bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple mechanisms of
N
-phosphonacetyl-
l
-aspartate resistance in human cell lines: Carbamyl-
P
synthetase/aspartate transcarbamylase/dihydro-orotase gene amplification is frequent only when chromosome 2 is rearranged
Authors: Smith Kathleen A. , Chernova Olga B. , Groves Richard P. , Stark Mary B. , Martínez José L. , Davidson Jeffrey N. , Trent Jeffrey M. , Patterson Thomas E. , Agarwal Archana , Duncan Pamela , Agarwal Munna L. , Stark George R.
Published: July 2002

Rodent cells resistant to
N
-phosphonacetyl-
l
-aspartate (PALA) invariably contain amplified carbamyl-
P
synthetase/aspartate transcarbamylase/dihydro-orotase (CAD) genes, usually in widely spaced tandem arrays present as extensions of the same chromosome arm that carries a single copy of CAD in normal cells. In contrast, amplification of CAD is very infrequent in several human tumor cell lines. Cell lines with minimal chromosomal rearrangement and with unrearranged copies of chromosome 2 rarely develop intrachromosomal amplifications of CAD. These cells frequently become resistant to PALA through a mechanism that increases the aspartate transcarbamylase activity with no increase in CAD copy number, or they obtain one extra copy of CAD by forming an isochromosome 2p or by retaining an extra copy of chromosome 2. In cells with multiple chromosomal aberrations and rearranged copies of chromosome 2, amplification of CAD as tandem arrays from rearranged chromosomes is the most frequent mechanism of PALA resistance. All of these different mechanisms of PALA resistance are blocked in normal human fibroblasts.
Estrogen regulates the rate of bone turnover but bone balance in ovariectomized rats is modulated by prevailing mechanical strain
Authors: Westerlind Kim C. , Wronski Thomas J. , Ritman Erik L. , Luo Zong-Ping , An Kai-Nan , Bell Norman H. , Turner Russell T.
Published: July 2002

Estrogen deficiency induced bone loss is associated with increased bone turnover in rats and humans. The respective roles of increased bone turnover and altered balance between bone formation and bone resorption in mediating estrogen deficiency-induced cancellous bone loss was investigated in ovariectomized rats. Ovariectomy resulted in increased bone turnover in the distal femur. However, cancellous bone was preferentially lost in the metaphysis, a site that normally experiences low strain energy. No bone loss was observed in the epiphysis, a site experiencing higher strain energy. The role of mechanical strain in maintaining bone balance was investigated by altering the strain history. Mechanical strain was increased and decreased in long bones of ovariectomized rats by treadmill exercise and functional unloading, respectively. Functional unloading was achieved during orbital spaceflight and following unilateral sciatic neurotomy. Increasing mechanical loading reduced bone loss in the metaphysis. In contrast, decreasing loading accentuated bone loss in the metaphysis and resulted in bone loss in the epiphysis. Finally, administration of estrogen to ovariectomized rats reduced bone loss in the unloaded and prevented loss in the loaded limb following unilateral sciatic neurotomy in part by reducing indices of bone turnover. These results suggest that estrogen regulates the rate of bone turnover, but the overall balance between bone formation and bone resorption is influenced by prevailing levels of mechanical strain.
Cryptic speciation and recombination in the aflatoxin-producing fungus
Aspergillus flavus
Authors: Geiser David M. , Pitt John I. , Taylor John W.
Published: July 2002

Aspergillus flavus
, like approximately one–third of ascomycete fungi, is thought to be cosmopolitan and clonal because it has uniform asexual morphology.
A. flavus
produces aflatoxin on nuts, grains, and cotton, and assumptions about its life history are being used to develop strategies for its biological control. We tested the assumptions of clonality and conspecificity in a sample of 31 Australian isolates by assaying restriction site polymorphisms from 11 protein encoding genes and DNA sequences from five of those genes.
A. flavus
isolates fell into two reproductively isolated clades (groups I and II). The lack of concordance among gene genealogies among isolates in one of the clades (group I) was consistent with a history of recombination. Our analysis included five strains of the closely related industrial fungus
A. oryzae
, all of which proved to be clonally related to group I.
A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels
Authors: Randolph Gwendalyn J. , Beaulieu Sylvie , Pope Melissa , Sugawara Isamu , Hoffman Lloyd , Steinman Ralph M. , Muller William A.
Published: July 2002

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.
Social stress and the reactivation of latent herpes simplex virus type 1
Authors: Padgett David A. , Sheridan John F. , Dorne Julianne , Berntson Gary G. , Candelora Jessica , Glaser Ronald
Published: July 2002

