bluuebunny/crossref_metadata_2025 huggingface dataset:
IDENTIFICATION OF A NOVEL CYTOCHROME P450 cDNA, CYP97E1, FROM THE MARINE DIATOM SKELETONEMA COSTATUM BACILLARIOPHYCEAE1
Authors: Yang Shao , Wu Rudolf S. S. , Mok Helen O. L. , Zhang Z. P. , Kong Richard Y. C.
Published: May 2003

Here we report identification of a 2269‐base pair full‐length cDNA, CYP97E1, encoding a novel cytochrome P450 protein from the marine diatom Skeletonema costatum. The CYP97E1 protein contains 659 amino acids (Mr 74,200) and is the largest P450 isoform described to date. Our BLAST homology search and parsimony analysis showed that CYP97E1 shared high sequence identity (>40%) and genetic relatedness, respectively, with the CYP97B isoforms from different plant species. CYP97E1 was predicted by PSORT (a protein localization site prediction program) to be a cytosolic protein. Northern hybridization analysis indicated that CYP97E1 expression in S. costatum was not significantly affected by 2,4‐dichlorophenol, suggesting that CYP97E1 may not be involved in 2,4‐dichlorophenol detoxification in this diatom.
Coincidence of synteny breakpoints with malignancy-related deletions on human chromosome 3
Authors: Kost-Alimova Maria , Kiss Hajnalka , Fedorova Ludmila , Yang Ying , Dumanski Jan P. , Klein George , Imreh Stefan
Published: May 2003

We have found previously that during tumor growth intact human chromosome 3 transferred into tumor cells regularly looses certain 3p regions, among them the ≈1.4-Mb common eliminated region 1 (CER1) at 3p21.3. Fluorescence
in situ
hybridization analysis of 12 mouse orthologous loci revealed that CER1 splits into two segments in mouse and therefore contains a murine/human conservation breakpoint region (CBR). Several breaks occurred in tumors within the region surrounding the CBR, and this sequence has features that characterize unstable chromosomal regions: deletions in yeast artificial chromosome clones, late replication, gene and segment duplications, and pseudogene insertions. Sequence analysis of the entire 3p12-22 revealed that other cancer-associated deletions (regions eliminated from monochromosomal hybrids carrying an intact chromosome 3 during tumor growth and homozygous deletions found in human tumors) colocalized nonrandomly with murine/human CBRs and were characterized by an increased number of local gene duplications and murine/human conservation mismatches (single genes that do not match into the conserved chromosomal segment). The CBR within CER1 contains a simple tandem TATAGA repeat capable of forming a 40-bp-long secondary hairpin-like structure. This repeat is nonrandomly localized within the other tumor-associated deletions and in the vicinity of 3p12-22 CBRs.
Differential expression of growth factors at the cellular level in virus-infected brain
Authors: Prosniak Mikhail , Zborek Anna , Scott Gwen S. , Roy Anirban , Phares Timothy W. , Koprowski Hilary , Hooper D. Craig
Published: May 2003

The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS.
The receptor kinases LePRK1 and LePRK2 associate in pollen and when expressed in yeast, but dissociate in the presence of style extract
Authors: Wengier Diego , Valsecchi Isabel , Cabanas María Laura , Tang Wei-hua , McCormick Sheila , Muschietti Jorge
Published: May 2003

After pollen grains germinate on the stigma, pollen tubes traverse the extracellular matrix of the style on their way to the ovules. We previously characterized two pollen-specific, receptor-like kinases, LePRK1 and LePRK2, from tomato (
Lycopersicon esculentum
). Their structure and immunolocalization pattern and the specific dephosphorylation of LePRK2 suggested that these kinases might interact with signaling molecules in the style extracellular matrix. Here, we show that LePRK1 and LePRK2 can be coimmunoprecipitated from pollen or when expressed together in yeast. In yeast, their association requires LePRK2 kinase activity. In pollen, LePRK1 and LePRK2 are found in an ≈400-kDa protein complex that persists on pollen germination, but this complex is disrupted when pollen is germinated
in vitro
in the presence of style extract. In yeast, the addition of style extract also disrupts the interaction between LePRK1 and LePRK2. Fractionation of the style extract reveals that the disruption activity is enriched in the 3- to 10-kDa fraction. A component(s) in this fraction also is responsible for the specific dephosphorylation of LePRK2. The style component(s) that dephosphorylates LePRK2 is likely to be a heat-stable peptide that is present in exudate from the style. The generally accepted model of receptor kinase signaling involves binding of a ligand to extracellular domains of receptor kinases and subsequent activation of the signaling pathway by receptor autophosphorylation. In contrast to this typical scenario, we propose that a putative style ligand transduces the signal in pollen tubes by triggering the specific dephosphorylation of LePRK2, followed by dissociation of the LePRK complex.
Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells
Authors: Kubota Hiroshi , Avarbock Mary R. , Brinster Ralph L.
Published: May 2003

