bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of key residues in the subdomain encoded by exons 8 and 9 of human inducible nitric oxide synthase: A critical role for Asp-280 in substrate binding and subunit interactions
Authors: Ghosh Dipak K. , Rashid Mohammad B. , Crane Brian , Taskar Varsha , Mast Molly , Misukonis Mary A. , Weinberg J. Brice , Eissa N. Tony
Published: July 2002

Human inducible nitric oxide synthase (iNOS) is active as a dimer of two identical subunits. Each subunit has an amino-terminal oxygenase domain that binds the substrate
l
-Arg and the cofactors heme and tetrahydrobiopterin and a carboxyl-terminal reductase domain that binds FMN, FAD, and NADPH. We previously demonstrated that a subdomain in the oxygenase domain encoded by exons 8 and 9 is important for dimer formation and NO synthesis. Further, we identified Trp-260, Asn-261, Tyr-267, and Asp-280 as key residues in that subdomain. In this study, using an
Escherichia coli
expression system, we produced, purified, and characterized wild-type iNOS and iNOS-Ala mutants. Using H
2
O
2
-supported oxidation of
N
ω
-hydroxy-
l
-Arg, we demonstrate that the iNOS mutants' inabilities to synthesize NO are due to selective defects in the oxygenase domain activity. Detailed characterization of the Asp-280–Ala mutant revealed that it retains a functional reductase domain, as measured by its ability to reduce cytochrome
c
. Gel permeation chromatography confirmed that the Asp-280–Ala mutant exists as a dimer, but, in contrast to wild-type iNOS, urea-generated monomers of the mutant fail to reassociate into dimers when incubated with
l
-Arg and tetrahydrobiopterin, suggesting inadequate subunit interaction. Spectral analysis reveals that the Asp-280–Ala mutant does not bind
l
-Arg. This indicates that, in addition to dimerization, proper subunit interaction is required for substrate binding. These data, by defining a critical role for Asp-280 in substrate binding and subunit interactions, give insights into the mechanisms of regulation of iNOS activity.
Induction of stereotypy in dopamine-deficient mice requires striatal D1 receptor activation
Authors: Chartoff Elena H. , Marck Brett T. , Matsumoto Alvin M. , Dorsa Daniel M. , Palmiter Richard D.
Published: July 2002

Motor stereotypies are abnormally repetitive behaviors that can develop with excessive dopaminergic stimulation and are features of some neurologic disorders. To investigate the mechanisms required for the induction of stereotypy, we examined the responses of dopamine-deficient (DD) mice to increasing doses of the dopamine precursor
l
-DOPA. DD mice lack the ability to synthesize dopamine (DA) specifically in dopaminergic neurons yet exhibit robust hyperlocomotion relative to wild-type (WT) mice when treated with
l
-DOPA, which restores striatal DA tissue content to ≈10% of WT levels. To further elevate brain DA content in DD mice, we administered the peripheral
l
-amino acid decarboxylase inhibitor carbidopa along with
l
-DOPA (C/
l
-DOPA). When striatal DA levels reached >50% of WT levels, a transition from hyperlocomotion to intense, focused stereotypy was observed that was correlated with an induction of
c-fos
mRNA in the ventrolateral and central striatum as well as the somatosensory cortex. WT mice were unaffected by C/
l
-DOPA treatments. A D1, but not a D2, receptor antagonist attenuated both the C/
l
-DOPA-induced stereotypy and the
c-fos
induction. Consistent with these results, stereotypy could be induced in DD mice by a D1, but not by a D2, receptor agonist, with neither agonist inducing stereotypy in WT mice. Intrastriatal injection of a D1 receptor antagonist ameliorated the stereotypy and
c-fos
induction by C/
l
-DOPA. These results indicate that activation of D1 receptors on a specific population of striatal neurons is required for the induction of stereotypy in DD mice.
Oral immunization with hepatitis B surface antigen expressed in transgenic plants
Authors: Kong Qingxian , Richter Liz , Yang Yu Fang , Arntzen Charles J. , Mason Hugh S. , Thanavala Yasmin
Published: July 2002

Oral immunogenicity of recombinant hepatitis B surface antigen
(HBsAg) derived from yeast (purified product) or in transgenic potatoes
(uncooked unprocessed sample) was compared. An oral adjuvant, cholera
toxin, was used to increase immune responses. Transgenic plant material
containing HBsAg was the superior means of both inducing a primary
immune response and priming the mice to respond to a subsequent
parenteral injection of HBsAg. Electron microscopy of transgenic plant
samples revealed evidence that the HBsAg accumulated intracellularly;
we conclude that natural bioencapsulation of the antigen may provide
protection from degradation in the digestive tract until plant cell
degradation occurs near an immune effector site in the gut. The
correlate of protection from hepatitis B virus infection is serum
antibody titers induced by vaccination; the protective level in humans
is 10 milliunits/ml or greater. Mice fed HBsAg-transgenic potatoes
produced HBsAg-specific serum antibodies that exceeded the protective
level and, on parenteral boosting, generated a strong long-lasting
secondary antibody response. We have also shown the effectiveness of
oral delivery by using a parenteral prime-oral boost immunization
schedule. The demonstrated success of oral immunization for hepatitis B
virus with an “edible vaccine” provides a strategy for
contributing a means to achieve global immunization for hepatitis B
prevention and eradication.
An initiation element in the yeastCUP1promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled
Authors: Leblanc Benoit P. , Benham Craig J. , Clark David J.
Published: July 2002