Psychological stress is thought to contribute to reactivation of latent herpes simplex virus (HSV). Although several animal models have been developed in an effort to reproduce different pathogenic aspects of HSV keratitis or labialis, until now, no good animal model existed in which application of a psychological laboratory stressor results in reliable reactivation of the virus. Reported herein, disruption of the social hierarchy within colonies of mice increased aggression among cohorts, activated the hypothalamic-pituitary-adrenal axis, and caused reactivation of latent HSV type 1 in greater than 40% of latently infected animals. However, activation of the hypothalamic-pituitary-adrenal axis using restraint stress did not activate the latent virus. Thus, the use of social stress in mice provides a good model in which to investigate the neuroendocrine mechanisms that underlie behaviorally mediated reactivation of latent herpesviruses.
Promotion of agonist activity of antiandrogens by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells
Authors: Miyamoto Hiroshi , Yeh Shuyuan , Wilding George , Chang Chawnshang
Published: July 2002

Although hormone therapy with antiandrogens has been widely used for the treatment of prostate cancer, some antiandrogens may act as androgen receptor (AR) agonists that may result in antiandrogen withdrawal syndrome. The molecular mechanism of this agonist response, however, remains unclear. Using mammalian two-hybrid assay, we report that antiandrogens, hydroxyflutamide, bicalutamide (casodex), cyproterone acetate, and RU58841, and other compounds such as genistein and RU486, can promote the interaction between AR and its coactivator, ARA70, in a dose-dependent manner. The chloramphenicol acetyltransferase assay further demonstrates that these antiandrogens and related compounds significantly enhance the AR transcriptional activity by cotransfection of AR and ARA70 in a 1:3 ratio into human prostate cancer DU145 cells. Our results suggest that the agonist activity of antiandrogens might occur with the proper interaction of AR and ARA70 in DU145 cells. These findings may provide a good model to develop better antiandrogens without agonist activity.
Chromosomal basis of X chromosome inactivation: Identification of a multigene domain in Xp11.21-p11.22 that escapes X inactivation
Authors: Miller Andrew P. , Willard Huntington F.
Published: July 2002

A number of genes have been identified that escape mammalian X chromosome inactivation and are expressed from both active and inactive X chromosomes. The basis for escape from inactivation is unknown and,a priori, could be a result of local factors that act in a gene-specific manner or of chromosomal control elements that act regionally. Models invoking the latter predict that such genes should be clustered in specific domains on the X chromosome, rather than distributed at random along the length of the X. To distinguish between these possibilities, we have constructed a transcription map composed of at least 23 distinct expressed sequences in an ≈5.5-megabase region on the human X chromosome spanning Xp11.21-p11.22. The inactivation status of these transcribed sequences has been determined in a somatic cell hybrid system and correlated with the position of the genes on the physical map. Although the majority of transcribed sequences in this region are subject to X inactivation, eight expressed sequences (representing at least six different genes) escape inactivation, and all are localized to within a region of less than 370 kb. Genes located both distal and proximal to this cluster are subject to inactivation, thereby defining a unique multigene domain on the proximal short arm that is transcriptionally active on the inactive X chromosome.
The hereditary renal cell carcinoma 3;8 translocation fuses
FHIT
to a
patched-
related gene,
TRC8
Authors: Gemmill Robert M. , West James D. , Boldog Ferenc , Tanaka Naotake , Robinson Linda J. , Smith David I. , Li Frederick , Drabkin Harry A.
Published: July 2002

The 3;8 chromosomal translocation, t(3;8)(p14.2;q24.1), was described in a family with classical features of hereditary renal cell carcinoma. Previous studies demonstrated that the 3p14.2 breakpoint interrupts the fragile histidine triad gene (
FHIT)
in its 5′ noncoding region. However, evidence that
FHIT
is causally related to renal or other malignancies is controversial. We now show that the 8q24.1 breakpoint region encodes a 664-aa multiple membrane spanning protein, TRC8, with similarity to the hereditary basal cell carcinoma/segment polarity gene,
patched
. This similarity involves two regions of
patched
, the putative sterol-sensing domain and the second extracellular loop that participates in the binding of sonic hedgehog. In the 3;8 translocation, TRC8 is fused to
FHIT
and is disrupted within the sterol-sensing domain. In contrast, the
FHIT
coding region is maintained and expressed. In a series of sporadic renal carcinomas, an acquired
TRC8
mutation was identified. By analogy to
patched
,
TRC8
might function as a signaling receptor and other pathway members, to be defined, are mutation candidates in malignant diseases involving the kidney and thyroid.
The neuregulin, glial growth factor 2, diminishes autoimmune demyelination and enhances remyelination in a chronic relapsing model for multiple sclerosis
Authors: Cannella Barbara , Hoban Carolyn J. , Gao Yan-Ling , Garcia-Arenas Renee , Lawson Deborah , Marchionni Mark , Gwynne David , Raine Cedric S.
Published: July 2002