Spermatogonial stem cells (SSCs) are responsible for maintaining spermatogenesis throughout life in the male by continuous production of daughter cells that differentiate into spermatozoa. However, no unique phenotypic markers to identify SSCs have been described. In this study, the SSC surface phenotype was characterized by using flow cytometric cell sorting in conjunction with a transplantation functional assay for SSCs. Highly enriched stem cell activity was found in the MHC class I (MHC-I)
–
Thy-1
+
c-kit
–
cell fraction of the mouse cryptorchid testis. There was little or no stem cell activity in any other fraction. The antigenic phenotype of the MHC-I
–
Thy-1
+
c-kit
–
SSCs was α6-integrin
+
CD24
+
αvintegrin
–
Sca-1
–
CD34
–
. Subsequently, testis side population (SP) cells, which are defined by a Hoechst dye efflux assay, were identified. Their surface phenotype was found to be MHC-I
+
Thy-1
–
Sca-1
+
, and the transplantation assay demonstrated that the testis SP and SSCs are distinct populations. In several other tissues, the SP has been shown to contain stem cells, but we found that this characteristic does not define SSCs. The identification of a surface phenotype that allows production of a highly enriched SSC population will facilitate functional and genomic studies and enable further comparison with other stem cells.
Positional cloning of the
young
mutation identifies an essential role for the Brahma chromatin remodeling complex in mediating retinal cell differentiation
Authors: Gregg Ronald G. , Willer Gregory B. , Fadool James M. , Dowling John E. , Link Brian A.
Published: May 2003

Zebrafish with the
young (yng)
mutation show a defect in retinal cell differentiation. Here we demonstrate that a mutation in a
brahma-related
gene (
brg1
) is responsible for the
yng
phenotype. Brahma homologues function as essential subunits for SWI/SNF-type chromatin remodeling complexes. Our analysis indicates that
brg1
is required for the wave of mitogen-activated protein kinase activity that precedes retinal cell differentiation. Using specific inhibitors of the mitogen-activated protein kinase pathway we show this signal has a direct role in retinal cell differentiation. Lastly, through investigations of mutants in other chromatin remodeling subunits, we provide genetic evidence for gene and tissue specificity of the Brahma chromatin remodeling complex.
Disruption of the estrogen receptor β gene in mice causes myeloproliferative disease resembling chronic myeloid leukemia with lymphoid blast crisis
Authors: Shim Gil-Jin , Wang Ling , Andersson Sandra , Nagy Noémi , Kis Loránd Levente , Zhang Qinghong , Mäkelä Sari , Warner Margaret , Gustafsson Jan-Åke
Published: May 2003

Proliferation of pluripotent, bone marrow stem cells, which develop to lymphoid and myeloid progenitors, is negatively regulated by estrogen. Although in estrogen deficiency and in estrogen receptor knockout mice there is significant alteration in bone marrow hematopoiesis, the effects of aging on estrogen receptor deficiencies in mice have not been investigated yet. In this study we show that by 1.5 years of age, estrogen receptor β knockout (ERβ–/–) mice develop pronounced splenomegaly that is much more severe in females than in males. Further characterization of these mice revealed myelogenous hyperplasia in bone marrow, an increase in the number of granulocytes and B lymphocytes in blood, lymphadenopathy, and infiltration of leukocytes in the liver and lung. Analysis by flow cytometry of the bone marrow cells revealed that the percentage and total number of Gr-1hi/Mac-1hi-positive granulocytes were increased by 15–30% and 100%, respectively. The numbers of B cells in the bone marrow and spleen were significantly higher in ERβ–/–mice than in WT littermates. Some of the ERβ–/–mice also had a severe lymphoproliferative phenotype. Thus the absence of ERβ results in a myeloproliferative disease resembling human chronic myeloid leukemia with lymphoid blast crisis. Our results indicate a previously unknown role for ERβ in regulating the differentiation of pluripotent hematopoietic progenitor cells and suggest that the ERβ–/–mouse is a potential model for myeloid and lymphoid leukemia. Furthermore, we suggest that ERβ agonists might have clinical value in the treatment of leukemia.
Retrofitting existing chemical scrubbers to biotrickling filters for H2S emission control
Authors: Gabriel David , Deshusses Marc A.
Published: May 2003

Biological treatment is a promising alternative to conventional air-pollution control methods, but thus far biotreatment processes for odor control have always required much larger reactor volumes than chemical scrubbers. We converted an existing full-scale chemical scrubber to a biological trickling filter and showed that effective treatment of hydrogen sulfide (H2S) in the converted scrubber was possible even at gas contact times as low as 1.6 s. That is 8–20 times shorter than previous biotrickling filtration reports and comparable to usual contact times in chemical scrubbers. Significant removal of reduced sulfur compounds, ammonia, and volatile organic compounds present in traces in the air was also observed. Continuous operation for >8 months showed stable performance and robust behavior for H2S treatment, with pollutant-removal performance comparable to that achieved by using a chemical scrubber. Our study demonstrates that biotrickling filters can replace chemical scrubbers and be a safer, more economical technique for odor control.
Molecular paleontology of transposable elements in the
Drosophila melanogaster
genome
Authors: Kapitonov Vladimir V. , Jurka Jerzy
Published: May 2003

