bluuebunny/crossref_metadata_2025 huggingface dataset:
Arabidopsis thaliana
ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein
Authors: Büttner Michael , Singh Karam B.
Published: July 2002

Ocs elements are a group of promoter sequences required for the expression of both pathogen genes in infected plants and plant defense genes. Genes for
ocs
element binding factors (OBFs), belonging to a specific class of basic-region leucine zipper (bZIP) transcription factors, have been isolated in a number of plants. Using protein–protein interaction screening with OBF4 we have isolated AtEBP, an
Arabidopsis
protein that contains a novel DNA-binding domain, the AP2/EREBP domain. One class of proteins that contain this domain are the tobacco ethylene-responsive element binding proteins (EREBPs). The EREBPs bind the GCC box that confers ethylene responsiveness to a number of pathogenesis related (PR) gene promoters.
AtEBP
expression is inducible by exogenous ethylene in wild-type plants and
AtEBP
transcripts are increased in the
ctr1-1
mutant, where ethylene-regulated pathways are constitutively active. Electrophoretic mobility-shift assay and DNase I footprint analysis revealed that AtEBP can specifically bind to the GCC box. Interestingly, the highest level of
AtEBP
expression was detected in callus tissue, where ocs elements are very active. Synergistic effects of the GCC box with ocs elements or the related G-box sequence have been previously observed, for example, in the ethylene-induced expression of a PR gene promoter. Our results suggest that cross-coupling between EREBP and bZIP transcription factors occurs and may therefore be important in regulating gene expression during the plant defense response.
A chemoattractant cytokine associated with granulomas in tuberculosis and silicosis
Authors: Nau Gerard J. , Guilfoile Patrick , Chupp Geoffrey L. , Berman Jeffrey S. , Kim Sue J. , Kornfeld Hardy , Young Richard A.
Published: July 2002

Chronic inflammation and granuloma formation are associated with mononuclear cell infiltrates and are characteristic pathologic responses in tuberculosis. To identify host cell genes involved in tuberculous pathology, we screened macrophage cDNA libraries for genes induced by mycobacterial infection. One gene isolated in this screen, osteopontin (also known as early T lymphocyte activation protein 1 or Eta-1), was of particular interest because it is a cytokine and macrophage chemoattractant. Further study revealed thatMycobacterium tuberculosisinfection of primary human alveolar macrophages causes a substantial increase in osteopontin gene expression. Osteopontin protein was identified by immunohistochemistry in macrophages, lymphocytes, and the extracellular matrix of pathologic tissue sections of patients with tuberculosis. Increased osteopontin expression also was found to be associated with silicosis, another granulomatous disease. The association of osteopontin with granulomatous pathology, together with the known properties of the protein, suggest that osteopontin may participate in granuloma formation. The strategy of identifying host genes whose expression is altered by infection thus can provide valuable clues to disease mechanisms and will be increasingly valuable as additional human genome sequences become available.
Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the
Drosophila
discs large tumor suppressor protein
Authors: Lee Siu Sylvia , Weiss Robert S. , Javier Ronald T.
Published: July 2002

The 9ORF1 gene encodes an adenovirus
E4
region oncoprotein that requires a C-terminal region for transforming activity. Screening a λgt11 cDNA expression library with a 9ORF1 protein probe yielded a novel cellular PDZ domain-containing protein, 9BP-1, which binds to wild-type, but not a transformation-defective, C-terminal, mutant 9ORF1 protein. The fact that PDZ domains complex with specific sequences at the free C-terminal end of some proteins led to the recognition that the 9ORF1 C-terminal region contained such a consensus-binding motif. This discovery prompted investigations into whether the 9ORF1 protein associates with additional cellular proteins having PDZ domains. It was found that the 9ORF1 protein interacts directly,
in vitro
and
in vivo
, with the PDZ domain-containing protein hDlg/SAP97 (DLG), which is a mammalian homolog of the
Drosophila
discs large tumor suppressor protein and which also binds the adenomatous polyposis coli tumor suppressor protein. Of interest, in forming complexes, the 9ORF1 protein preferentially associated with the second PDZ domain of DLG, similar to adenomatous polyposis coli protein. Human T cell leukemia virus type 1 Tax and most oncogenic human papillomavirus E6 oncoproteins also possessed PDZ domain-binding motifs at their C termini and, significantly, human T cell leukemia virus type 1 Tax and human papillomavirus 18 E6 proteins bound DLG
in vitro
. Considering the requirement of the 9ORF1 C-terminal region in transformation, these findings suggest that interactions with the cellular factor DLG may contribute to the tumorigenic potentials of several different human virus oncoproteins.
Highly conservative reciprocal translocations formed by apparent joining of exchanged DNA double-strand break ends
Authors: Wang Peng , Zhou Rui-Hua , Zou Ying , Jackson-Cook Colleen K. , Povirk Lawrence F.
Published: July 2002