Glial growth factor 2 (GGF2) is a neuronal signal that promotes the proliferation and survival of the oligodendrocyte, the myelinating cell of the central nervous system (CNS). The present study examined whether recombinant human GGF2 (rhGGF2) could effect clinical recovery and repair to damaged myelin in chronic relapsing experimental autoimmune encephalomyelitis (EAE) in the mouse, a major animal model for the human demyelinating disease, multiple sclerosis. Mice with EAE were treated with rhGGF2 during both the acute and relapsing phases. Clinically, GGF2 treatment delayed signs, decreased severity, and resulted in statistically significant reductions in relapse rate. rhGGF2-treated groups displayed CNS lesions with more remyelination than in controls. This correlated with increased mRNA expression of myelin basic protein exon 2, a marker for remyelination, and with an increase in the CNS of the regulatory cytokine, interleukin 10, at both the RNA and protein levels. Thus, a beneficial effect of a neurotrophic growth factor has been demonstrated on the clinical, pathologic, and molecular manifestations of autoimmune demyelination, an effect that was associated with increased expression of a T helper 2 cytokine. rhGGF2 treatment may represent a novel approach to the treatment of multiple sclerosis.
A method for directed evolution and functional cloning of enzymes
Authors: Pedersen Henrik , Hölder Swen , Sutherlin Daniel P. , Schwitter Urs , King David S. , Schultz Peter G.
Published: July 2002

A general scheme is described for the
in vitro
evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.
Deletion of long-range regulatory elements upstream of
SOX9
causes campomelic dysplasia
Authors: Wunderle Véronique M. , Critcher Ricky , Hastie Nicholas , Goodfellow Peter N. , Schedl Andreas
Published: July 2002

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal. Patients present with either heterozygous mutations in the
SOX9
gene or chromosome rearrangements mapping at least 50 kb upstream of
SOX9.
Whereas mutations in
SOX9
ORF cause haploinsufficiency, the effects of translocations 5′ to
SOX9
are unclear. To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of
SOX9
, we generated mice transgenic for human
SOX9-lacZ
yeast artificial chromosomes containing variable amounts of DNA sequences upstream of
SOX9
. We show that elements necessary for
SOX9
expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of
SOX9,
similar to those observed in CD patients, leads to a substantial reduction of
SOX9
expression, particularly in chondrogenic tissues. These data demonstrate that important regulatory elements are scattered over a large region upstream of
SOX9
and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5′ to
SOX9
.
Nonsynonymous substitution in abalone sperm fertilization genes exceeds substitution in introns and mitochondrial DNA
Authors: Metz Edward C. , Robles-Sikisaka Refugio , Vacquier Victor D.
Published: July 2002

Strong positive Darwinian selection acts on two sperm fertilization proteins, lysin and 18-kDa protein, from abalone (
Haliotis
). To understand the phylogenetic context for this dramatic molecular evolution, we obtained sequences of mitochondrial cytochrome
c
oxidase subunit I (mtCOI), and genomic sequences of lysin, 18-kDa, and a G protein subunit. Based on mtDNA differentiation, four north Pacific abalone species diverged within the past 2 million years (Myr), and remaining north Pacific species diverged over a period of 4–20 Myr. Between-species nonsynonymous differences in lysin and 18-kDa exons exceed nucleotide differences in introns by 3.5- to 24-fold. Remarkably, in some comparisons nonsynonymous substitutions in lysin and 18-kDa genes exceed synonymous substitutions in mtCOI. Lysin and 18-kDa intron/exon segments were sequenced from multiple red abalone individuals collected over a 1,200-km range. Only two nucleotide changes and two sites of slippage variation were detected in a total of >29,000 nucleotides surveyed. However, polymorphism in mtCOI and a G protein intron was found in this species. This finding suggests that positive selection swept one lysin allele and one 18-kDa allele to fixation. Similarities between mtCOI and lysin gene trees indicate that rapid adaptive evolution of lysin has occurred consistently through the history of the group. Comparisons with mtCOI molecular clock calibrations suggest that nonsynonymous substitutions accumulate 2–50 times faster in lysin and 18-kDa genes than in rapidly evolving mammalian genes.
Diversity components of impending primate extinctions
Authors: Jernvall Jukka , Wright Patricia C.
Published: July 2002

