bluuebunny/crossref_metadata_2025 huggingface dataset:
Olfaction: Transient expression of a putative odorant receptor in the avian notochord
Authors: Nef Serge , Nef Patrick
Published: July 2002

In vertebrates, odors are thought to be detected by a multigene family encoding several hundreds of seven-transmembrane-domain G-protein-coupled receptors found in fish, rat, mouse, dog, and human. Recently, the putative odorant receptor (OR) gene family in the chicken has been characterized. Twelve members have been isolated and subdivided into six subfamilies. Herein, we have further characterized the chicken olfactory receptor subfamily 7 (COR7) composed of two highly related genes (named COR7a and COR7b) which are 98.5% identical. By
in situ
hybridization experiments, both COR7a and COR7b transcripts were detected in the olfactory epithelium from embryonic day 6 (E6) to the new born stage. Within the olfactory epithelium, the spatial distribution of COR7a and COR7b labeled cells was random. We also observed that every individual positive cell did not coexpress the COR7a and COR7b genes. Interestingly, the COR7b gene was found to be transiently expressed in the notochord from E2 to E6, whereas COR7a or any of the other known members of the COR gene family were not detected in this mesodermal tissue. These data suggest that, in addition to its potential role as an OR in the olfactory system, COR7b may also have a function in the notochord that is essential for the dorsoventral organization of the neural tube and of the somitic mesoderm. We also discuss the possible role(s) of a putative OR present in both the notochord and the sensory olfactory epithelium.
How do owls localize interaurally phase-ambiguous signals?
Authors: Saberi Kourosh , Farahbod Haleh , Konishi Masakazu
Published: July 2002

Owls and other animals, including humans, use the difference in arrival time of sounds between the ears to determine the direction of a sound source in the horizontal plane. When an interaural time difference (ITD) is conveyed by a narrowband signal such as a tone, human beings may fail to derive the direction represented by that ITD. This is because they cannot distinguish the true ITD contained in the signal from its phase equivalents that are ITD ±
nT
, where
T
is the period of the stimulus tone and
n
is an integer. This uncertainty is called phase-ambiguity. All ITD-sensitive neurons in birds and mammals respond to an ITD and its phase equivalents when the ITD is contained in narrowband signals. It is not known, however, if these animals show phase-ambiguity in the localization of narrowband signals. The present work shows that barn owls (
Tyto alba
) experience phase-ambiguity in the localization of tones delivered by earphones. We used sound-induced head-turning responses to measure the sound-source directions perceived by two owls. In both owls, head-turning angles varied as a sinusoidal function of ITD. One owl always pointed to the direction represented by the smaller of the two ITDs, whereas a second owl always chose the direction represented by the larger ITD (i.e., ITD −
T
).
Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington disease gene
Authors: Cha Jang-Ho J. , Kosinski Christoph M. , Kerner Julie A. , Alsdorf Stephen A. , Mangiarini Laura , Davies Stephen W. , Penney John B. , Bates Gillian P. , Young Anne B.
Published: July 2002

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9–11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [
3
H]glutamate receptor binding, protein immunoblotting, and
in situ
hybridization. Ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while
N
-methyl-
d
-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not γ-aminobutyric acid receptors. D
1
-like and D
2
-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice.
In situ
hybridization indicated that mGluR and D
1
dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.
Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase
Authors: Taagepera Salme , McDonald David , Loeb Jonathan E. , Whitaker Laura L. , McElroy Anita K. , Wang Jean Y. J. , Hope Thomas J.
Published: July 2002

The ubiquitously expressed nonreceptor tyrosine kinase c-Abl contains three nuclear localization signals, however, it is found in both the nucleus and the cytoplasm of proliferating fibroblasts. A rapid and transient loss of c-Abl from the nucleus is observed upon the initial adhesion of fibroblasts onto a fibronectin matrix, suggesting the possibility of nuclear export [Lewis, J., Baskaran, R., Taagepera, S., Schwartz, M. & Wang, J. (1996)
Proc. Natl. Acad. Sci. USA
93, 15174–15179]. Here we show that the C terminus of c-Abl does indeed contain a functional nuclear export signal (NES) with the characteristic leucine-rich motif. The c-Abl NES can functionally complement an NES-defective HIV Rev protein (RevΔ3NI) and can mediate the nuclear export of glutathione-
S
-transferase. The c-Abl NES function is sensitive to the nuclear export inhibitor leptomycin B. Mutation of a single leucine (L1064A) in the c-Abl NES abrogates export function. The NES-mutated c-Abl, termed c-Abl NES(−), is localized exclusively to the nucleus. Treatment of cells with leptomycin B also leads to the nuclear accumulation of wild-type c-Abl protein. The c-Abl NES(−) is not lost from the nucleus when detached fibroblasts are replated onto fibronectin matrix. Taken together, these results demonstrate that c-Abl shuttles continuously between the nucleus and the cytoplasm and that the rate of nuclear import and export can be modulated by the adherence status of fibroblastic cells.
Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation
Authors: Jin Jianguo , Kunapuli Satya P.
Published: July 2002