We report here a superfamily of “cut and paste” DNA transposons called
Transib
. These transposons populate the
Drosophila melanogaster
and
Anopheles gambiae
genomes, use a transposase that is not similar to any known proteins, and are characterized by 5-bp target site duplications. We found that the fly genome, which was thought to be colonized by the
P
element <100 years ago, harbors ≈5 million year (Myr)-old fossils of
ProtoP
, an ancient ancestor of the
P
element. We also show that
Hoppel
, a previously reported transposable element (TE), is a nonautonomous derivate of
ProtoP
. We found that the “rolling-circle”
Helitron
transposons identified previously in plants and worms populate also insect genomes. Our results indicate that
Helitron
s were horizontally transferred into the fly or/and mosquito genomes. We have also identified a most abundant TE in the fly genome,
DNAREP1
_
DM
, which is an ≈10-Myr-old footprint of a
Penelope
-like retrotransposon. We estimated that TEs are three times more abundant than reported previously, making up ≈22% of the whole genome. The chromosomal and age distributions of TEs in
D. melanogaster
are very similar to those in
Arabidopsis thaliana
. Both genomes contain only relatively young TEs (<20 Myr old), constituting a main component of paracentromeric regions.
Global perception in small brains: Topological pattern recognition in honey bees
Authors: Chen Lin , Zhang Shaowu , Srinivasan Mandyam V.
Published: May 2003

A series of experiments with honey bees demonstrate that their small brains nevertheless possess the ability for topological perception. Bees rapidly learned to discriminate patterns that are topologically different, and they generalized the learned cue to other novel patterns. By contrast, discrimination of topologically equivalent patterns was learned much more slowly and not as well. Thus, although the global nature of topological properties makes their computation difficult, topology may be a fundamental component of the vocabulary by which visual systems represent and characterize objects.
Dictyostelium
and
Acanthamoeba
myosin II assembly domains go to the cleavage furrow of
Dictyostelium
myosin II-null cells
Authors: Shu Shi , Liu Xiong , Korn Edward D.
Published: May 2003

How myosin II localizes to the cleavage furrow of dividing cells is largely unknown. We show here that a 283-residue protein, assembly domain (AD)1, corresponding to the AD in the tail of
Dictyostelium
myosin II assembles into bundles of long tubules when expressed in myosin II-null cells and localizes to the cleavage furrow of dividing cells. AD1 mutants that do not polymerize
in vitro
do not go to the cleavage furrow
in vivo
. An assembly-competent polypeptide corresponding to the C-terminal 256 residues of
Acanthamoeba
myosin II also goes to the cleavage furrow of
Dictyostelium
myosin II-null cells. When overexpressed in wild-type cells, AD1 colocalizes with endogenous myosin II (possibly as a copolymer) in interphase, motile, and dividing cells and under caps of Con A receptors but has no effect on myosin II-dependent functions. These results suggest that neither a specific sequence, other than that required for polymerization, nor interaction with other proteins is required for localization of myosin II to the cleavage furrow.
Mate choice theory and the mode of selection in sexual populations
Authors: Carson Hampton L.
Published: May 2003

Indirect new data imply that mate and/or gamete choice are major selective forces driving genetic change in sexual populations. The system dictates nonrandom mating, an evolutionary process requiring both revised genetic theory and new data on heritability of characters underlying Darwinian fitness. Successfully reproducing individuals represent rare selections from among vigorous, competing survivors of preadult natural selection. Nonrandom mating has correlated demographic effects: reduced effective population size, inbreeding, low gene flow, and emphasis on deme structure. Characters involved in choice behavior at reproduction appear based on quantitative trait loci. This variability serves selection for fitness within the population, having only an incidental relationship to the origin of genetically based reproductive isolation between populations. The claim that extensive hybridization experiments withDrosophilaindicate that selection favors a gradual progression of “isolating mechanisms” is flawed, because intra-group random mating is assumed. Over deep time, local sexual populations are strong, independent genetic systems that use rich fields of variable polygenic components of fitness. The sexual reproduction system thus particularizes, in small subspecific populations, the genetic basis of the grand adaptive sweep of selective evolutionary change, much as Darwin proposed.
Fatty acid synthesis is essential in embryonic development: Fatty acid synthase null mutants and most of the heterozygotes die
in utero
Authors: Chirala Subrahmanyam S. , Chang Hua , Matzuk Martin , Abu-Elheiga Lutfi , Mao Jianqiang , Mahon Kathleen , Finegold Milton , Wakil Salih J.
Published: May 2003

In animals, including humans, the source of long-chain saturated fatty acids is
de novo
synthesis, which is mediated by fatty acid synthase (FAS), ingested food, or both. To understand the importance of
de novo
fatty acid synthesis, we generated FAS knockout mice. The heterozygous FAS mutants (
Fasn
+/–
) are ostensibly normal. In
Fasn
+/–
mice the levels of FAS mRNA and the FAS activity are ≈50% and 35% lower, respectively, than those of WT mice; hence, FAS levels are affected by gene dosage. When the
Fasn
+/–
mutant mice were interbred,
Fasn
–/–
mice were not produced; thus, FAS is essential during embryonic development. Furthermore, the number of
Fasn
+/–
progeny obtained was 70% less than predicted by Mendelian inheritance, indicating partial haploid insufficiency. Even when one of the parents was WT, the estimated loss of heterozygous progeny was 60%. This loss of
Fasn
+/–
pups appeared to be strain-specific and became more pronounced as the heterozygous females produced more litters. Most of the
Fasn
–/–
mutant embryos died before implantation and the
Fasn
+/–
embryos died at various stages of their development. Feeding the breeders a diet rich in saturated fatty acids did not prevent the loss of homoor heterozygotes. These observations are very important in considering teratogenic consequences of drugs aimed at inhibiting FAS activity, to reduce either obesity or the growth of cancerous tissues.
Sexual transmission of the [Het-s] prion leads to meiotic drive in
Podospora anserina
Authors: Dalstra Henk J. P. , Swart Klaas , Debets Alfons J. M. , Saupe Sven J. , Hoekstra Rolf F.
Published: May 2003