Many extant species are at risk to go extinct. This impending loss of species is likely to cause changes in future ecosystem functions. Ecological components of diversity, such as dietary or habitat specializations, can be used to estimate the impact of extinctions on ecosystem functions. As an approach to estimate the impact of future extinctions, we tested interdependency between ecological and taxonomic change based on current predictions of extinction rates in primates. We analyzed the ecological characteristics of extant primate faunas having species in various categories of endangerment of extinction and forecasted the future primate faunas as if they were paleontological faunas. Predicting future faunas combines the wealth of ecological information on living primates with large, fossil record-like changes in diversity. Predicted extinction patterns of living primates in Africa, Asia, Madagascar, and South America show that changes in ecology differ among the regions in ways that are not reducible to taxonomic measures. The ecological effects of primate extinctions are initially least severe in South America and larger in Asia and Africa. Disproportionately larger ecological changes are projected for Madagascar. The use of taxonomy as a proxy for ecology can mislead when estimating competence of future primate ecosystems.
Pro-phenol oxidase activating proteinase from an insect,
Manduca sexta:
A bacteria-inducible protein similar to
Drosophila
easter
Authors: Jiang Haobo , Wang Yang , Kanost Michael R.
Published: July 2002

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm,
Manduca sexta
, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the
easter
gene from
Drosophila melanogaster
. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with
Escherichia coli
. Sequence data and
3
H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.
Regulation of endothelial monocyte-activating polypeptide II release by apoptosis
Authors: Knies Ulrike E. , Behrensdorf Heike A. , Mitchell Christopher A. , Deutsch Urban , Risau Werner , Drexler Hannes C. A. , Clauss Matthias
Published: July 2002

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.
Comparative studies on mammalian
Hoxc8
early enhancer sequence reveal a baleen whale-specific deletion of a cis-acting element
Authors: Shashikant Cooduvalli S. , Kim Chang B. , Borbély Marc A. , Wang Wayne C. H. , Ruddle Frank H.
Published: July 2002

Variations in regulatory regions of developmental control genes have been implicated in the divergence of axial morphologies. To find potentially significant changes in cis-regulatory regions, we compared nucleotide sequences and activities of mammalian
Hoxc8
early enhancers. The nucleotide sequence of the early enhancer region is extremely conserved among mammalian clades, with five previously described cis-acting elements, A–E, being invariant. However, a 4-bp deletion within element C of the
Hoxc8
early enhancer sequence is observed in baleen whales. When assayed in transgenic mouse embryos, a baleen whale enhancer (unlike other mammalian enhancers) directs expression of the reporter gene to more posterior regions of the neural tube but fails to direct expression to posterior mesoderm. We suggest that regulation of
Hoxc8
in baleen whales differs from other mammalian species and may be associated with variation in axial morphology.
Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge
Authors: Belyakov Igor M. , Derby Michael A. , Ahlers Jeffrey D. , Kelsall Brian L. , Earl Patricia , Moss Bernard , Strober Warren , Berzofsky Jay A.
Published: July 2002

Mucosal tissues are major sites of HIV entry and initial infection. Thus, the induction of a mucosal cytotoxic T lymphocyte (CTL) response is an important feature for an effective HIV vaccine. However, little is known about approaches to induce such a protective CTL response in the mucosa. Here for the first time we show that intrarectal immunization with a synthetic, multideterminant HIV peptide plus cholera toxin adjuvant induced long-lasting, antigen-specific CTL memory in both the inductive (Peyer’s patch) and effector (lamina propria) mucosal sites, as well as in systemic sites (spleen), whereas systemic immunization induced specific CTL only in the spleen. Cholera toxin adjuvant, while enhancing the response, was not essential. The CTL recognized target cells either pulsed with HIV peptide or expressing endogenous whole envelope glycoprotein ofMr160,000 (gp160). Exploring the requirements for CTL induction, we show that mucosal CTL responses are both interleukin 12 and interferon-γ dependent by using antibody-treated and knock-out mice. Finally, to determine whether a mucosal response is actually protective against local mucosal challenge with virus, we show that intrarectal immunization with the synthetic HIV peptide vaccine protected mice against infection via mucosal challenge with a recombinant vaccinia virus expressing HIV-1IIIB gp160. These studies provide an approach to development of an HIV vaccine that induces CTL immunity in the mucosal and systemic immune systems and protects against mucosal infection with a virus expressing HIV-1 gp160.
Acceleration of amyloid protein A amyloidosis by amyloid-like synthetic fibrils
Authors: Johan Katarzyna , Westermark Gunilla , Engström Ulla , Gustavsson Åsa , Hultman Per , Westermark Per
Published: July 2002