ADP is an important platelet agonist causing shape change and aggregation required for physiological hemostasis. We recently demonstrated the existence of two distinct G protein-coupled ADP receptors on platelets, one coupled to phospholipase C, P2Y1, and the other to inhibition of adenylyl cyclase, P2T
AC
. In this study, using specific antagonists for these two receptors, we demonstrated that concomitant intracellular signaling from both the P2T
AC
and P2Y1 receptors is essential for ADP-induced platelet aggregation. Inhibition of signaling through either receptor, by specific antagonists, is sufficient to block ADP-induced platelet aggregation. Furthermore, signaling through the P2T
AC
receptor could be replaced by activation of α
2A
-adrenergic receptors. On the other hand, activation of serotonin receptors supplements signaling through the P2Y1 receptor. Moreover, this mechanism of ADP-induced platelet aggregation could be mimicked by coactivation of two non-ADP receptors coupled to G
i
and G
q
, neither of which can cause platelet aggregation by itself. We propose that platelet aggregation results from concomitant signaling from both the G
i
and G
q
, a mechanism by which G protein-coupled receptors elicit a physiological response.
Mutagenicity of arsenic in mammalian cells: Role of reactive oxygen species
Authors: Hei Tom K. , Liu Su X. , Waldren Charles
Published: July 2002

Arsenite, the trivalent form of arsenic present in the environment, is a known human carcinogen that lacked mutagenic activity in bacterial and standard mammalian cell mutation assays. We show herein that when evaluated in an assay (A
L
cell assay), in which both intragenic and multilocus mutations are detectable, that arsenite is in fact a strong dose-dependent mutagen and that it induces mostly large deletion mutations. Cotreatment of cells with the oxygen radical scavenger dimethyl sulfoxide significantly reduces the mutagenicity of arsenite. Thus, the carcinogenicity of arsenite can be explained at least in part by it being a mutagen that depends on reactive oxygen species for its activity.
Rice (
Oryza sativa
) centromeric regions consist of complex DNA
Authors: Dong Fenggao , Miller Joseph T. , Jackson Scott A. , Wang Guo-Liang , Ronald Pamela C. , Jiang Jiming
Published: July 2002

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence
in situ
hybridization. Six of the elements are middle repetitive, and their copy numbers range from ≈50 to ≈300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented ≈6,000 times in the rice genome and is detected only in
Oryza
species. Fiber-fluorescence
in situ
hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and
Arabidopsis thaliana
chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.
The CC chemokine 6Ckine binds the CXC chemokine receptor CXCR3
Authors: Soto Hortensia , Wang Wei , Strieter Robert M. , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Hedrick Joseph , Zlotnik Albert
Published: July 2002

We cloned the mouse homologue of the chemokine receptor CXCR3, which is located in mouse chromosome X. We screened a large panel of chemokines for their ability to induce a calcium flux in mouse CXCR3-transfected cells and identified a new ligand for this receptor, the recently reported CC chemokine 6Ckine. This represents an example of a CC chemokine, which binds to a CXC chemokine receptor. Like other ligands of this receptor, 6Ckine has angiostatic properties. 6Ckine is known to chemoattract T cells. In line with this, CXCR3 is expressed preferentially in Th1 cells and in lymphoid organs of the IL-10−/−mouse that develops chronic colitis. Its ability to attract T cells as well as its angiostatic properties suggest that 6Ckine may be an effective anti-tumor agent.
Antarctic fish hemoglobins: Evidence for adaptive evolution at subzero temperature
Authors: Bargelloni Luca , Marcato Stefania , Patarnello Tomaso
Published: July 2002

Notothenioids represent a large group of marine teleosts that are mostly endemic to the Antarctic Ocean. In this environment, the low metabolic demand and the high oxygen concentration reduce the need for hemoglobin(s) [Hb(s)]. The extreme condition is represented by the icefish (Channichthyidae, Notothenioidei), the only vertebrates that lack Hb. We obtained the nucleotide sequence coding for the β-globin chain of the single major Hb form in six red-blooded notothenioids. These included
Gymnodraco acuticeps
, one of the closest species to the Hb-less icefish, which is also the only known fish having a single Hb without Bohr effect. This species shows a higher rate of nonsynonymous substitutions (
K
A
), in contrast with the homogeneity of synonymous substitution (
K
S
) rates, and
K
A
/
K
S
ratios significantly greater than one in the majority of comparisons. These results are suggestive of positive selection, diversifying the single major Hb toward specialized functions. A single Hb that is free to diversify means that its role in routine oxygen transport can be reduced in the presence of a combination of physiological, ecological, and environmental factors. Although a reduced “routine” function for Hb, as is apparent in
G. acuticeps
, might, indeed, evoke the lack of Hb in icefish, evidence of diversifying selection reported here is at variance with the hypothesis of a simple trend from a single Hb toward the Hb-less condition.
Evolutionary parameters of the transcribed mammalian genome: An analysis of 2,820 orthologous rodent and human sequences
Authors: Makałowski Wojciech , Boguski Mark S.
Published: July 2002