Purified RNA polymerase II initiated transcription from the yeastCUP1promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of theCUP1promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observedin vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: theCUP1initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, theCUP1promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.
Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity
Authors: Holmes Fiona E. , Mahoney Sally , King Von R. , Bacon Andrea , Kerr Niall C. H. , Pachnis Vassilis , Curtis Rory , Priestley John V. , Wynick David
Published: July 2002

The neuropeptide galanin is expressed developmentally in the dorsal
root ganglion (DRG) and is rapidly up-regulated 120-fold after
peripheral nerve section in the adult. Here we report that adult mice
carrying a loss-of-function mutation in the galanin gene have a 13%
reduction in the number of cells in the DRG associated with a 24%
decrease in the percentage of neurons that express substance P. These
deficits are associated with a 2.8- and 2.6-fold increase in the number
of apoptotic cells in the DRG at postnatal days 3 and 4,
respectively. After crush injury to the sciatic nerve, the rate of
peripheral nerve regeneration is reduced by 35% with associated
long-term functional deficits. Cultured DRG neurons from adult mutant
mice demonstrate similar deficits in neurite number and length. These
results identify a critical role for galanin in the development and
regeneration of sensory neurons.
Cytochrome
c
binding to Apaf-1: The effects of dATP and ionic strength
Authors: Purring-Koch Cherie , McLendon George
Published: July 2002

In the apoptosis pathway in mammals, cytochrome
c
and dATP are critical cofactors in the activation of
caspase 9 by Apaf-1. Until now, the detailed sequence of events in
which these cofactors interact has been unclear. Here, we show through
fluorescence polarization experiments that cytochrome
c
can bind to Apaf-1 in the absence of dATP; when dATP is added to the
cytochrome
c
·Apaf-1 complex, further assembly occurs
to produce the apoptosome. These findings, along with the discovery
that the exposed heme edge of cytochrome
c
is involved
in the cytochrome
c
·Apaf-1 interaction, are confirmed
through enhanced chemiluminescence visualization of native PAGE gels
and through acrylamide fluorescence quenching experiments. We also
report here that the cytochrome
c
·Apaf-1 interaction
depends highly on ionic strength, indicating that there is a strong
electrostatic interaction between the two proteins.
The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80
Authors: Tung Kuei-Shu , Hong Eun-Jin Erica , Roeder G. Shirleen
Published: July 2002

In budding yeast, many mutants defective in meiotic recombination
and chromosome synapsis undergo checkpoint-mediated arrest at the
pachytene stage of meiotic prophase. We recovered the
NDT80
gene in a screen for genes whose overexpression
bypasses the pachytene checkpoint. Ndt80 is a meiosis-specific
transcription factor that promotes expression of genes required for
exit from pachytene and entry into meiosis I. Herein, we show that the
Ndt80 protein accumulates and is extensively phosphorylated during
meiosis in wild type but not in cells arrested at the pachytene
checkpoint. Our results indicate that inhibition of Ndt80 activity is
one mechanism used to achieve pachytene arrest.
Structural requirements of
endo
polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)
Authors: Federici L. , Caprari C. , Mattei B. , Savino C. , Di Matteo A. , De Lorenzo G. , Cervone F. , Tsernoglou D.
Published: July 2002

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them,
endo
polygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 Å resolution of PG from the phytopathogenic fungus
Fusarium moniliforme
(
Fm
PG). The structure of
Fm
PG was useful to study the mode of interaction of the enzyme with PGIP-2 from
Phaseolus vulgaris.
Several amino acids of
Fm
PG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding.
Increased sensitivity to cocaine by cholinergic cell ablation in nucleus accumbens
Authors: Hikida Takatoshi , Kaneko Satoshi , Isobe Tomohiro , Kitabatake Yasuji , Watanabe Dai , Pastan Ira , Nakanishi Shigetada
Published: July 2002