Amyloid protein A (AA) amyloidosis is a consequence of some long-standing inflammatory conditions, and subsequently, an N-terminal fragment of the acute phase protein serum AA forms β-sheet fibrils that are deposited in different tissues. It is unknown why only some individuals develop AA amyloidosis. In the mouse model, AA amyloidosis develops after ≈25 days of inflammatory challenge. This lag phase can be shortened dramatically by administration of a small amount of amyloid extract containing an as yet undefined amyloid-enhancing factor. In the present study, we show that preformed amyloid-like fibrils made from short synthetic peptides corresponding to parts of several different amyloid fibril proteins exert amyloidogenic enhancing activity when given i.v. to mice at the induction of inflammation. We followed i.v. administered, radiolabeled, heterologous, synthetic fibrils to the lung and to the perifollicular area in the spleen and found that new AA–amyloid fibrils developed on these preformed fibrils. Our findings thus show that preformed, synthetic, amyloid-like fibrils have an
in vivo
nidus activity and that amyloid-enhancing activity may occur, at least in part, through this mechanism. Our findings also show that fibrils of a heterologous chemical nature exert amyloid-enhancing activity.
Structural requirements of tRNA
Lys
for its import into yeast mitochondria
Authors: Entelis Nina S. , Kieffer Sandrine , Kolesnikova Olga A. , Martin Robert P. , Tarassov Ivan A.
Published: July 2002

In the yeast
Saccharomyces cerevisiae
, one of the two cytoplasmic lysine tRNAs, tRNA
CUU
Lys
, is partially associated with the mitochondrial matrix. Mitochondrial import of this tRNA requires binding to the precursor of the mitochondrial lysyl-tRNA synthetase, pre-MSK, and aminoacylation by the cytoplasmic lysyl-tRNA synthetase, KRS, appears to be a prerequisite for this binding. The second lysine isoacceptor tRNA
mnm
Lys

5

s
2

UUU
{where 5-[(methylamino)-methyl]-2-thiouridine is mnm
5
s
2
U} is exclusively localized in the cytoplasm. To study import determinants within the tRNA
CUU
Lys
molecule, we introduced a panel of replacements in the original sequences of the imported and nonimported lysine tRNAs that correspond to domains or individual residues that differ between these two isoacceptors. The mutant transcripts were tested for import, aminoacylation, and binding to pre-MSK. Import and aminoacylation efficiencies correlate well for the majority of mutant transcripts. However, some poorly aminoacylated transcripts were rather efficiently imported. Surprisingly, these transcripts retained binding capacity to pre-MSK. In fact, all imported transcripts retained pre-MSK binding capacity but nonimported versions did not, suggesting that this binding, rather than aminoacylation, is essential for import. Substitution of the anticodon arm of tRNA
CUU
Lys
with that of tRNA
mnm
Lys

5

s
2

UUU
abolished import without affecting aminoacylation. A version of tRNA
mnm
Lys

5

s
2

UUU
with an anticodon CUU was efficiently imported
in vitro
and was also found to be imported
in vivo
. This implies that the anticodon arm, especially position 34, is important for recognition by the import machinery. A nicked tRNA
CUU
Lys
transcript is still imported but its import requires reannealing of the two tRNA moieties, which implies that tRNA
CUU
Lys
is imported as a folded molecule.
Rack-induced metal binding vs. flexibility: Met121His azurin crystal structures at different pH
Authors: Messerschmidt Albrecht , Prade Lars , Kroes Sandra J. , Sanders–Loehr Joann , Huber Robert , Canters Gerard W.
Published: July 2002