Chromosomal translocations induced by ionizing radiation and radiomimetic drugs are thought to arise by incorrect joining of DNA double-strand breaks. To dissect such misrepair events at a molecular level, large-scale, bleomycin-induced rearrangements in the
aprt
gene of Chinese hamster ovary D422 cells were mapped, the breakpoints were sequenced, and the original non-
aprt
parental sequences involved in each rearrangement were recovered from nonmutant cells. Of seven rearrangements characterized, six were reciprocal exchanges between
aprt
and unrelated sequences. Consistent with a mechanism involving joining of exchanged double-strand break ends, there was, in most cases, no homology between the two parental sequences, no overlap in sequences retained at the two newly formed junctions, and little or no loss of parental sequences (usually ≤2 bp) at the breakpoints. The breakpoints were strongly correlated (
P
< 0.0001) with expected sites of bleomycin-induced, double-strand breaks. Fluorescence
in situ
hybridization indicated that, in six of the mutants, the rearrangement was accompanied by a chromosomal translocation at the
aprt
locus, because upstream and downstream flanking sequences were detected on separate chromosomes. The results suggest that repair of free radical-mediated, double-strand breaks in confluence-arrested cells is effected by a conservative, homology-independent, end-joining pathway that does not involve single-strand intermediate and that misjoining of exchanged ends by this pathway can directly result in chromosomal translocations.
Defective γ-aminobutyric acid type B receptor-activated inwardly rectifying K
+
currents in cerebellar granule cells isolated from
weaver
and
Girk2
null mutant mice
Authors: Slesinger Paul A. , Stoffel Markus , Jan Yuh Nung , Jan Lily Y.
Published: July 2002

Stimulation of inhibitory neurotransmitter receptors, such as γ-aminobutyric acid type B (GABA
B
) receptors, activates G protein-gated inwardly rectifying K
+
channels (GIRK) which, in turn, influence membrane excitability. Seizure activity has been reported in a
Girk2
null mutant mouse lacking GIRK2 channels but showing normal cerebellar development as well as in the
weaver
mouse, which has mutated GIRK2 channels and shows abnormal development. To understand how the function of GIRK2 channels differs in these two mutant mice, we compared the G protein-activated inwardly rectifying K
+
currents in cerebellar granule cells isolated from
Girk2
null mutant and
weaver
mutant mice with those from wild-type mice. Activation of GABA
B
receptors in wild-type granule cells induced an inwardly rectifying K
+
current, which was sensitive to pertussis toxin and inhibited by external Ba
2+
ions. The amplitude of the GABA
B
receptor-activated current was severely attenuated in granule cells isolated from both
weaver
and
Girk2
null mutant mice. By contrast, the G protein-gated inwardly rectifying current and possibly the agonist-independent basal current appeared to be less selective for K
+
ions in
weaver
but not
Girk2
null mutant granule cells. Our results support the hypothesis that a nonselective current leads to the
weaver
phenotype. The loss of GABA
B
receptor-activated GIRK current appears coincident with the absence of GIRK2 channel protein and the reduction of GIRK1 channel protein in the
Girk2
null mutant mouse, suggesting that GABA
B
receptors couple to heteromultimers composed of GIRK1 and GIRK2 channel subunits.
Analysis of genomic alterations in benign, atypical, and anaplastic meningiomas: Toward a genetic model of meningioma progression
Authors: Weber Ruthild G. , Boström Jan , Wolter Marietta , Baudis Michael , Collins V. Peter , Reifenberger Guido , Lichter Peter
Published: July 2002