In the filamentous fungus
Podospora anserina
, two phenomena are associated with polymorphism at the
het-s
locus, vegetative incompatibility and ascospore abortion. Two
het-s
alleles occur naturally,
het-s
and
het-S
. The
het-s
encoded protein is a prion propagating as a self-perpetuating amyloid aggregate. When prion-infected [Het-s] hyphae fuse with [Het-S] hyphae, the resulting heterokaryotic cells necrotize. [Het-s] and [Het-S] strains are sexually compatible. When, however, a female [Het-s] crosses with [Het-S], a significant percentage of
het-S
spores abort, in a way similar to spore killing in Neurospora and Podospora. We report here that sexual transmission of the [Het-s] prion after nonisogamous mating in the reproductive cycle of Podospora is responsible for the killing of
het-S
spores. Progeny of crosses between isogenic strains with distinct wild-type or introduced, ectopic
het-s
/
S
alleles were cytologically and genetically analyzed. The effect of
het-s
/
S
overexpression, ectopic
het-s
/
S
expression, absence of
het-s
expression, loss of [Het-s] prion infection, and the distribution patterns of HET-s/S-GFP proteins were categorized during meiosis and ascospore formation. This study unveiled a
het-S
spore-killing system that is governed by dosage of and interaction between the [Het-s] prion and the HET-S protein. Due to this property of the [Het-s] prion, the
het-s
allele acts as a meiotic drive element favoring maintenance of the prion-forming allele in natural populations.
Compression forces generated by actin comet tails on lipid vesicles
Authors: Giardini Paula A. , Fletcher Daniel A. , Theriot Julie A.
Published: May 2003

Polymerizing networks of actin filaments generate force for a variety of movements in living cells, including protrusion of filopodia and lamellipodia, intra- and intercellular motility of certain bacterial and viral pathogens, and motility of endocytic vesicles and other membrane-bound organelles. During actin-based motility, coexisting populations of actin filaments exert both pushing and retarding forces on the moving cargo. To examine the distribution and magnitude of forces generated by actin, we have developed a model system where large artificial lipid vesicles coated with the protein ActA from the bacterial pathogen
Listeria monocytogenes
are propelled by actin polymerization in cytoplasmic extract. We find that motile vesicles associated with actin comet tails are significantly deformed due to an inward compression force exerted by actin polymerization orthogonal to the direction of motion, which is >10-fold greater in magnitude than the component of the force exerted in the direction of motion. Furthermore, there is a spatial segregation of the pushing and retarding forces, such that pushing predominates along the sides of the vesicle, although retarding forces predominate at the rear. We estimate that the total net (pushing minus retarding) force generated by the actin comet tail is ≈0.4–4 nN. In addition, actin comet tail formation is associated with polarization of the ActA protein on the fluid vesicle surface, which may reinforce the persistence of unidirectional motion by helping to maintain a persistent asymmetry of actin filament density.
Susceptibility to tuberculosis: A locus on mouse chromosome 19 (Trl-4) regulates Mycobacterium tuberculosis replication in the lungs
Authors: Mitsos Loukia-Maria , Cardon Lon R. , Ryan Lynn , LaCourse Ronald , North Robert J. , Gros Philippe
Published: May 2003

The mouse DBA/2 (D2) strain is very susceptible to infection with virulent
Mycobacterium tuberculosis
, whereas C57BL/6 (B6) is much more resistant. Infection of D2 and B6 mice with
M. tuberculosis
H37Rv by the respiratory route is biphasic: during the first 3 weeks, there is rapid bacterial growth in the lung of both strains, whereas beyond this point replication stops in B6 but continues in D2, causing rapidly fatal pulmonary disease. To identify the genes regulating growth of
M. tuberculosis
in the lungs of these two strains, 98 informative (B6 × D2) F
2
mice were infected by the respiratory route with
M. tuberculosis
H37Rv (2 × 10
2
colony-forming units), and the extent of bacterial replication in the lungs at 90 days was used as a quantitative measure of susceptibility in a whole-genome scan. Quantitative trait locus mapping identified a major locus on chromosome 19 (
T
uberculosis
r
esistance
l
ocus-4,
Trl-4
; logarithm of odds 5.6), which regulated pulmonary replication of
M. tuberculosis
and accounted for 25% of the phenotypic variance. B6 alleles at
Trl-4
were inherited in an incompletely dominant fashion and associated with reduced bacterial replication. An additional effect of a locus (
Trl-3
), previously shown to affect survival to i.v. infection with
M. tuberculosis
, was also noted. F
2
mice homozygous for B6 alleles at both
Trl-3
and
Trl-4
were as resistant as B6 parents, whereas mice homozygous for D2 alleles were as susceptible as D2 parents. These results suggest a strong genetic interaction between
Trl-3
and
Trl-4
in regulating pulmonary replication of
M. tuberculosis
.
Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana
Authors: Laule Oliver , Fürholz Andreas , Chang Hur-Song , Zhu Tong , Wang Xun , Heifetz Peter B. , Gruissem Wilhelm , Lange Markus
Published: May 2003