The rack-induced bonding mechanism of metals to proteins is a useful concept for explaining the generation of metal sites in electron transfer proteins, such as the blue copper proteins, that are designed for rapid electron transfer. The trigonal pyramidal structure imposed by the protein with three strong equatorial ligands (one Cys and two His) provides a favorable geometry for both cuprous and cupric oxidation states. However, the crystal structures of the Met121His mutant of azurin from
Alcaligenes denitrificans
at pH 6.5 (1.89- and 1.91-Å resolutions) and pH 3.5 (2.45-Å resolution) show that the preformed metal binding cavity in the protein is more flexible than expected. At high pH (6.5), the Cu site retains the same three equatorial ligands as in the wild-type azurin and adds His121 as a fourth strong ligand, creating a tetrahedral copper site geometry with a green color referred to as 1.5 type. In the low pH (3.5) structure, the protonation of His121 causes a conformational change in residues 117–123, moving His121 away from the copper. The empty coordination site is occupied by an oxygen atom of a nitrate molecule of the buffer solution. This axial ligand is coordinated less strongly, generating a distorted tetrahedral copper geometry with a blue color and spectroscopic properties of a type-1 site. These crystal structures demonstrate that blue copper proteins are flexible enough to permit a range of movement of the Cu atom along the axial direction of the trigonal pyramid.
An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells
Authors: Siemeister Gerhard , Schirner Michael , Reusch Petra , Barleon Bernhard , Marmé Dieter , Martiny-Baron Georg
Published: July 2002

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.
Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals
Authors: Ramana Chilakamarti V. , Boldogh Istvan , Izumi Tadahide , Mitra Sankar
Published: July 2002

Apurinic/apyrimidinic (AP) endonuclease (APE; EC4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3′-phosphoesterase activity removes 3′ blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by other genotoxicants—e.g., UV light and alkylating agents. Increased expression of APE mRNA and protein was observed both in the HeLa S3 tumor line and in WI 38 primary fibroblasts, and it was accompanied by translocation of the endonuclease to the nucleus. ROS-treated cells showed a significant increase in resistance to the cytotoxicity of such ROS generators as H2O2and bleomycin, but not to UV light. This “adaptive response” appears to result from enhanced repair of cytotoxic DNA lesions due to an increased activity of APE-1, which may be limiting in the base excision repair process for ROS-induced toxic lesions.
SDS capillary gel electrophoresis of proteins in microfabricated channels
Authors: Yao Shao , Anex Deon S. , Caldwell W. Brett , Arnold Don W. , Smith Katherine B. , Schultz Peter G.
Published: July 2002

Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Two-dimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures.
Influence of drinking water and diet on the stable-hydrogen isotope ratios of animal tissues
Authors: Hobson Keith A. , Atwell Lisa , Wassenaar Leonard I.
Published: July 2002

Despite considerable interest in using stable-hydrogen isotope ratio (δD) measurements in ecological research, it was previously unknown whether hydrogen derived from drinking water, in addition to that derived from diet, contributed to the nonexchangeable hydrogen in animal tissues. We raised four experimental groups of quail (
Coturnix coturnix japonica
) from hatch on two isotopically distinct diets (mean nonexchangeable δD: −146 and −60‰, Vienna Standard Mean Ocean Water Standard) and drinking waters (mean δD: −130 and +196‰, Vienna Standard Mean Ocean Water Standard). Here we show that both dietary and drinking water hydrogen are incorporated into nonexchangeable hydrogen in both metabolically active (i.e., muscle, liver, blood, fat) and inactive (i.e., feather, nail) tissues. Approximately 20% of hydrogen in metabolically active quail tissues and 26–32% of feathers and nail was derived from drinking water. Our findings suggest environmental interpretations of δD values from modern and fossil animal tissues may need to account for potentially large isotopic differences between drinking water and food and require a good understanding of the physiological ecology of study organisms.
Mechanisms of inactivation of mismatch repair genes in human colorectal cancer cell lines: The predominant role of hMLH1
Authors: Wheeler J. M. D. , Beck N. E. , Kim H. C. , Tomlinson I. P. M. , Mortensen N. J. McC. , Bodmer W. F.
Published: July 2002

Fifteen to twenty-five percent of sporadic colorectal carcinomas are replication error (RER) positive. Because the frequency of mutations in the mismatch repair genes (hMLH1 and hMSH2) is low in these tumors, we have investigated the role of mutational inactivation, methylation of the promoter region, and loss of heterozygosity (LOH) as a possible explanation for the mutator phenotype of RER+ colorectal cancer cell lines. Genomic DNA was extracted from a panel of 49 human colorectal cancer cell lines. The RER status was determined by amplification of BAT-26. All exons of hMLH1 and hMSH2 were amplified with the PCR and screened by using single-strand conformational polymorphism and direct sequencing. The methylation status was ascertained by methylation-specific PCR after bisulfite modification of DNA. Western blotting for hMLH1 was performed on methylated cell lines before and after the addition of the demethylating agent 5-azacytidine. LOH was sought by
genescan
analysis of amplified CA repeat markers and indirectly by determining the number of homozygotes in the cell lines and human random controls. Twelve cell lines from ten tumors (24%) were RER+. Hypermethylation of the hMLH1 promoter occurred in five of ten (50%) RER+ tumors, whereas three of thirty-two (6%) RER tumors showed partial methylation. None of the fully methylated cell lines expressed hMLH1, although all reexpressed hMLH1 after treatment with 5-azacytidine. There was no LOH in the RER+ tumors in either hMLH1 or hMSH2. Our results suggest that mutations of hMLH1 together with hypermethylation of the promoter region, but not LOH, are the cause of the mutator phenotype in the majority (70%) of RER+ tumors.
MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia
Authors: Alessandrini Alessandro , Namura Shobu , Moskowitz Michael A. , Bonventre Joseph V.
Published: July 2002

