genes. The reactivation of silenced, transduced genes has implications for gene therapy. Efficient viral gene transfer followed by drug treatment to relieve suppression may provide a powerful combination for treatment of various genetic and infectious diseases.