We have rigorously defined 2,820 orthologous mRNA and protein sequence pairs from rats, mice, and humans. Evolutionary rate analyses indicate that mammalian genes are evolving 17–30% more slowly than previous textbook values. Data are presented on the average properties of mRNA and protein sequences, on variations in sequence conservation in coding and noncoding regions, and on the absolute and relative frequencies of repetitive elements and splice sites in untranslated regions of mRNAs. Our data set contains 1,880 unique human/rodent sequence pairs that represent about 2–4% of all mammalian genes. Of the 1,880 human orthologs, 70% are present on a new gene map of the human genome, thus providing a valuable resource for cross-referencing human and rodent genomes. In addition to comparative mapping, these results have practical applications in the interpretation of noncoding sequence conservation between syntenic regions of human and mouse genomic sequence, and in the design and calibration of gene expression arrays.
A peptidoglycan recognition protein in innate immunity conserved from insects to humans
Authors: Kang Daiwu , Liu Gang , Lundström Annika , Gelius Eva , Steiner Håkan
Published: July 2002

Innate nonself recognition must rely on common structures of invading microbes. In a differential display screen for up-regulated immune genes in the moth
Trichoplusia ni
we have found mechanisms for recognition of bacterial cell wall fragments. One bacteria-induced gene encodes a protein that, after expression in the baculovirus system, was shown to be a peptidoglycan recognition protein (PGRP). It binds strongly to Gram-positive bacteria. We have also cloned the corresponding cDNA from mouse and human and shown this gene to be expressed in a variety of organs, notably organs of the immune system—i.e., bone marrow and spleen. In addition, purified recombinant murine PGRP was shown to possess peptidoglycan affinity. From our results and the sequence homology, we conclude that PGRP is a ubiquitous protein involved in innate immunity, conserved from insects to humans.
Differential expression of two isopentenyl pyrophosphate isomerases and enhanced carotenoid accumulation in a unicellular chlorophyte
Authors: Sun Zairen , Cunningham Francis X. , Gantt Elisabeth
Published: July 2002

The enzyme isopentenyl pyrophosphate (IPP) isomerase catalyzes the reversible isomerization of IPP to produce dimethylallyl pyrophosphate, the initial substrate leading to the biosynthesis of carotenoids and many other long-chain isoprenoids. Expression of IPP isomerase, and of two enzymes specific to the carotenoid pathway (lycopene β-cyclase and β-carotene-C-4-oxygenase), was followed in the green unicellular alga
Haematococcus pluvialis
after exposure to high illumination. This alga uniquely accumulates carotenoids in the cytoplasm and in late developmental stages turns deep-red in color because of accumulation of ketocarotenoids in the cytosol. The carotenoid/chlorophyll ratio increased 3-fold in wild type and 6-fold in a precocious carotenoid-accumulating mutant (Car-3) within 24 h after increasing the illumination from 20 to 150 μmol photon m
−2
⋅s
−1
. Two cDNAs encoding IPP isomerase in
Haematococcus
,
ipiHp1
and
ipiHp2,
were identified. Although otherwise highly similar (95% identity overall), the predicted sequence of
ipiHp1
contained a 12-aa region not found in that of
ipiHp2
. This was reflected by a size difference between two polypeptides of 34 and 32.5 kDa, both of which reacted with an antibody to the product of
ipiHp1
. We suggest that the 32.5-kDa form is involved with the carotenoid accumulation in the cytoplasm, since the 32.5-kDa polypeptide was preferentially up-regulated by high light preceding the carotenoid increase and only this form was detected in red cysts.
The
PEBP2
β
MYH11
fusion created by Inv(16)(p13;q22) in myeloid leukemia impairs neutrophil maturation and contributes to granulocytic dysplasia
Authors: Kogan Scott C. , Lagasse Eric , Atwater Susan , Bae Suk-chul , Weissman Irving , Ito Yoshiaki , Bishop J. Michael
Published: July 2002

Chromosomal translocations involving the genes encoding the alpha and beta subunits of the Pebp2/Cbf transcription factor have been associated with human acute myeloid leukemia and the preleukemic condition, myelodysplasia. Inv(16)(p13;q22) fuses the gene encoding the beta subunit of Pebp2 to the
MYH11
gene encoding a smooth muscle myosin heavy chain (Smmhc). To examine the effect of the inv(16)(p13;q22) on myelopoiesis, we used the
hMRP8
promoter element to generate transgenic mice expressing the Pebp2βSmmhc chimeric fusion protein in myeloid cells. Neutrophil maturation was impaired in
PEBP2βMYH11
transgenic mice. Although the transgenic mice had normal numbers of circulating neutrophils, their bone marrow contained increased numbers of immature neutrophilic cells, which exhibited abnormal characteristics. In addition,
PEBP2βMYH11
inhibited neutrophilic differentiation in colonies derived from hematopoietic progenitors. Coexpression of both
PEBP2βMYH11
and activated
NRAS
induced a more severe phenotype characterized by abnormal nuclear morphology indicative of granulocytic dysplasia. These results show that
PEBP2βMYH11
can impair neutrophil development and provide evidence that alterations of Pebp2 can contribute to the genesis of myelodysplasia.
Aberrant methylation of
p16
INK4a
is an early event in lung cancer and a potential biomarker for early diagnosis
Authors: Belinsky Steven A. , Nikula Kristen J. , Palmisano William A. , Michels Ruth , Saccomanno Geno , Gabrielson Edward , Baylin Stephen B. , Herman James G.
Published: July 2002