Chronic exposure to cocaine causes long-lasting behavioral changes associated with cocaine reinforcement and addiction. An important neural substrate for cocaine addiction is the nucleus accumbens (NAc), which receives dopaminergic input from the ventral tegmental area. Although the neural circuit of the NAc is controlled by several other neurotransmitters, their involvement in cocaine addiction remains elusive. In this investigation, we ablated cholinergic interneurons from the adult NAc with immunotoxin-mediated cell targeting and examined the role of acetylcholine transmitter in adaptive behavioral changes associated with cocaine reinforcement and addiction. Acute exposure to cocaine induced abnormal rotation in unilaterally cholinergic cell-eliminated mice. This abnormal turning was enhanced by repeated exposure of cocaine. In bilaterally cholinergic cell-eliminated mice, chronic cocaine administration induced a prominent and progressive increase in locomotor activity. Moreover, these mice showed robust conditioned place preference with a lower dose of cocaine, compared with wild-type littermates. This investigation demonstrates that acetylcholine in the NAc plays a key role in both acute and chronic actions of cocaine.
Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations
Authors: Smith Leslie , Plug Annemieke , Thayer Mathew
Published: July 2002

Chromosomal rearrangements are found in virtually all types of human cancers. We show that certain chromosome translocations display a delay in mitotic chromosome condensation that is associated with a delay in the mitosis-specific phosphorylation of histone H3. This delay in mitotic condensation is preceded by a delay in both the initiation as well as the completion of chromosome replication. In addition, chromosomes with this phenotype participate in numerous secondary translocations and rearrangements. Chromosomes with this phenotype were detected in five of seven tumor-derived cell lines and in five of thirteen primary tumor samples. These data suggest that certain chromosomal rearrangements found in tumor cells cause a significant delay in replication timing of the entire chromosome that subsequently results in delayed mitotic chromosome condensation and ultimately in chromosomal instability.
Structure of a quinohemoprotein amine dehydrogenase with an uncommon redox cofactor and highly unusual crosslinking
Authors: Datta Saumen , Mori Youichi , Takagi Kazuyoshi , Kawaguchi Katsunori , Chen Zhi-Wei , Okajima Toshihide , Kuroda Shun'ichi , Ikeda Tokuji , Kano Kenji , Tanizawa Katsuyuki , Mathews F. Scott
Published: July 2002

The crystal structure of the heterotrimeric quinohemoprotein amine
dehydrogenase from
Paracoccus denitrificans
has been
determined at 2.05-Å resolution. Within an 82-residue subunit
is contained an unusual redox cofactor, cysteine tryptophylquinone
(CTQ), consisting of an orthoquinone-modified tryptophan side chain
covalently linked to a nearby cysteine side chain. The subunit is
surrounded on three sides by a 489-residue, four-domain subunit that
includes a diheme cytochrome
c
. Both subunits sit on the
surface of a third subunit, a 337-residue seven-bladed β-propeller
that forms part of the enzyme active site. The small catalytic subunit
is internally crosslinked by three highly unusual covalent
cysteine to aspartic or glutamic acid thioether linkages in addition to
the cofactor crossbridge. The catalytic function of the enzyme as well
as the biosynthesis of the unusual catalytic subunit is discussed.
The Abl-related gene (Arg) nonreceptor tyrosine kinase uses two F-actin-binding domains to bundle F-actin
Authors: Wang Yinxiang , Miller Ann L. , Mooseker Mark S. , Koleske Anthony J.
Published: July 2002

Abl family nonreceptor tyrosine kinases regulate
cellular morphogenesis and motility through functional interactions
with the actin cytoskeleton. Although Abl family kinases are known to
contain filamentous (F)-actin-binding domains at their C termini, it is
unclear how Abl family kinases regulate the structure and/or
function of the actin cytoskeleton. We show here that the Abl-related
kinase Arg binds with positive cooperativity to F-actin
in
vitro
with binding saturating at a ratio of one Arg/two actin
molecules. Measurements of the F-actin-binding properties of Arg
deletion mutants led to the identification of a second, previously
uncharacterized internal F-actin-binding domain in Arg. Purified Arg
can bundle F-actin
in vitro
, and this bundling activity
requires both F-actin-binding domains. An Arg-yellow fluorescent
protein fusion protein can induce the formation of actin-rich
structures at the lamellipodia of Swiss 3T3 fibroblasts. Both of Arg's
F-actin-binding domains are necessary and sufficient for the formation
of these actin-rich structures. Together, our data suggest that Arg can
use its F-actin-bundling activity to directly regulate actin
cytoskeletal structure
in vivo
.
Seven Wnt homologues in
Drosophila
: A case study of the developing tracheae
Authors: Llimargas Marta , Lawrence Peter A.
Published: July 2002