Nineteen benign [World Health Organization (WHO) grade I; MI], 21 atypical (WHO grade II; MII), and 19 anaplastic (WHO grade III; MIII) sporadic meningiomas were screened for chromosomal imbalances by comparative genomic hybridization (CGH). These data were supplemented by molecular genetic analyses of selected chromosomal regions and genes. With increasing malignancy grade, a marked accumulation of genomic aberrations was observed; i.e., the numbers (mean ± SEM) of total alterations detected per tumor were 2.9 ± 0.7 for MI, 9.2 ± 1.2 for MII, and 13.3 ± 1.9 for MIII. The most frequent alteration detected in MI was loss on 22q (58%). In MII, aberrations most commonly identified were losses on 1p (76%), 22q (71%), 14q (43%), 18q (43%), 10 (38%), and 6q (33%), as well as gains on 20q (48%), 12q (43%), 15q (43%), 1q (33%), 9q (33%), and 17q (33%). In MIII, most of these alterations were found at similar frequencies. However, an increase in losses on 6q (53%), 10 (68%), and 14q (63%) was observed. In addition, 32% of MIII demonstrated loss on 9p. Homozygous deletions in the
CDKN2A
gene at 9p21 were found in 4 of 16 MIII (25%). Highly amplified DNA sequences were mapped to 12q13–q15 by CGH in 1 MII. Southern blot analysis of this tumor revealed amplification of
CDK4
and
MDM2
. By CGH, DNA sequences from 17q were found to be amplified in 1 MII and 8 MIII, involving 17q23 in all cases. Despite the high frequency of chromosomal aberrations in the MII and MIII investigated, none of these tumors showed mutations in exons 5–8 of the
TP53
gene. On the basis of the most common aberrations identified in the various malignancy grades, a model for the genomic alterations associated with meningioma progression is proposed.
Essential role of the tyrosine kinase substrate phospholipase C-γ1 in mammalian growth and development
Authors: Ji Qun-sheng , Winnier Glenn E. , Niswender Kevin D. , Horstman Debra , Wisdom Ron , Magnuson Mark A. , Carpenter Graham
Published: July 2002

The activation of many tyrosine kinases leads to the phosphorylation and activation of phospholipase C-γ1 (PLC-γ1). To examine the biological function of this protein, homologous recombination has been used to selectively disrupt the
Plcg1
gene in mice. Homozygous disruption of
Plcg1
results in embryonic lethality at approximately embryonic day (E) 9.0. Histological analysis indicates that
Plcg1
(−/−) embryos appear normal at E 8.5 but fail to continue normal development and growth beyond E 8.5–E9.0. These results clearly demonstrate that PLC-γ1 with, by inference, its capacity to mobilize second messenger molecules is an essential signal transducing molecule whose absence is not compensated by other signaling pathways or other genes encoding PLC isozymes.
Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages
Authors: Fazio Sergio , Babaev Vladimir R. , Murray Alisa B. , Hasty Alyssa H. , Carter Kathy J. , Gleaves Linda A. , Atkinson James B. , Linton MacRae F.
Published: July 2002

Macrophage-derived foam cells express apolipoprotein E (apoE) abundantly in atherosclerotic lesions. To examine the physiologic role of apoE secretion by the macrophage in atherogenesis, bone marrow transplantation was used to reconstitute C57BL/6 mice with macrophages that were either null or wild type for the apoE gene. After 13 weeks on an atherogenic diet, C57BL/6 mice reconstituted with apoE null marrow developed 10-fold more atherosclerosis than controls in the absence of significant differences in serum cholesterol levels or lipoprotein profiles. ApoE expression was absent in the macrophage-derived foam cells of C57BL/6 mice reconstituted with apoE null marrow. Thus, lack of apoE expression by the macrophage promotes foam cell formation. These data support a protective role for apoE expression by the macrophage in early atherogenesis.
A transgenic mouse model for measles virus infection of the brain
Authors: Rall Glenn F. , Manchester Marianne , Daniels Lia R. , Callahan Eric M. , Belman Alec R. , Oldstone Michael B. A.
Published: July 2002