The
p16
INK4a
(
p16
) tumor suppressor gene can be inactivated by promoter region hypermethylation in many tumor types including lung cancer, the leading cause of cancer-related deaths in the U.S. We have determined the timing of this event in an animal model of lung carcinogenesis and in human squamous cell carcinomas (SCCs). In the rat, 94% of adenocarcinomas induced by the tobacco specific carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone were hypermethylated at the
p16
gene promoter; most important, this methylation change was frequently detected in precursor lesions to the tumors: adenomas, and hyperplastic lesions. The timing for
p16
methylation was recapitulated in human SCCs where the
p16
gene was coordinately methylated in 75% of carcinoma
in situ
lesions adjacent to SCCs harboring this change. Moreover, the frequency of this event increased during disease progression from basal cell hyperplasia (17%) to squamous metaplasia (24%) to carcinoma
in situ
(50%) lesions. Methylation of
p16
was associated with loss of expression in both tumors and precursor lesions indicating that both alleles were functionally inactivated. The potential of using assays for aberrant
p16
methylation to identify disease and/or risk was validated by detection of this change in sputum from three of seven patients with cancer and 5 of 26 cancer-free individuals at high risk. These studies show for the first time that an epigenetic alteration, aberrant methylation of the
p16
gene, can be an early event in lung cancer and may constitute a new biomarker for early detection and monitoring of prevention trials.
The anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the
Escherichia coli
methionine aminopeptidase
Authors: Lowther W. Todd , McMillen Debra A. , Orville Allen M. , Matthews Brian W.
Published: July 2002

Methionine aminopeptidase (MetAP) exists in two forms (type I and type II), both of which remove the N-terminal methionine from proteins. It previously has been shown that the type II enzyme is the molecular target of fumagillin and ovalicin, two epoxide-containing natural products that inhibit angiogenesis and suppress tumor growth. By using mass spectrometry, N-terminal sequence analysis, and electronic absorption spectroscopy we show that fumagillin and ovalicin covalently modify a conserved histidine residue in the active site of the MetAP from
Escherichia coli
, a type I enzyme. Because all of the key active site residues are conserved, it is likely that a similar modification occurs in the type II enzymes. This modification, by occluding the active site, may prevent the action of MetAP on proteins or peptides involved in angiogenesis. In addition, the results suggest that these compounds may be effective pharmacological agents against pathogenic and resistant forms of
E. coli
and other microorganisms.
Charge transfer and transport in DNA
Authors: Jortner Joshua , Bixon Mordechai , Langenbacher Thomas , Michel-Beyerle Maria E.
Published: July 2002

We explore charge migration in DNA, advancing two distinct mechanisms of charge separation in a donor (d)–bridge ({Bj})–acceptor (a) system, where {Bj} = B1,B2, … , BNare the N-specific adjacent bases of B-DNA: (i) two-center unistep superexchange induced charge transfer, d*{Bj}a → d∓{Bj}a±, and (ii) multistep charge transport involves charge injection from d* (or d+) to {Bj}, charge hopping within {Bj}, and charge trapping by a. For off-resonance coupling, mechanismiprevails with the charge separation rate and yield exhibiting an exponential dependence ∝ exp(−βR) on the d-a distance (R). Resonance coupling results in mechanismiiwith the charge separation lifetime τ ∝ Nηand yield Y ≃ (1 + δ̄ Nη)−1exhibiting a weak (algebraic) N and distance dependence. The power parameter η is determined by charge hopping random walk. Energetic control of the charge migration mechanism is exerted by the energetics of the ion pair state d∓B1±B2 … BNa relative to the electronically excited donor doorway state d*B1B2 … BNa. The realization of charge separation via superexchange or hopping is determined by the base sequence within the bridge. Our energetic–dynamic relations, in conjunction with the energetic data for d*/d−and for B/B+, determine the realization of the two distinct mechanisms in different hole donor systems, establishing the conditions for “chemistry at a distance” after charge transport in DNA. The energetic control of the charge migration mechanisms attained by the sequence specificity of the bridge is universal for large molecular-scale systems, for proteins, and for DNA.
A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia
Authors: Kawasaki Hiroaki , Springett Gregory M. , Toki Shinichiro , Canales Juan J. , Harlan Patricia , Blumenstiel Justin P. , Chen Emy J. , Bany I. Amy , Mochizuki Naoki , Ashbacher Amy , Matsuda Michiyuki , Housman David E. , Graybiel Ann M.
Published: July 2002