The MEK1 (
M
AP kinase/
E
RK
k
inase)/ERK (
e
xtracellular-signal-
r
esponsive
k
inase) pathway has been implicated in cell growth and differentiation [Seger, R. & Krebs, E. G. (1995)
FASEB J.
9, 726–735]. Here we show that the MEK/ERK pathway is activated during focal cerebral ischemia and may play a role in inducing damage. Treatment of mice 30 min before ischemia with the MEK1-specific inhibitor PD98059 [Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T. & Saltiel, A. R. (1995)
J. Biol. Chem.
270, 27489–27494] reduces focal infarct volume at 22 hr after ischemia by 55% after transient occlusion of the middle cerebral artery. This is accompanied by a reduction in phospho-ERK1/2 immunohistochemical staining. MEK1 inhibition also results in reduced brain damage 72 hr after ischemia, with focal infarct volume reduced by 36%. This study indicates that the MEK1/ERK pathway contributes to brain injury during focal cerebral ischemia and that PD98059, a MEK1-specific antagonist, is a potent neuroprotective agent.
Crystal structure of SQD1, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose
Authors: Mulichak Anne M. , Theisen Michael J. , Essigmann Bernd , Benning Christoph , Garavito R. Michael
Published: July 2002

The SQD1 enzyme is believed to be involved in the biosynthesis of the sulfoquinovosyl headgroup of plant sulfolipids, catalyzing the transfer of SO
3
−
to UDP-glucose. We have determined the structure of the complex of SQD1 from
Arabidopsis thaliana
with NAD
+
and the putative substrate UDP-glucose at 1.6-Å resolution. Both bound ligands are completely buried within the binding cleft, along with an internal solvent cavity which is the likely binding site for the, as yet, unidentified sulfur-donor substrate. SQD1 is a member of the short-chain dehydrogenase/reductase (SDR) family of enzymes, and its structure shows a conservation of the SDR catalytic residues. Among several highly conserved catalytic residues, Thr-145 forms unusually short hydrogen bonds with both susceptible hydroxyls of UDP-glucose. A His side chain may also be catalytically important in the sulfonation.
Cannabinoid CB1 receptors and ligands in vertebrate retina: Localization and function of an endogenous signaling system
Authors: Straiker Alex , Stella Nephi , Piomelli Daniele , Mackie Ken , Karten Harvey J. , Maguire Greg
Published: July 2002

CB1, a cannabinoid receptor enriched in neuronal tissue, was found in high concentration in retinas of rhesus monkey, mouse, rat, chick, goldfish, and tiger salamander by using a subtype-specific polyclonal antibody. Immunolabeling was detected in the two synaptic layers of the retina, the inner and outer plexiform layers, of all six species examined. In the outer plexiform layer, CB1 was located in and/or on cone pedicles and rod spherules. Labeling was detected in some amacrine cells of all species and in the ganglion cells and ganglion cell axons of all species except fish. In addition, sparse labeling was found in the inner and/or outer segments of the photoreceptors of monkey, mouse, rat, and chick. Using GC/MS to detect possible endogenous cannabinoids, we found 3 nmol of 2-arachidonylglycerol per g of tissue, but no anandamide was detectable. Cannabinoid receptor agonists induced a dramatic reduction in the amplitude of voltage-gated L-type calcium channel currents in identified retinal bipolar cells. The presence and distribution of the CB1 receptor, the large amounts of 2-arachidonylglycerol found, and the effects of cannabinoids on calcium channel activity in bipolar cells suggest a substantive role for an endogenous cannabinoid signaling system in retinal physiology, and perhaps vision in general.
A bZIP factor, TRAB1, interacts with VP1 and mediates abscisic acid-induced transcription
Authors: Hobo Tokunori , Kowyama Yasuo , Hattori Tsukaho
Published: July 2002