Sequencing of the
Drosophila
genome has revealed
that there are “silent” homologues of many important
genes—family members that were not detected by classic genetic
approaches. Why have so many homologues been conserved during
evolution? Perhaps each one has a different but important function in
every system. Perhaps each one works independently in a different part
of the body. Or, perhaps some are redundant. Here, we take one well
known gene family and analyze how the individual members contribute to
the making of one system, the tracheae. There are seven
DWnt
genes in the
Drosophila
genome, including
wingless
(
wg).
The
wg
gene helps to pattern the developing trachea but is
not responsible for all Wnt functions there. We test each one of the
seven
DWnts
in several ways and find evidence that
wg
and
DWnt2
can function in the
developing trachea: when both genes are removed together, the phenotype
is identical or very similar to that observed when the Wnt
pathway is shut down.
DWnt2
is expressed near the
tracheal cells in the embryo in a different pattern to
wg
but is also transduced through the canonical
Wnt
pathway. We find that the seven
DWnt
genes vary in their effectiveness in specific tissues, such as the
tracheae, and, moreover, the epidermis and the tracheae respond to
DWnt2 and Wg differently. We suggest that the main advantage of
retaining a number of similar genes is that it allows more subtle forms
of control and more flexibility during evolution.
Genetically engineered macrophages expressing IFN-γ restore alveolar immune function inscidmice
Authors: Wu Min , Hussain Shabbir , He Ying-Hui , Pasula Rajamouli , Smith Patricia A. , Martin William J.
Published: July 2002

Reversal of immunodeficiency in the lung by gene therapy is limited in part by the difficulty of transfecting lung cellsin vivo. Many options exist for successfully transfecting cellsin vitro, but they are not easily adapted to thein vivocondition. To overcome this limitation, we transduced macrophagesin vitrowith the murine IFN-γ (mIFN-γ) gene and intratracheally delivered the macrophages to express mIFN-γin vivo. A recombinant retroviral vector pSF91 system was modified to encode mIFN-γ and enhanced green fluorescent protein (EGFP). A murine macrophage cell line J774A.1 transduced with the retroviral supernatant increased secretion from undetectable levels to 131.6 ± 4.2 μg/ml mIFN-γ at 24 hin vitro. The mIFN-γ-producing macrophages were intratracheally instilled into mechanically ventilatedscidmice. mIFN-γ levels in the bronchoalveolar lavage increased from undetectable levels at baseline to 158.8 ± 5.1 pg/ml at 48 h (P< 0.001). Analysis of the lavaged cells for EGFP expression revealed that EGFP expression was directly proportional to the number of transduced macrophages instilled into the lung. Immune function was partially restored in the alveolar spaces ofscidmice with evidence of enhanced MHC class II antigen expression and increased phagocytosis (P< 0.05). Tumor necrosis factor α was increased from undetectable at baseline to 103.5 ± 11.4 pg/ml. In contrast, i.p. administration of the engineered macrophages did not enhance IFN-γ levels in the lung. Our study suggests airway delivery of genetically engineered macrophages expressing mIFN-γ gene can partially restore significant immune activity in the lungs of immunodeficient mice.
Dynamic control of auditory activity during sleep: Correlation between song response and EEG
Authors: Nick Teresa A. , Konishi Masakazu
Published: July 2002

The song nucleus high vocal center (HVC) sends neural signals for song production and receives auditory input. By using electroencephalography (EEG) to objectively identify wake/sleep state, we show that HVC auditory responses change with physiological states. Comparison of EEG and HVC records revealed that HVC response to auditory stimuli is greatest during slow-wave sleep. During slow-wave sleep, HVC neurons responded preferentially to the bird's own song. Strikingly, both spontaneous and forced waking during sleep caused HVC auditory responses to cease within milliseconds of an EEG-measured state change. State-dependent phenomena in downstream nuclei, such as robustus archistriatalis, are likely to be derivatives of those in HVC.
Role of SEREX-defined immunogenic wild-type cellular molecules in the development of tumor-specific immunity
Authors: Nishikawa Hiroyoshi , Tanida Koji , Ikeda Hiroaki , Sakakura Miho , Miyahara Yoshihiro , Aota Takumi , Mukai Katsumi , Watanabe Masato , Kuribayashi Kagemasa , Old Lloyd J. , Shiku Hiroshi
Published: July 2002

Recognition of altered self-antigens in tumor cells by lymphocytes forms the basis for antitumor immune responses. The effector cells in most experimental tumor systems are CD8+T cells that recognize MHC class I binding peptides derived from molecules with altered expression in tumor cells. Although the need for CD4+helper T cells in regulating CD8+T cells has been documented, their target epitopes and functional impact in antitumor responses remain unclear. We examined whether broadly expressed wild-type molecules in murine tumor cells eliciting humoral immunity contributed to the generation of CD8+T cells and protective antitumor immune responses to unrelated tumor-specific antigens [mutated ERK2 (mERK2) and c-erbB2/HER/neu(HER2)]. The immunogenic wild-type molecules, presumably dependent on recognition by CD4+helper T cells, were defined by serological analysis of recombinant cDNA expression libraries (SEREX) using tumor-derived λ phage libraries screened with IgG antibodies of hosts bearing transplanted 3-methylchoranthrene-induced tumors. Coimmunization of mice with plasmids encoding SEREX-defined murine wild-type molecules and mERK2 or HER2 led to a profound increase in CD8+T cells specific for mERK2 or HER2 peptides. This heightened response depended on CD4+T cells and copresentation of SEREX-defined molecules and CD8+T cell epitopes. In tumor protection assays, immunization with SEREX-defined wild-type molecules and mERK2 resulted in an inhibition of pulmonary metastasis, which was not achieved by immunization with mERK2 alone.
Prenatal exposure to high levels of glucocorticoids increases the susceptibility of cerebellar granule cells to oxidative stress-induced cell death
Authors: Ahlbom Eva , Gogvadze Vladimir , Chen Ming , Celsi Gianni , Ceccatelli Sandra
Published: July 2002