Ras proteins, key regulators of growth, differentiation, and malignant transformation, recently have been implicated in synaptic function and region-specific learning and memory functions in the brain. Rap proteins, members of the Ras small G protein superfamily, can inhibit Ras signaling through the Ras/Raf-1/mitogen-activated protein (MAP) kinase pathway or, through B-Raf, can activate MAP kinase. Rap and Ras proteins both can be activated through guanine nucleotide exchange factors (GEFs). Many Ras GEFs, but to date only one Rap GEF, have been identified. We now report the cloning of a brain-enriched gene, CalDAG-GEFI, which has substrate specificity for Rap1A, dual binding domains for calcium (Ca
2+
) and diacylglycerol (DAG), and enriched expression in brain basal ganglia pathways and their axon-terminal regions. Expression of CalDAG-GEFI activates Rap1A and inhibits Ras-dependent activation of the Erk/MAP kinase cascade in 293T cells. Ca
2+
ionophore and phorbol ester strongly and additively enhance this Rap1A activation. By contrast, CalDAG-GEFII, a second CalDAG-GEF family member that we cloned and found identical to RasGRP [Ebinu, J. O., Bottorff, D. A., Chan, E. Y. W., Stang, S. L., Dunn, R. J. & Stone, J. C. (1998)
Science
280, 1082–1088], exhibits a different brain expression pattern and fails to activate Rap1A, but activates H-Ras, R-Ras, and the Erk/MAP kinase cascade under Ca
2+
and DAG modulation. We propose that CalDAG-GEF proteins have a critical neuronal function in determining the relative activation of Ras and Rap1 signaling induced by Ca
2+
and DAG mobilization. The expression of CalDAG-GEFI and CalDAG-GEFII in hematopoietic organs suggests that such control may have broad significance in Ras/Rap regulation of normal and malignant states.
Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase
Authors: Lipman Richard S. A. , Hou Ya-Ming
Published: July 2002

Aminoacyl-tRNA synthetases catalyze aminoacylation of tRNAs by joining an amino acid to its cognate tRNA. The selection of the cognate tRNA is jointly determined by separate structural domains that examine different regions of the tRNA. The cysteine-tRNA synthetase of
Escherichia coli
has domains that select for tRNAs containing U73, the GCA anticodon, and a specific tertiary structure at the corner of the tRNA L shape. The
E. coli
enzyme does not efficiently recognize the yeast or human tRNA
Cys
, indicating the evolution of determinants for tRNA aminoacylation from
E. coli
to yeast to human and the coevolution of synthetase domains that interact with these determinants. By successively modifying the yeast and human tRNA
Cys
to ones that are efficiently aminoacylated by the
E. coli
enzyme, we have identified determinants of the tRNA that are important for aminoacylation but that have diverged in the course of evolution. These determinants provide clues to the divergence of synthetase domains. We propose that the domain for selecting U73 is conserved in evolution. In contrast, we propose that the domain for selecting the corner of the tRNA L shape diverged early, after the separation between
E. coli
and yeast, while that for selecting the GCA-containing anticodon loop diverged late, after the separation between yeast and human.
A p53-dependent S-phase checkpoint helps to protect cells from DNA damage in response to starvation for pyrimidine nucleotides
Authors: Agarwal Munna L. , Agarwal Archana , Taylor William R. , Chernova Olga , Sharma Yogesh , Stark George R.
Published: July 2002

Normal mammalian cells arrest primarily in G
1
in response to
N-
(phosphonacetyl)-
l
-aspartate (PALA), which starves them for pyrimidine nucleotides, and do not generate or tolerate amplification of the CAD gene, which confers resistance to PALA. Loss of p53, accompanied by loss of G
1
arrest, permits CAD gene amplification and the consequent formation of PALA-resistant colonies. We have found rat and human cell lines that retain wild-type p53 but have lost the ability to arrest in G
1
in response to PALA. However, these cells still fail to give PALA-resistant colonies and are protected from DNA damage through the operation of a second checkpoint that arrests them reversibly within S-phase. This S-phase arrest, unmasked in the absence of the G
1
checkpoint, is dependent on p53 and independent of p21/waf1.
Impaired metabolic modulation of α-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle
Authors: Thomas Gail D. , Sander Mikael , Lau Kim S. , Huang Paul L. , Stull James T. , Victor Ronald G.
Published: July 2002