In plants, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, the central intermediates in the biosynthesis of isoprenoids, is compartmentalized: the mevalonate (MVA) pathway, which is localized to the cytosol, is responsible for the synthesis of sterols, certain sesquiterpenes, and the side chain of ubiquinone; in contrast, the recently discovered MVA-independent pathway, which operates in plastids, is involved in providing the precursors for monoterpenes, certain sesquiterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and plastoquinone. Specific inhibitors of the MVA pathway (lovastatin) and the MVA-independent pathway (fosmidomycin) were used to perturb biosynthetic flux in
Arabidopsis thaliana
seedlings. The interaction between both pathways was studied at the transcriptional level by using GeneChip (Affymetrix) microarrays and at the metabolite level by assaying chlorophylls, carotenoids, and sterols. Treatment of seedlings with lovastatin resulted in a transient decrease in sterol levels and a transient increase in carotenoid as well as chlorophyll levels. After the initial drop, sterol amounts in lovastatin-treated seedlings recovered to levels above controls. As a response to fosmidomycin treatment, a transient increase in sterol levels was observed, whereas chlorophyll and carotenoid amounts decreased dramatically when compared with controls. At 96 h after fosmidomycin addition, the levels of all metabolites assayed (sterols, chlorophylls, and carotenoids) were substantially lower than in controls. Interestingly, these inhibitor-mediated changes were not reflected in altered gene expression levels of the genes involved in sterol, chlorophyll, and carotenoid metabolism. The lack of correlation between gene expression patterns and the accumulation of isoprenoid metabolites indicates that posttranscriptional processes may play an important role in regulating flux through isoprenoid metabolic pathways.
Structural and biochemical studies of p21RasS-nitrosylation and nitric oxide-mediated guanine nucleotide exchange
Authors: Williams Jason G. , Pappu Kamesh , Campbell Sharon L.
Published: May 2003

Ras is a guanine nucleotide-binding protein that cycles between inactive GDP-bound and active GTP-bound states to regulate a diverse array of cellular processes, including cell growth, apoptosis, and differentiation. The guanine nucleotide-bound state of Ras is tightly maintained by regulatory factors to promote regulated growth control. A class of regulatory molecules that lead to Ras activation are guanine nucleotide exchange factors (GEFs). Ras GEFs bind to Ras and facilitate GDP release, followed by GTP incorporation and Ras activation. Nitric oxide (NO) has also been shown to promote guanine nucleotide exchange (GNE) on Ras and increase cellular Ras-GTP levels, but the process by which NO-mediated GNE occurs is not clear. We initiated NMR structural and biochemical studies to elucidate how nitrosylation of Ras might lead to enhanced GNE. Surprisingly, our studies show that stable S-nitrosylation of Ras at Cys-118, does not affect the structure of Ras, its association with the Ras-binding domain of Raf (a downstream effector of Ras), or GNE rates relative to non-nitrosylated Ras. We have found, however, that the actual chemical process of nitrosylation, rather than the end-product of Ras S-nitrosylation, accounts for the enhanced GNE that we have observed and that has been previously observed by others.
γ-Secretase is a membrane protein complex comprised of presenilin, nicastrin, aph-1, and pen-2
Authors: Kimberly W. Taylor , LaVoie Matthew J. , Ostaszewski Beth L. , Ye Wenjuan , Wolfe Michael S. , Selkoe Dennis J.
Published: May 2003

γ-Secretase catalyzes the intramembrane proteolysis of Notch, β-amyloid precursor protein, and other substrates as part of a new signaling paradigm and as a key step in the pathogenesis of Alzheimer's disease. This unusual protease has eluded identification, though evidence suggests that the presenilin heterodimer comprises the catalytic site and that a highly glycosylated form of nicastrin associates with it. The formation of presenilin heterodimers from the holoprotein is tightly gated by unknown limiting cellular factors. Here we show that Aph-1 and Pen-2, two recently identified membrane proteins genetically linked to γ-secretase, associate directly with presenilin and nicastrin in the active protease complex. Coexpression of all four proteins leads to marked increases in presenilin heterodimers, full glycosylation of nicastrin, and enhanced γ-secretase activity. These findings suggest that the four membrane proteins comprise the limiting components of γ-secretase and coassemble to form the active enzyme in mammalian cells.
Gastrointestinal stromal tumors in a mouse model by targeted mutation of the Kit receptor tyrosine kinase
Authors: Sommer Gunhild , Agosti Valter , Ehlers Imke , Rossi Ferdinand , Corbacioglu Selim , Farkas Judith , Moore Malcolm , Manova Katia , Antonescu Cristina R. , Besmer Peter
Published: May 2003