There is growing concern that prenatal exposure to excessive
glucocorticoids may have deleterious effects on the development of
various organs, including the nervous system. This study aimed at
evaluating whether prenatal exposure to high levels of glucocorticoids
might produce long-term effects on neuronal cell survival. Pregnant
rats were injected i.p. with 0.1 mg/kg dexamethasone (DEX) from day
14 postconception, and cerebellar granule cells (CGC) were prepared
from 1-week-old rats from DEX-treated and control dams. After 7 days in
culture, cells were exposed to H
2
O
2
,
methylmercury, or colchicine at concentrations known to induce
apoptotic cell death. After exposure to
H
2
O
2
or methylmercury, both inducing oxidative
stress, the number of apoptotic cells was significantly higher
in DEX- than in control-CGC. Because mitochondria play a key role in
apoptosis, mitochondrial function was investigated, and a
decrease in the threshold level of Ca
2+
necessary for
induction of mitochondrial permeability transition, in Ca
2+
accumulation rate, and in oxygen consumption was detected in DEX-CGC.
Moreover, the activity of the antioxidant enzyme catalase was
significantly decreased in DEX-CGC. A similar decrease in catalase
activity was observed in cerebellar homogenate from newborn and
40-day-old DEX-rats. In conclusion, these results indicate that
prenatal exposure to high levels of glucocorticoids induces
long-lasting changes in CGC rendering them more sensitive to oxidative
stress. With the increasing use of multiple doses of glucocorticoids in
preterm infants, the possibility that prenatal exposure to excess
glucocorticoids may lead to long-term neurological consequences becomes
a relevant issue.
Molecular characteristics of non-small cell lung cancer
Authors: Nacht Mariana , Dracheva Tatiana , Gao Yuhong , Fujii Takeshi , Chen Yidong , Player Audrey , Akmaev Viatcheslav , Cook Brian , Dufault Michael , Zhang Mindy , Zhang Wen , Guo MingZhou , Curran John , Han Sean , Sidransky David , Buetow Kenneth , Madden Stephen L. , Jen Jin
Published: July 2002

We used hierarchical clustering to examine gene
expression profiles generated by serial analysis of gene expression
(SAGE) in a total of nine normal lung epithelial cells and non-small
cell lung cancers. Separation of normal and tumor, as well as
histopathological subtypes, was evident by using the 3,921 most
abundant transcript tags. This distinction remained when only 115
highly differentially expressed tags were used. Furthermore, these 115
transcript tags clustered into groups suggestive of the unique
biological and pathological features of the different tissues examined.
Adenocarcinomas were characterized by high-level expression of small
airway-associated or immunologically related proteins, whereas squamous
cell carcinomas overexpressed genes involved in cellular detoxification
or antioxidation. The messages of two p53-regulated genes,

p21
WAF1/CIP1

and
14-3-3σ
, were consistently underexpressed in the
adenocarcinomas, suggesting that the p53 pathway itself might be
compromised in this cancer type. Gene expression patterns observed by
SAGE were consistent with results obtained by quantitative real-time
PCR or cDNA array analyses by using a total of 43 lung tumor and normal
samples. Thus, although derived from only a few tissue libraries, gene
expression profiles obtained by using SAGE most likely represent an
unbiased yet distinctive molecular signature for the most common forms
of human lung cancer.
Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells.
Authors: Diamond G , Russell J P , Bevins C L
Published: July 2002

Mammals continually confront microbes at mucosal surfaces. A current model suggests that epithelial cells contribute to defense at these sites, in part through the production of broad-spectrum antibiotic peptides. Previous studies have shown that invertebrates can mount a host defense response characterized by the induction in epithelia] cells of a variety of antibiotic proteins and peptides when they are challenged with microorganisms, bacterial cell wall/membrane components, or traumatic injury [Boman, H.G. & Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103-126J. However, factors that govern the expression of similar defense molecules in mammalian epithelial cells are poorly understood. Here, a 13-fold induction of the endogenous gene encoding tracheal antimicrobial peptide was found to characterize a host response of tracheal epithelia] cells (TECs) exposed to bacterial lipopolysaccharide (LPS). Northern blot data indicated that TECs express CD14, a well-characterized LPS-binding protein known to mediate many LPS responses. A monoclonal antibody to CD14 blocked the observed tracheal antimicrobial peptide induction by LPS under serum-free conditions. Together the data support that CD14 of epithelial cell origin mediates the LPS induction of an antibiotic peptide gene in TECs, providing evidence for the active participation of epithelial cells in the host's local defense response to bacteria. Furthermore, the data allude to a conservation of this host response in evolution and suggest that a similar inducible pathway of host defense is prevalent at mucosal surfaces of mammals.
Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and developmental success.
Authors: Navara C S , First N L , Schatten G
Published: July 2002