The neuronal isoform of nitric oxide synthase (nNOS) is highly expressed in mammalian skeletal muscle, but its functional role has not been defined. NO has been implicated in the local metabolic regulation of blood flow in contracting skeletal muscle in part by antagonizing sympathetic vasoconstriction. We therefore hypothesized that nNOS in skeletal muscle is the source of the NO mediating the inhibition of sympathetic vasoconstriction in contracting muscle. In the
mdx
mouse, a model of Duchenne muscular dystrophy in which dystrophin deficiency results in greatly reduced expression of nNOS in skeletal muscle, we found that the normal ability of skeletal muscle contraction to attenuate α-adrenergic vasoconstriction is defective. Similar results were obtained in mutant mice that lack the gene encoding nNOS. Together these data suggest a specific role for nNOS in the local metabolic inhibition of α-adrenergic vasoconstriction in active skeletal muscle.
A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery
Authors: Murphy John E. , Uno Tetsuo , Hamer Janice D. , Cohen Fred E. , Dwarki Varavani , Zuckermann Ronald N.
Published: July 2002

A family of N-substituted glycine oligomers (peptoids) of defined length and sequence are shown to condense plasmid DNA into small particles, protect it from nuclease degradation, and efficiently mediate the transfection of several cell lines. The oligomers were discovered by screening a combinatorial library of cationic peptoids that varied in length, density of charge, side-chain shape, and hydrophobicity. Transfection activity and peptoid–DNA complex formation are shown to be highly dependent on the peptoid structure. The most active peptoid is a 36-mer that contains 12 cationic aminoethyl side chains. This molecule can be synthesized efficiently from readily available building blocks. The peptoid condenses plasmid DNA into uniform particles 50–100 nm in diameter and mediates the transfection of a number of cell lines with efficiencies greater than or comparable to DMRIE-C, Lipofectin, and Lipofectamine. Unlike many cationic lipids, peptoids are capable of working in the presence of serum.
Comparative genetics in the grasses
Authors: Gale Michael D. , Devos Katrien M.
Published: July 2002

Genetic mapping of wheat, maize, and rice and other grass species
with common DNA probes has revealed remarkable conservation of gene
content and gene order over the 60 million years of radiation of
Poaceae.
The linear organization of genes in some nine
different genomes differing in basic chromosome number from 5 to 12 and
nuclear DNA amount from 400 to 6,000 Mb, can be described in terms of
only 25 “rice linkage blocks.” The extent to which this
intergenomic colinearity is confounded at the micro level by gene
duplication and micro-rearrangements is still an open question.
Nevertheless, it is clear that the elucidation of the organization of
the economically important grasses with larger genomes, such as maize
(2
n
= 10, 4,500 Mb DNA), will, to a greater or
lesser extent, be predicted from sequence analysis of smaller genomes
such as rice, with only 400 Mb, which in turn may be greatly aided by
knowledge of the entire sequence of
Arabidopsis
, which
may be available as soon as the turn of the century. Comparative
genetics will provide the key to unlock the genomic secrets of crop
plants with bigger genomes than
Homo sapiens.
A cysteine-rich domain of the “mannose” receptor mediates GalNAc-4-SO
4
binding
Authors: Fiete Dorothy J. , Beranek Mary C. , Baenziger Jacques U.
Published: July 2002

A critical element of lutropin bioactivity
in vivo
is its rapid removal from the blood by a receptor, located in hepatic endothelial cells, that recognizes the terminal sulfated carbohydrate structure SO
4
-4-GalNAcβ1,4GlcNAcβ1,2Manα (S4GGnM). We have previously shown that the macrophage mannose (Man)-receptor cDNA directs the synthesis of a protein that binds oligosaccharides with either terminal S4GGnM or terminal Man, at independent sites. We now show that the cysteine-rich (Cys-Rich) domain at the N terminus of the Man/S4GGnM receptor accounts for binding of oligosaccharides with terminal GalNAc-4-SO
4
, whereas calcium-dependent carbohydrate recognition domains (CRDs) account for binding of ligands containing terminal Man. The Cys-Rich domain is thus a previously unrecognized carbohydrate binding motif. Cys-Rich domains have been described on the three other members of the endocytic C-type lectin family of receptors. The structural relationship of these receptors to the Man/S4GGnM receptor raises the possibility that their Cys-Rich domains also bind carbohydrate moieties and contribute to their function.
HFE gene knockout produces mouse model of hereditary hemochromatosis
Authors: Zhou Xiao Yan , Tomatsu Shunji , Fleming Robert E. , Parkkila Seppo , Waheed Abdul , Jiang Jinxing , Fei Ying , Brunt Elizabeth M. , Ruddy David A. , Prass Cynthia E. , Schatzman Randall C. , O’Neill Rosemary , Britton Robert S. , Bacon Bruce R. , Sly William S.
Published: July 2002