Oncogenic Kit mutations are found in somatic gastrointestinal (GI) stromal tumors (GISTs) and mastocytosis. A mouse model for the study of constitutive activation of Kit in oncogenesis has been produced by a knock-in strategy introducing a Kit exon 11-activating mutation into the mouse genome based on a mutation found in a case of human familial GIST syndrome. Heterozygous mutant
Kit
V558
Δ
/+ mice develop symptoms of disease and eventually die from pathology in the GI tract. Patchy hyperplasia of Kit-positive cells is evident within the myenteric plexus of the entire GI tract. Neoplastic lesions indistinguishable from human GISTs were observed in the cecum of the mutant mice with high penetrance. In addition, mast cell numbers in the dorsal skin were increased. Therefore
Kit
V558
Δ
/+ mice reproduce human familial GISTs, and they may be used as a model for the study of the role and mechanisms of Kit in neoplasia. Importantly, these results demonstrate that constitutive Kit signaling is critical and sufficient for induction of GIST and hyperplasia of interstitial cells of Cajal.
Molecular genetic analysis of the glycosyltransferase Fringe in
Drosophila
Authors: Correia Trudy , Papayannopoulos Venizelos , Panin Vladislav , Woronoff Pamela , Jiang Jin , Vogt Thomas F. , Irvine Kenneth D.
Published: May 2003

Fringe proteins are β1,3-
N
-acetylglucosaminyltransferases that modulate signaling through Notch receptors by modifying O-linked fucose on epidermal growth factor domains. Fringe is highly conserved, and comparison among 18 different Fringe proteins from 11 different species identifies a core set of 84 amino acids that are identical among all Fringes. Fringe is only distantly related to other glycosyltransferases, but analysis of the predicted
Drosophila
proteome identifies a set of four sequence motifs shared among Fringe and other putative β1,3-glycosyltransferases. To gain functional insight into these conserved sequences, we genetically and molecularly characterized 14 point mutations in
Drosophila fringe
. Most nonsense mutations act as recessive antimorphs, raising the possibility that Fringe may function as a dimer. Missense mutations identify two distinct motifs that are conserved among β1,3-glycosyltransferases, and that can be modeled onto key motifs in the crystallographic structures of bovine β1,4-galactosyltransferase 1 and human glucuronyltransferase I. Other missense mutations map to amino acids that are conserved among Fringe proteins, but not among other glycosyltransferases, and thus may identify structural motifs that are required for unique aspects of Fringe activity.
Direct stimulation of naïve T cells by membrane vesicles from antigen-presenting cells: Distinct roles for CD54 and B7 molecules
Authors: Hwang Inkyu , Shen Xuefei , Sprent Jonathan
Published: May 2003

T cell stimulation usually requires direct contact with viable antigen-presenting cells (APCs). However, we show here that small exosome-like membrane vesicles shed from APCs can be recognized by naïve CD8+T cells in the absence of viable APCs. T cell antigen receptor-dependent binding of vesicles by CD8+cells is MHC class I/peptide-specific and requires that the vesicles coexpress intercellular adhesion molecule 1 (ICAM-1, CD54), although not B7 (B7-1). In the absence of B7, T cell binding of vesicles is nonimmunogenic. By contrast, vesicles expressing both ICAM-1 and B7 are strongly immunogenic and cause purified APC-depleted CD8+cells to mount peptide-specific proliferative responses and differentiate into effector cells.
Mice lacking methyl-CpG binding protein 1 have deficits in adult neurogenesis and hippocampal function
Authors: Zhao Xinyu , Ueba Tetsuya , Christie Brian R. , Barkho Basam , McConnell Michael J. , Nakashima Kinichi , Lein Edward S. , Eadie Brennan D. , Willhoite Andrew R. , Muotri Alysson R. , Summers Robert G. , Chun Jerold , Lee Kuo-Fen , Gage Fred H.
Published: May 2003

DNA methylation-mediated epigenetic regulation plays critical roles in regulating mammalian gene expression, but its role in normal brain function is not clear. Methyl-CpG binding protein 1 (MBD1), a member of the methylated DNA-binding protein family, has been shown to bind methylated gene promoters and facilitate transcriptional repression
in vitro
. Here we report the generation and analysis of MBD1
-/-
mice. MBD1
-/-
mice had no detectable developmental defects and appeared healthy throughout life. However, we found that MBD1
-/-
neural stem cells exhibited reduced neuronal differentiation and increased genomic instability. Furthermore, adult MBD1
-/-
mice had decreased neurogenesis, impaired spatial learning, and a significant reduction in long-term potentiation in the dentate gyrus of the hippocampus. Our findings indicate that DNA methylation is important in maintaining cellular genomic stability and is crucial for normal neural stem cell and brain functions.
A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen
Authors: Kushner Nicholas , Zhang Dong , Touzjian Neal , Essex Max , Lieberman Judy , Lu Yichen
Published: May 2003