This study describes a paternal effect on sperm aster size and microtubule organization during bovine fertilization. Immunocytochemistry using tubulin antibodies quantitated with confocal microscopy was used to measure the diameter of the sperm aster and assign a score (0-3) based on the degree of radial organization (0, least organized; 3, most organized). Three bulls (A-C) were chosen based on varying fertility (A, lowest fertility; C, highest fertility) as assessed by nonreturn to estrus after artificial insemination and in vitro embryonic development to the blastocyst stage. The results indicate a statistically significant bull-dependent difference in diameter of the sperm aster and in the organization of the sperm astral microtubules. Insemination from bull A resulted in an average sperm aster diameter of 101.4 microm (76.3% of oocyte diameter). This significantly differs (P < or = 0.0001) from the average sperm aster diameters produced after inseminations from bull B (78.2 microm; 60.8%) or bull C (77.9 microm; 57.8%), which themselves displayed no significant differences. The degree of radial organization of the sperm aster was also bull-dependent. Sperm asters organized by bull A-derived sperm had an average quality score of 1.8, which was higher than that of bull B (1.4; P < or = 0.0005) or bull C (1.2; P < or = 0.0001). Results with bulls B and C were also significantly different (P < or = 0.025). These results indicate that the paternally derived portion of the centrosome varies among males and that this variation affects male fertility, the outcome of early development, and, therefore, reproductive success.
Host-parasite dynamics and outgrowth of virus containing a single K70R amino acid change in reverse transcriptase are responsible for the loss of human immunodeficiency virus type 1 RNA load suppression by zidovudine.
Authors: de Jong M D , Veenstra J , Stilianakis N I , Schuurman R , Lange J M , de Boer R J , Boucher C A
Published: July 2002

The association between human immunodeficiency virus type I (HIV-1) RNA load changes and the emergence of resistant virus variants was investigated in 24 HIV-1-infected asymptomatic persons during 2 years of treatment with zidovudine by sequentially measuring serum HIV-1 RNA load and the relative amounts of HIV-1 RNA containing mutations at reverse transcriptase (RT) codons 70 (K-->R), 41 (M-->L), and 215 (T-->Y/F). A mean maximum decline in RNA load occurred during the first month, followed by a resurgence between 1 and 3 months, which appeared independent of drug-resistance. Mathematical modeling suggests that this resurgence is caused by host-parasite dynamics, and thus reflects infection of the transiently increased numbers of CD4+ lymphocytes. Between 3 and 6 months of treatment, the RNA load returned to baseline values, which was associated with the emergence of virus containing a single lysine to arginine amino acid change at RT codon 70, only conferring an 8-fold reduction in susceptibility. Despite the relative loss of RNA load suppression, selection toward mutations at RT codons 215 and 41 continued. Identical patterns were observed in the mathematical model. While host-parasite dynamics and outgrowth of low-level resistant virus thus appear responsible for the loss of HIV-1 RNA load suppression, zidovudine continues to select for alternative mutations, conferring increasing levels of resistance.
Genetic interactions affecting touch sensitivity in Caenorhabditis elegans.
Authors: Gu G , Caldwell G A , Chalfie M
Published: July 2002

At least 13 genes (mec-1, mec-2, mec-4-10, mec-12, mec-14, mec-15, and mec-18) are needed for the response to gentle touch by 6 touch receptor neurons in the nematode Caenorhabditis elegans. Several, otherwise recessive alleles of some of these genes act as dominant enhancer mutations of temperature-sensitive alleles of mec-4, mec-5, mec-6, mec-12, and mec-15. Screens for additional dominant enhancers of mec-4 and mec-5 yielded mutations in previously known genes. In addition, some mec-7 alleles showed allele-specific, dominant suppression of the mec-15 touch-insensitive (Mec) phenotype. The dominant enhancement and suppression exhibited by these mutations suggest that the products of several touch genes interact. These results are consistent with a model, supported by the known sequences of these genes, that almost all of the touch function genes contribute to the mechanosensory apparatus.
An antibody-interleukin 2 fusion protein overcomes tumor heterogeneity by induction of a cellular immune response.
Authors: Becker J C , Varki N , Gillies S D , Furukawa K , Reisfeld R A
Published: July 2002