Hereditary hemochromatosis (HH) is a common autosomal recessive disease characterized by increased iron absorption and progressive iron storage that results in damage to major organs in the body. Recently, a candidate gene for HH called HFE encoding a major histocompatibility complex class I-like protein was identified by positional cloning. Nearly 90% of Caucasian HH patients have been found to be homozygous for the same mutation (C282Y) in the HFE gene. To test the hypothesis that the HFE gene is involved in regulation of iron homeostasis, we studied the effects of a targeted disruption of the murine homologue of the HFE gene. The HFE-deficient mice showed profound differences in parameters of iron homeostasis. Even on a standard diet, by 10 weeks of age, fasting transferrin saturation was significantly elevated compared with normal littermates (96 ± 5% vs. 77 ± 3%,
P
< 0.007), and hepatic iron concentration was 8-fold higher than that of wild-type littermates (2,071 ± 450 vs. 255 ± 23 μg/g dry wt,
P
< 0.002). Stainable hepatic iron in the HFE mutant mice was predominantly in hepatocytes in a periportal distribution. Iron concentrations in spleen, heart, and kidney were not significantly different. Erythroid parameters were normal, indicating that the anemia did not contribute to the increased iron storage. This study shows that the HFE protein is involved in the regulation of iron homeostasis and that mutations in this gene are responsible for HH. The knockout mouse model of HH will facilitate investigation into the pathogenesis of increased iron accumulation in HH and provide opportunities to evaluate therapeutic strategies for prevention or correction of iron overload.
Bridging of β-catenin and glycogen synthase kinase-3β by Axin and inhibition of β-catenin-mediated transcription
Authors: Sakanaka Chie , Weiss Joseph B. , Williams Lewis T.
Published: July 2002

Axin antagonizes the developmental effects of Wnt in vertebrates. We show here that Axin simultaneously binds two components of the Wnt pathway, β-catenin and its negative regulator glycogen synthase kinase-3β. In mammalian cells, Axin inhibits Wnt-1 stimulation of β-catenin/lymphoid enhancer factor 1-dependent transcription. Axin also blocks β-catenin-mediated transcription in colon cancer cells that have a mutation in the adenomatous polyposis coli gene. These findings suggest that Axin, by forming a complex with β-catenin and glycogen synthase kinase-3β, can block signaling stimulated by Wnt or by adenomatous polyposis coli mutations.
Epoxidation of olefins by cytochrome P450: Evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant
Authors: Vaz Alfin D. N. , McGinnity Dermot F. , Coon Minor J.
Published: July 2002

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and
cis
- or
trans
-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO
2
H)
3+
and oxenoid-iron (FeO)
3+
, can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.
Budesonide epimer R or dexamethasone selectively inhibit platelet-activating factor-induced or interleukin 1β-induced DNA binding activity ofcis-acting transcription factors and cyclooxygenase-2 gene expression in human epidermal keratinocytes
Authors: Lukiw Walter J. , Pelaez Ricardo Palacios , Martinez Jorge , Bazan Nicolas G.
Published: July 2002

To further understand the molecular mechanism of glucocorticoid action on gene expression, DNA-binding activities of thecis-acting transcription factors activator protein 1 (AP1), AP2, Egr1 (zif268), NF-κB, the signal transducers and activators of transcription proteins gamma interferon activation site (GAS), Sis-inducible element, and the TATA binding protein transcription factor II D (TFIID) were examined in human epidermal keratinocytes. The cytokine interleukin 1β (IL-1β) and platelet-activating factor (PAF), both potent mediators of inflammation, were used as triggers for gene expression. Budesonide epimer R (BUDeR) and dexamethasone (DEX) were studied as potential antagonists. BUDeR or DEX before IL-1β- or PAF-mediated gene induction elicited strong inhibition of AP1-, GAS-, and in particular NF-κB-DNA binding (P< 0.001, ANOVA). Only small effects were noted on AP2, Egr1 (zif268), and Sis-inducible element-DNA binding (P> 0.05). No significant effect was noted on the basal transcription factor TFIID recognition of TATA-containing core promoter sequences (P> 0.68). To test the hypothesis that changingcis-acting transcription factor binding activity may be involved in inflammatory-response related gene transcription, RNA message abundance for human cyclooxygenase (COX)-1 and -2 (E.C.1.14.99.1) was assessed in parallel by using reverse transcription–PCR. Although the COX-1 gene was found to be expressed at constitutively low levels, the TATA-containing COX-2 gene, which contains AP1-like, GAS, and NF-κB DNA-binding sites in its immediate promoter, was found to be strongly induced by IL-1β or PAF (P< 0.001). BUDeR and DEX both suppressed COX-2 RNA message generation; however, no correlation was associated with TFIID–DNA binding. These results suggest that on stimulation by mediators of inflammation, although the basal transcription machinery remains intact, modulation ofcis-activating transcription factor AP1, GAS, and NF-κB-DNA binding by the glucocorticoids BUDeR and DEX play important regulatory roles in the extent of specific promoter activation and hence the expression of key genes involved in the inflammatory response.
Virulence of antibiotic-resistant
Salmonella typhimurium
Authors: Björkman Johanna , Hughes Diarmaid , Andersson Dan I.
Published: July 2002