Anthrax protective antigen (PA) is a 735-aa polypeptide that facilitates the exit of anthrax lethal factor (LF) from the endosome to the cytosol where the toxin acts. We recently found, however, that a fusion protein of the detoxified N-terminal domain of lethal factor (LFn) with a foreign peptide could induce CD8 T cell immune responses in the absence of PA. Because CD8 T cells recognize peptides derived from proteins degraded in the cytosol, this result suggests that lethal factor may be capable of entering the cytosol independently of PA. To investigate this further, the intracellular trafficking of an LFn-enhanced green fluorescent protein fusion protein (LFn-GFP) in the presence or absence of PA was examined by using confocal microscopy. LFn-GFP is able to enter the cytosol without PA. Moreover, it efficiently colocalizes with the proteosome 20s subunit, which degrades proteins into peptides for presentation to CD8 T cells by the MHC class I pathway. We further demonstrate that in the presence of an immune adjuvant LFn fusion protein without PA is able to effectively elicit anti-HIV cytotoxic T lymphocyte in inbred mice. These results indicate that LFn may be used without PA in a protein vaccine as a carrier to deliver antigens into the cytosol for efficient induction of T lymphocyte responses. Furthermore, these results enable us to propose a modified molecular mechanism of anthrax lethal toxin.
Sympatric speciation as a consequence of male pregnancy in seahorses
Authors: Jones Adam G. , Moore Glenn I. , Kvarnemo Charlotta , Walker DeEtte , Avise John C.
Published: May 2003

The phenomenon of male pregnancy in the family Syngnathidae (seahorses, pipefishes, and sea dragons) undeniably has sculpted the course of behavioral evolution in these fishes. Here we explore another potentially important but previously unrecognized consequence of male pregnancy: a predisposition for sympatric speciation. We present microsatellite data on genetic parentage that show that seahorses mate size-assortatively in nature. We then develop a quantitative genetic model based on these empirical findings to demonstrate that sympatric speciation indeed can occur under this mating regime in response to weak disruptive selection on body size. We also evaluate phylogenetic evidence bearing on sympatric speciation by asking whether tiny seahorse species are sister taxa to large sympatric relatives. Overall, our results indicate that sympatric speciation is a plausible mechanism for the diversification of seahorses, and that assortative mating (in this case as a result of male parental care) may warrant broader attention in the speciation process for some other taxonomic groups as well.
Structural basis of albumin–thyroxine interactions and familial dysalbuminemic hyperthyroxinemia
Authors: Petitpas Isabelle , Petersen Charles E. , Ha Chung-Eun , Bhattacharya Ananyo A. , Zunszain Patricia A. , Ghuman Jamie , Bhagavan Nadhipuram V. , Curry Stephen
Published: May 2003

Human serum albumin (HSA) is the major protein component of blood plasma and serves as a transporter for thyroxine and other hydrophobic compounds such as fatty acids and bilirubin. We report here a structural characterization of HSA–thyroxine interactions. Using crystallographic analyses we have identified four binding sites for thyroxine on HSA distributed in subdomains IIA, IIIA, and IIIB. Mutation of residue R218 within subdomain IIA greatly enhances the affinity for thyroxine and causes the elevated serum thyroxine levels associated with familial dysalbuminemic hyperthyroxinemia (FDH). Structural analysis of two FDH mutants of HSA (R218H and R218P) shows that this effect arises because substitution of R218, which contacts the hormone bound in subdomain IIA, produces localized conformational changes to relax steric restrictions on thyroxine binding at this site. We have also found that, although fatty acid binding competes with thyroxine at all four sites, it induces conformational changes that create a fifth hormone-binding site in the cleft between domains I and III, at least 9 Å from R218. These structural observations are consistent with binding data showing that HSA retains a high-affinity site for thyroxine in the presence of excess fatty acid that is insensitive to FDH mutations.
Identification of receptors for pig endogenous retrovirus
Authors: Ericsson Thomas A. , Takeuchi Yasuhiro , Templin Christian , Quinn Gary , Farhadian Shelli F. , Wood James C. , Oldmixon Beth A. , Suling Kristen M. , Ishii Jennifer K. , Kitagawa Yoshinori , Miyazawa Takayuki , Salomon Daniel R. , Weiss Robin A. , Patience Clive
Published: May 2003

Xenotransplantation of porcine tissues has the potential to treat a wide variety of major health problems including organ failure and diabetes. Balanced against the potential benefits of xenotransplantation, however, is the risk of human infection with a porcine microorganism. In particular, the transmission of porcine endogenous retrovirus (PERV) is a major concern [Chapman, L. E. & Bloom, E. T. (2001)
J. Am. Med. Assoc.
285, 2304–2306]. Here we report the identification of two, sequence-related, human proteins that act as receptors for PERV-A, encoded by genes located on chromosomes 8 and 17. We also describe homologs from baboon and porcine cells that also are active as receptors. Conversely, activity could not be demonstrated with a syntenic murine receptor homolog. Sequence analysis indicates that PERV-A receptors [human PERV-A receptor (HuPAR)-1, HuPAR-2, baboon PERV-A receptor 2, and porcine PERV-A receptor] are multiple membrane-spanning proteins similar to receptors for other gammaretroviruses. Expression is widespread in human tissues including peripheral blood mononuclear cells, but their biological functions are unknown. The identification of the PERV-A receptors opens avenues of research necessary for a more complete assessment of the retroviral risks of pig to human xenotransplantation.
Copper chelation represses the vascular response to injury
Authors: Mandinov Lazar , Mandinova Anna , Kyurkchiev Stanimir , Kyurkchiev Dobroslav , Kehayov Ivan , Kolev Vihren , Soldi Raffaella , Bagala Cinzia , de Muinck Ebo D. , Lindner Volkhard , Post Mark J. , Simons Michael , Bellum Stephen , Prudovsky Igor , Maciag Thomas
Published: May 2003