In addition to the rash, fever, and upper respiratory tract congestion that are the hallmarks of acute measles virus (MV) infection, invasion of the central nervous system (CNS) can occur, establishing a persistent infection primarily in neurons. The recent identification of the human membrane glycoprotein, CD46, as the MV receptor allowed for the establishment of transgenic mice in which the CD46 gene was transcriptionally regulated by a neuron-specific promoter. Expression of the measles receptor rendered primary CD46-positive neurons permissive to infection with MV–Edmonston. Notably, viral transmission within these cultures occurred in the absence of extracellular virus, presumably via neuronal processes. No infection was seen in nontransgenic mice inoculated intracerebrally with MV–Edmonston. In contrast, scattered neurons were infected following inoculation of transgenic adults, and an impressive widespread neuronal infection was established in transgenic neonates. The neonatal infection resulted in severe CNS disease by 3–4 weeks after infection. Illness was characterized initially by awkward gait and a lack of mobility, and in later stages seizures leading to death. These results show that expression of the MV receptor on specific murine cells (neurons)
in vivo
is absolutely essential to confer both susceptibility to infection and neurologic disease by this human virus. The disparity in clinical findings between neonatal and adult transgenic mice indicates that differences exist between the developing and mature CNS with respect to MV infection and pathogenesis.
A 12
R
-lipoxygenase in human skin: Mechanistic evidence, molecular cloning, and expression
Authors: Boeglin William E. , Kim Richard B. , Brash Alan R.
Published: July 2002

A recognized feature of psoriasis and other proliferative dermatoses is accumulation in the skin of the unusual arachidonic acid metabolite, 12
R
-hydroxyeicosatetraenoic acid (12
R
-HETE). This hydroxy fatty acid is opposite in chirality to the product of the well-known 12
S
-lipoxygenase and heretofore in mammals is known only as a product of cytochrome P450s. Here we provide mechanistic evidence for a lipoxygenase route to 12
R
-HETE in human psoriatic tissue and describe a 12
R
-lipoxygenase that can account for the biosynthesis. Initially we demonstrated retention of the C-12 deuterium of octadeuterated arachidonic acid in its conversion to 12
R
-HETE in incubations of psoriatic scales, indicating the end product is not formed by isomerization from 12
S
-H(P)ETE via the 12-keto derivative. Secondly, analysis of product formed from [10
R
-
3
H] and [10
S
-
3
H]-labeled arachidonic acids revealed that 12
R
-HETE synthesis is associated with stereospecific removal of the pro-
R
hydrogen from the 10-carbon of arachidonate. This result is compatible with 12
R
-lipoxygenase-catalyzed formation of 12
R
-HETE and not with a P450-catalyzed route to 12
R
-HETE in psoriatic scales. We cloned a lipoxygenase from human keratinocytes; the cDNA and deduced amino acid sequences share ≤50% identity to other human lipoxygenases. This enzyme, when expressed in Hela cells, oxygenates arachidonic acid to 12-HPETE, >98% 12
R
in configuration. The 12
R
-lipoxygenase cDNA is detectable by PCR in psoriatic scales and as a 2.5-kilobase mRNA by Northern analysis of keratinocytes. Identification of this enzyme extends the known distribution of
R
-lipoxygenases to humans and presents an additional target for potential therapeutic interventions in psoriasis.
Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2
Authors: Kamijo Takehiko , Weber Jason D. , Zambetti Gerard , Zindy Frederique , Roussel Martine F. , Sherr Charles J.
Published: July 2002

The
INK4a-ARF
locus encodes two proteins, p16
INK4a
and p19
ARF
, that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively. Disruption of this locus by deletions or point mutations is a common event in human cancer, perhaps second only to the loss of p53. Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we determined that mouse p19
ARF
can interact directly with p53, as well as with the p53 regulator mdm2. ARF can bind p53-DNA complexes, and it depends upon functional p53 to transcriptionally induce mdm2 and the cyclin-dependent kinase inhibitor p21
Cip1
, and to arrest cell proliferation. Binding of p19
ARF
to p53 requires the ARF N-terminal domain (amino acids 1–62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19
ARF
in wild type or
ARF
-null mouse embryo fibroblasts increases the half-life of p53 from 15 to ≈75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra-physiologic levels after introduction into
ARF
-null NIH 3T3 cells or mouse embryo fibroblasts, the p53 protein is handicapped in inducing this checkpoint response. In this setting, reintroduction of p19
ARF
restores p53’s ability to induce p21
Cip1
and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53-dependent function through an additional mechanism.
Differential thymic selection outcomes stimulated by focal structural alteration in peptide/major histocompatibility complex ligands
Authors: Ghendler Yoseph , Teng Mai-kun , Liu Jin-huan , Witte Torsten , Liu Ju , Kim Ki Seok , Kern Petra , Chang Hsiu-Ching , Wang Jia-huai , Reinherz Ellis L.
Published: July 2002