Antibody-based therapies for cancer rely on the expression of defined antigens on neoplastic cells. However, most tumors display heterogeneity in the expression of such antigens. We demonstrate here that antibody-targeted interleukin 2 delivery overcomes this problem by induction of a host immune response. Immunohistochemical analysis demonstrated that the antibody-interleukin 2 fusion protein-induced eradication of established tumors is mediated by host immune cells, particularly CD8+ T cells. Because of this cellular immune response, antibody-directed interleukin 2 therapy is capable to address established metastases displaying substantial heterogeneity in expression of the targeted antigen. This effector mechanism further enables the induction of partial regressions of large subcutaneous tumors that exceeded more than 5% of the body weight. These observations indicate that antibody-directed cytokine delivery offers an effective new tool for cancer therapy.
Eradication of large colon tumor xenografts by targeted delivery of maytansinoids.
Authors: Liu C , Tadayoni B M , Bourret L A , Mattocks K M , Derr S M , Widdison W C , Kedersha N L , Ariniello P D , Goldmacher V S , Lambert J M , Blättler W A , Chari R V
Published: July 2002

The maytansinoid drug DM1 is 100- to 1000-fold more cytotoxic than anticancer drugs that are currently in clinical use. The immunoconjugate C242-DM1 was prepared by conjugating DM1 to the monoclonal antibody C242, which recognizes a mucin-type glycoprotein expressed to various extents by human colorectal cancers. C242-DM1 was found to be highly cytotoxic toward cultured colon cancer cells in an antigen-specific manner and showed remarkable antitumor efficacy in vivo. C242-DM1 cured mice bearing subcutaneous COLO 205 human colon tumor xenografts (tumor size at time of treatment 65-130 mm3), at doses that showed very little toxicity and were well below the maximum tolerated dose. C242-DM1 could even effect complete regressions or cures in animals with large (260- to 500-mm3) COLO 205 tumor xenografts. Further, C242-DM1 induced complete regressions of subcutaneous LoVo and HT-29 colon tumor xenografts that express the target antigen in a heterogeneous manner. C242-DM1 represents a new generation of immunoconjugates that may yet fulfill the promise of effective cancer therapy through antibody targeting of cytotoxic agents.
The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain.
Authors: Schüddekopf K , Schorpp M , Boehm T
Published: July 2002

Mutations in the whn gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/ winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene revealed the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. As the whnrnu mutant protein lacks this domain, our results indicate that a transactivation function is essential for the activity of the whn transcription factor.
Organization and expression of canine olfactory receptor genes.
Authors: Issel-Tarver L , Rine J
Published: July 2002

Four members of the canine olfactory receptor gene family were characterized. The predicted proteins shared 40-64% identity with previously identified olfactory receptors. The four subfamilies identified in Southern hybridization experiments had as few as 2 and as many as 20 members. All four genes were expressed exclusively in olfactory epithelium. Expression of multiple members of the larger subfamilies was detected, suggesting that most if not all of the cross-hybridizing bands in genomic Southern blots represented actively transcribed olfactory receptor genes. Analysis of large DNA fragments using Southern blots of pulsed-field gels indicated that subfamily members were clustered together, and that two of the subfamilies were closely linked in the dog genome. Analysis of the four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.
Multiple paternity in two natural populations (orchard and vineyard) of Drosophila.
Authors: Ochando M D , Reyes A , Ayala F J
Published: July 2002

Male mating success is an important fitness component in Drosophila. The seminal fluid conveyed with the sperm inhibits the proclivity of the female to remate and reduces her fitness. Nevertheless, females may remate before they have exhausted the sperm from the first male and consequently use sperm from both males. We have studied concurrent multiple paternity (CMP) in two Drosophila melanogaster populations, from an apple orchard and a vineyard just after harvest. CMP is high in both populations, somewhat greater than 50%; but it is not significantly higher in the vineyard, where the population density is much greater than in the orchard. Population density had been thought to be an important determinant of CMP incidence. We have used four gene loci coding for enzymes as independent markers for detecting CMP.
The effects of stress on social preferences are sexually dimorphic in prairie voles.
Authors: DeVries A C , DeVries M B , Taymans S E , Carter C S
Published: July 2002

Prairie voles (Microtus ochrogaster) are monogamous rodents that form pair bonds characterized by a preference for a familiar social partner. In male prairie voles, exposure to either the stress of swimming or exogenous injections of corticosterone facilitate the development of a social preference for a female with which the male was paired after injection or swimming. Conversely, adrenalectomy inhibits partner preference formation in males and the behavioral effects of adrenalectomy are reversed by corticosterone replacement. In female prairie voles, swim stress interferes with the development of social preferences and corticosterone treatments inhibit the formation of partner preferences, while adrenalectomized females form preferences more quickly than adrenally intact controls. Because sex differences in both behavior and physiology are typically reduced in monogamous species, we initially predicted that male and female prairie voles would exhibit similar behavioral responses to corticosterone. However, our findings suggest an unanticipated sexual dimorphism in the physiological processes modulating social preferences. This dimorphic involvement of stress hormones in pair bonding provides a proximate mechanism for regulating social organization, while permitting males and females to adapt their reproductive strategies in response to environmental challenges.
A positive genetic selection for disrupting protein–protein interactions: Identification of CREB mutations that prevent association with the coactivator CBP
Authors: Shih Hsiu-ming , Goldman Phyllis S. , DeMaggio Anthony J. , Hollenberg Stanley M. , Goodman Richard H. , Hoekstra Merl F.
Published: July 2002