The induction of an acute inflammatory response followed by the release of polypeptide cytokines and growth factors from peripheral blood monocytes has been implicated in mediating the response to vascular injury. Because the Cu
2
+
-binding proteins IL-1α and fibroblast growth factor 1 are exported into the extracellular compartment in a stress-dependent manner by using intracellular Cu
2
+
to facilitate the formation of S100A13 heterotetrameric complexes and these signal peptideless polypeptides have been implicated as regulators of vascular injury
in vivo
, we examined the ability of Cu
2
+
chelation to repress neointimal thickening in response to injury. We observed that the oral administration of the Cu
2
+
chelator tetrathiomolybdate was able to reduce neointimal thickening after balloon injury in the rat. Interestingly, although immunohistochemical analysis of control neointimal sections exhibited prominent staining for MAC1, IL-1α, S100A13, and the acidic phospholipid phosphatidylserine, similar sections obtained from tetrathiomolybdate-treated animals did not. Further, adenoviral gene transfer of the IL-1 receptor antagonist during vascular injury also significantly reduced the area of neointimal thickening. Our data suggest that intracellular copper may be involved in mediating the response to injury
in vivo
by its ability to regulate the stress-induced release of IL-1α by using the nonclassical export mechanism employed by human peripheral blood mononuclear cells
in vitro
.
The working stroke upon myosin–nucleotide complexes binding to actin
Authors: Steffen Walter , Smith David , Sleep John
Published: May 2003

For many years, it has been known that myosin binds to actin tightly, but it had not been possible to devise a muscle fiber experiment to determine whether this binding energy is directly coupled to the working stroke of the actomyosin crossbridge cycle. Addressing the question at the single-molecule level with optical tweezers allows the problem to be resolved. We have compared the working stroke on the binding of four myosin complexes (myosin, myosin-ADP, myosin-pyrophosphate, and myosin-adenyl-5′yl imidodiphosphate) with that observed while hydrolyzing ATP. None of the four was observed to give a working stroke significantly different from zero. A working stroke (5.4 nm) was observed only with ATP, which indicates that the other states bind to actin in a rigor-like conformation and that myosin products (M.ADP.P
i
), the state that binds to actin during ATPase activity, binds in a different, prestroke conformation. We conclude that myosin, while dissociated from actin, must be able to take up at least two mechanical conformations and show that our results are consistent with these conformations corresponding to the two states characterized at high resolution, which are commonly referred to in terms of having open and closed nucleotide binding pockets.
Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice
Authors: Hendricks Carrie A. , Almeida Karen H. , Stitt Molly S. , Jonnalagadda Vidya S. , Rugo Rebecca E. , Kerrison G. Foster , Engelward Bevin P.
Published: May 2003

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length
EYFP
coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute
EYFP
coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 ± 0.1 per 10
6
cell divisions. Interestingly, the rate is ≈10-fold greater in fibroblasts derived from embryonic tissue. We observe an ≈15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both
in vitro
and
in vivo
.
Saccharomyces cerevisiae
chromatin-assembly factors that act during DNA replication function in the maintenance of genome stability
Authors: Myung Kyungjae , Pennaneach Vincent , Kats Ellen S. , Kolodner Richard D.
Published: May 2003

Some spontaneous gross chromosomal rearrangements (GCRs) seem to result from DNA-replication errors. The chromatin-assembly factor I (CAF-I) and replication-coupling assembly factor (RCAF) complexes function in chromatin assembly during DNA replication and repair and could play a role in maintaining genome stability. Inactivation of CAF-I or RCAF increased the rate of accumulating different types of GCRs including translocations and deletion of chromosome arms with associated
de novo
telomere addition. Inactivation of CAF-I seems to cause damage that activates the DNA-damage checkpoints, whereas inactivation of RCAF seems to cause damage that activates the DNA-damage and replication checkpoints. Both defects result in increased genome instability that is normally suppressed by these checkpoints, RAD52-dependent recombination, and PIF1-dependent inhibition of
de novo
telomere addition. Treatment of CAF-I- or RCAF-defective cells with methyl methanesulfonate increased the induction of GCRs compared with that seen for a wild-type strain. These results indicate that coupling of chromatin assembly to DNA replication and DNA repair is critical to maintaining genome stability.
Specific protein methylation defects and gene expression perturbations in coactivator-associated arginine methyltransferase 1-deficient mice
Authors: Yadav Neelu , Lee Jaeho , Kim Jeesun , Shen Jianjun , Hu Mickey C.-T. , Aldaz C. Marcelo , Bedford Mark T.
Published: May 2003

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we show that embryos with a targeted disruption of CARM1 are small in size and die perinatally. The methylation of two known CARM1 substrates, poly(A)-binding protein (PABP1) and the transcriptional cofactor p300, was abolished in knockout embryos and cells. However, CARM1-dependent methylation of histone H3 was not observed. Furthermore, estrogen-responsive gene expression was aberrant in
Carm1
–
/
–
fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. These findings provide genetic evidence for the essential role of CARM1 in estrogen-mediated transcriptional activation.