The transcription factor VP1 regulates maturation and dormancy in plant seeds by activating genes responsive to the stress hormone abscisic acid (ABA). Although activation involves ABA-responsive elements (ABREs), VP1 itself does not specifically bind ABREs. Instead, we have identified and cloned a basic region leucine zipper (bZIP) factor, TRAB1, that interacts with both VP1 and ABREs. Transcription from a chimeric promoter with GAL4-binding sites was ABA-inducible if cells expressed a GAL4 DNA-binding domain∷TRAB1 fusion protein. Results indicate that TRAB1 is a true trans-acting factor involved in ABA-regulated transcription and reveal a molecular mechanism for the VP1-dependent, ABA-inducible transcription that controls maturation and dormancy in plant embryos.
The small GTPase RalA targets filamin to induce filopodia
Authors: Ohta Yasutaka , Suzuki Nobuchika , Nakamura Shun , Hartwig John H. , Stossel Thomas P.
Published: July 2002

The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA’s effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.
CORE-SINEs: Eukaryotic short interspersed retroposing elements with common sequence motifs
Authors: Gilbert Nicolas , Labuda Damian
Published: July 2002

A 65-bp “core” sequence is dispersed in hundreds of thousands copies in the human genome. This sequence was found to constitute the central segment of a group of short interspersed elements (SINEs), referred to as mammalian-wide interspersed repeats, that proliferated before the radiation of placental mammals. Here, we propose that the core identifies an ancient tRNA-like SINE element, which survived in different lineages such as mammals, reptiles, birds, and fish, as well as mollusks, presumably for >550 million years. This element gave rise to a number of sequence families (CORE-SINEs), including mammalian-wide interspersed repeats, whose distinct 3′ ends are shared with different families of long interspersed elements (LINEs). The evolutionary success of the generic CORE-SINE element can be related to the recruitment of the internal promoter from highly transcribed host RNA as well as to its capacity to adapt to changing retropositional opportunities by sequence exchange with actively amplifying LINEs. It reinforces the notion that the very existence of SINEs depends on the cohabitation with both LINEs and the host genome.
Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein
Authors: Jiang Jianzhong , Zhang Ying , Krainer Adrian R. , Xu Rui-Ming
Published: July 2002

Human p32 (also known as SF2-associated p32, p32/TAP, and gC1qR) is a conserved eukaryotic protein that localizes predominantly in the mitochondrial matrix. It is thought to be involved in mitochondrial oxidative phosphorylation and in nucleus–mitochondrion interactions. We report the crystal structure of p32 determined at 2.25 Å resolution. The structure reveals that p32 adopts a novel fold with seven consecutive antiparallel β-strands flanked by one N-terminal and two C-terminal α-helices. Three monomers form a doughnut-shaped quaternary structure with an unusually asymmetric charge distribution on the surface. The implications of the structure on previously proposed functions of p32 are discussed and new specific functional properties are suggested.
Endoplasmic reticulum retention is a common defect associated with tyrosinase-negative albinism
Authors: Halaban Ruth , Svedine Sherri , Cheng Elaine , Smicun Yoel , Aron Rebecca , Hebert Daniel N.
Published: July 2002

Tyrosinase is a melanocyte-specific enzyme critical for the synthesis of melanin, a process normally restricted to a post-Golgi compartment termed the melanosome. Loss-of-function mutations in tyrosinase are the cause of oculocutaneous albinism, demonstrating the importance of the enzyme in pigmentation. In the present study, we explored the possibility that trafficking of albino tyrosinase from the endoplasmic reticulum (ER) to the Golgi apparatus and beyond is disrupted. Toward this end, we analyzed the common albino mouse mutation Tyr(C85S), the frequent human albino substitution TYR(T373K), and the temperature-sensitive tyrosinase TYR(R402Q)/Tyr(H402A) found in humans and mice, respectively. Intracellular localization was monitored in albino melanocytes carrying the native mutation, as well as in melanocytes ectopically expressing green fluorescent protein-tagged tyrosinase. Enzymatic characterization of complex glycans and immunofluorescence colocalization with organelle-specific resident proteins established that all four mutations produced defective proteins that were retained in the ER. TYR(R402Q)/Tyr(H402A) Golgi processing and transport to melanosomes were promoted at the permissive temperature of 32°C, but not at the nonpermissive 37°C temperature. Furthermore, evidence of protein misfolding was demonstrated by the prolonged association of tyrosinase mutants with calnexin and calreticulin, known ER chaperones that play a key role in the quality-control processes of the secretory pathway. From these results we concluded that albinism, at least in part, is an ER retention disease.