TheEscherichia coli tet-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein–protein interactions can be identified by a positive selection. This assay, designated the “split-hybrid system,” contains a two-component reporter. The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator binding sites upstream ofHIS3. Interaction of one protein fused to the LexA DNA binding domain with a second protein fused to the VP16 activation domain results in TetR expression. TetR subsequently binds to thetetoperators, blocking the expression ofHIS3and preventing yeast growth in media lacking histidine. The utility of the split-hybrid system was analyzed by examining the phosphorylation-dependent interaction of CREB and its coactivator CREB binding protein (CBP). CREB and CBP associate through an interaction that depends upon CREB phosphorylation at Ser-133. Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybrid strains. The split-hybrid system was used to identify other CREB mutations that disrupt its association with CBP. These mutations localized around the site of CREB phosphorylation, indicating that only a small portion of the CREB activation domain is required for CBP interaction. The yeast split-hybrid system should be useful in identifying mutations, proteins, peptides, and drugs that disrupt protein–protein interactions.
The anatomy of T-cell activation and tolerance.
Authors: Mondino A , Khoruts A , Jenkins M K
Published: July 2002

The mammalian immune system must specifically recognize and eliminate foreign invaders but refrain from damaging the host. This task is accomplished in part by the production of a large number of T lymphocytes, each bearing a different antigen receptor to match the enormous variety of antigens present in the microbial world. However, because antigen receptor diversity is generated by a random mechanism, the immune system must tolerate the function of T lymphocytes that by chance express a self-reactive antigen receptor. Therefore, during early development, T cells that are specific for antigens expressed in the thymus are physically deleted. The population of T cells that leaves the thymus and seeds the secondary lymphoid organs contains helpful cells that are specific for antigens from microbes but also potentially dangerous T cells that are specific for innocuous extrathymic self antigens. The outcome of an encounter by a peripheral T cell with these two types of antigens is to a great extent determined by the inability of naive T cells to enter nonlymphoid tissues or to be productively activated in the absence of inflammation.
Occurrence of plastid RNA editing in all major lineages of land plants
Authors: Freyer Regina , Kiefer-Meyer Marie-Christine , Kössel Hans
Published: July 2002

RNA editing changes posttranscriptionally single nucleotides in chloroplast-encoded transcripts. Although much work has been done on mechanistic and functional aspects of plastid editing, little is known about evolutionary aspects of this RNA processing step. To gain a better understanding of the evolution of RNA editing in plastids, we have investigated the editing patterns in
ndh
B and
rbc
L transcripts from various species comprising all major groups of land plants. Our results indicate that RNA editing occurs in plastids of bryophytes, fern allies, true ferns, gymnosperms, and angiosperms. Both editing frequencies and editing patterns show a remarkable degree of interspecies variation. Furthermore, we have found that neither plastid editing frequencies nor the editing pattern of a specific transcript correlate with the phylogenetic tree of the plant kingdom. The poor evolutionary conservation of editing sites among closely related species as well as the occurrence of single species-specific editing sites suggest that the differences in the editing patterns and editing frequencies are probably due both to independent loss and to gain of editing sites. In addition, our results indicate that RNA editing is a relatively ancient process that probably predates the evolution of land plants. This supposition is in good agreement with the phylogenetic data obtained for plant mitochondrial RNA editing, thus providing additional evidence for common evolutionary roots of the two plant organellar editing systems.
Three novel families of miniature inverted-repeat transposable elements are associated with genes of the yellow fever mosquito,Aedes aegypti
Authors: Tu Zhijian
Published: July 2002

Three novel families of transposable elements,Wukong,Wujin, andWuneng, are described in the yellow fever mosquito,Aedes aegypti. Their copy numbers range from 2,100 to 3,000 per haploid genome. There are high degrees of sequence similarity within each family, and many structural but not sequence similarities between families. The common structural characteristics include small size, no coding potential, terminal inverted repeats, potential to form a stable secondary structure, A+T richness, and putative 2- to 4-bp A+T-biased specific target sites. Evidence of previous mobility is presented for theWukongelements. Elements of these three families are associated with 7 of 16 fully or partially sequencedAe. aegyptigenes. Characteristics of these mosquito elements indicate strong similarities to the miniature inverted-repeat transposable elements (MITEs) recently found to be associated with plant genes. MITE-like elements have also been reported in two species ofXenopusand inHomo sapiens. This characterization of multiple families of highly repetitive MITE-like elements in an invertebrate extends the range of these elements in eukaryotic genomes. A hypothesis is presented relating genome size and organization to the presence of highly reiterated MITE families. The association of MITE-like elements withAe. aegyptigenes shows the same bias toward noncoding regions as in plants. This association has potentially important implications for the evolution of gene regulation.