We show that most
Salmonella typhimurium
mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice. Of seven resistant mutants examined, six were avirulent and one was similar to the wild type in competition experiments in mice. The avirulent-resistant mutants rapidly accumulated various types of compensatory mutations that restored virulence without concomitant loss of resistance. Such second-site compensatory mutations were more common then reversion to the sensitive wild type. We infer from these results that a reduction in the use of antibiotics might not result in the disappearance of the resistant bacteria already present in human and environmental reservoirs. Thus, second-site compensatory mutations could increase the fitness of resistant bacteria and allow them to persist and compete successfully with sensitive strains even in an antibiotic-free environment.
Homology-directed repair is a major double-strand break repair pathway in mammalian cells
Authors: Liang Feng , Han Mingguang , Romanienko Peter J. , Jasin Maria
Published: July 2002

Mammalian cells have been presumed to repair potentially lethal chromosomal double-strand breaks (DSBs) in large part by processes that do not require homology to the break site. This contrasts with
Saccharomyces cerevisiae
where the major DSB repair pathway is homologous recombination. Recently, it has been determined that DSBs in genomic DNA in mammalian cells can stimulate homologous recombination as much as 3 or 4 orders of magnitude, suggesting that homology-directed repair may play an important role in the repair of chromosomal breaks. To determine whether mammalian cells use recombinational repair at a significant level, we have analyzed the spectrum of repair events at a defined chromosomal break by using direct physical analysis of repair products. When an endonuclease-generated DSB is introduced into one of two direct repeats, homologous repair is found to account for 30–50% of observed repair events. Both noncrossover and deletional homologous repair products are detected, at approximately a 1:3 ratio. These results demonstrate the importance of homologous recombination in the repair of DSBs in mammalian cells. In the remaining observed repair events, DSBs are repaired by nonhomologous processes. The nonhomologous repair events generally result in small deletions or insertions at the break site, although a small fraction of events result in larger chromosomal rearrangements. Interestingly, in two insertions, GT repeats were integrated at one of the broken chromosome ends, suggesting that DSB repair can contribute to the spread of microsatellite sequences in mammalian genomes.
In situ
determination of transient pK
a
changes of internal amino acids of bacteriorhodopsin by using time-resolved attenuated total reflection Fourier-transform infrared spectroscopy
Authors: Zscherp Christian , Schlesinger Ramona , Tittor Jörg , Oesterhelt Dieter , Heberle Joachim
Published: July 2002

Active proton transfer through membrane proteins is accomplished by shifts in the acidity of internal amino acids, prosthetic groups, and water molecules. The recently introduced step-scan attenuated total reflection Fourier-transform infrared (ATR/FT-IR) spectroscopy was employed to determine transient pK
a
changes of single amino acid side chains of the proton pump bacteriorhodopsin. The high pK
a
of D96 (>12 in the ground state) drops to 7.1 ± 0.2 (in 1 M KCl) during the lifetime of the N intermediate, quantitating the role of D96 as the internal proton donor of the retinal Schiff base. We conclude from experiments on the pH dependence of the proton release reaction and on point mutants where each of the glutamates on the extracellular surface has been exchanged that besides D85 no other carboxylic group changes its protonation state during proton release. However, E194 and E204 interact with D85, the primary proton acceptor of the Schiff base proton. The C⩵O stretching vibration of D85 undergoes a characteristic pH-dependent shift in frequency during the M state of wild-type bacteriorhodopsin with a pK
a
of 5.2 (±0.3) which is abolished in the single-site mutants E194Q and E204Q and the quadruple mutant E9Q/E74Q/E194Q/E204Q. The double mutation E9Q/E74Q does not affect the lifetime of the intermediates, ruling out any participation of these residues in the proton transfer chain of bacteriorhodopsin. This study demonstrates that transient changes in acidity of single amino acid residues can be quantified
in situ
with infrared spectroscopy.
Convergent evolution of
Trichomonas vaginalis
lactate dehydrogenase from malate dehydrogenase
Authors: Wu Gang , Fiser András , ter Kuile Benno , Šali Andrej , Müller Miklós
Published: July 2002

Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist
Trichomonas vaginalis
and some but not all other trichomonad species. The derived amino acid sequence of
T. vaginalis
LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two
T. vaginalis
sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently.
Rapid and reversible relocalization of heat shock factor 1 within seconds to nuclear stress granules
Authors: Jolly Caroline , Usson Yves , Morimoto Richard I.
Published: July 2002

Heat shock factor 1 (HSF1) is essential for the stress-induced expression of heat shock genes. On exposure to heat shock, HSF1 localizes within seconds to discrete nuclear granules. On recovery from heat shock, HSF1 rapidly dissipates from these stress granules to a diffuse nucleoplasmic distribution, typical of unstressed cells. Subsequent reexposure to heat shock results in the rapid relocalization of HSF1 to the same stress granules with identical kinetics. Although the appearance of HSF1 stress granules corresponds to the hyperphosphorylated, trimeric DNA-binding state of HSF1 and correlates temporally with the inducible transcription of heat shock genes, they are also present in heat-shocked mitotic cells that are devoid of transcription. This finding suggests a role for HSF1 stress granules as a nuclear compartment for the temporal regulation and spatial organization of HSF1 activity and reveals new features of the dynamics of nuclear organization.