The T lineage repertoire is shaped by T cell receptor (TCR)-dependent positive and negative thymic selection processes. Using TCR-transgenic (N15tg) β
2
-microglobulin-deficient (β
2
m
−/−
) RAG-2
−/−
H-2
b
mice specific for the VSV8 (RGYVYQGL) octapeptide bound to K
b
, we identified a single weak agonist peptide variant V4L (L4) inducing phenotypic and functional T cell maturation. The cognate VSV8 peptide, in contrast, triggers negative selection. The crystal structure of L4/K
b
was determined and refined to 2.1 Å for comparison with the VSV8/K
b
structure at similar resolution. Aside from changes on the p4 side chain of L4 and the resulting alteration of the exposed K
b
Lys-66 side chain, these two structures are essentially identical. Hence, a given TCR recognizes subtle distinctions between highly related ligands, resulting in dramatically different selection outcomes. Based on these finding and the recent structural elucidation of the N15-VSV8/K
b
complex, moreover, it appears that the germ-line Vα repertoire contributes in a significant way to positive selection.
Interaction of HIV-1 Nef with the cellular dileucine-based sorting pathway is required for CD4 down-regulation and optimal viral infectivity
Authors: Craig Heather M. , Pandori Mark W. , Guatelli John C.
Published: July 2002

The HIV-1 Nef protein is important for pathogenesis, enhances viral infectivity, and regulates the sorting of at least two cellular transmembrane proteins, CD4 and major histocompatibility complex (MHC) class I. Although several lines of evidence support the hypothesis that the Nef protein interacts directly with the cellular protein sorting machinery, the sorting signal in HIV-1 Nef has not been identified. By using a competition assay that functionally discriminates between dileucine-based and tyrosine-based sorting signals, we have categorized the motif through which Nef interacts with the sorting machinery as dileucine-based. Inspection of diverse Nef proteins from HIV-1, HIV-2, and simian immunodeficiency virus revealed a well-conserved sequence in the central region of the C-terminal, solvent-exposed loop of Nef (E/DXXXLφ) that conforms to the consensus sequence of the dileucine-based sorting motifs found in cellular transmembrane proteins. This sequence in Nef
NL4-3
, ENTSLL, functioned as an endocytosis signal when appended to the cytoplasmic tail of a heterologous protein. The leucine residues in this motif were required for the interaction of full-length Nef with the dileucine-based sorting pathway and were required for Nef-mediated down-regulation of CD4. These leucine residues were also required for optimal viral infectivity. These data indicate that a dileucine-based sorting signal in Nef is utilized to address the cellular sorting machinery. The data also suggest that an influence on the distribution of cellular transmembrane proteins may mechanistically unite two previously distinct properties of Nef: down-regulation of CD4 and enhancement of viral infectivity.
The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation
Authors: Huber Michael , Helgason Cheryl D. , Damen Jacqueline E. , Liu Ling , Humphries R. Keith , Krystal Gerald
Published: July 2002

To clarify the role that the src homology 2-containing inositol phosphatase (SHIP) plays in mast cell degranulation, the gene for SHIP was disrupted by homologous recombination in embryonic stem cells. Bone-marrow-derived mast cells from SHIP+/+, +/−, and −/− F2littermates were compared. SHIP−/− mast cells were found to be far more prone to degranulation, after the crosslinking of IgE preloaded cells, than SHIP+/− or +/+ cells. Intriguingly, IgE alone also stimulated massive degranulation in SHIP−/− but not in +/+ mast cells. This degranulation with IgE alone, which may be due to low levels of IgE aggregates, correlated with a higher and more sustained intracellular calcium level than that observed with SHIP+/+ cells and was dependent upon the entry of extracellular calcium. Immunoprecipitation studies revealed that the addition of IgE alone to normal mast cells stimulates multiple cascades, which are prevented from progressing to degranulation by SHIP. PI 3-kinase inhibitor studies suggested that IgE-induced activation of PI 3-kinase is upstream of the entry of extracellular calcium and that SHIP restricts this entry by hydrolyzing phosphatidylinositol 3,4,5-trisphosphate. These results show the critical role that SHIP plays in setting the threshold for degranulation and that SHIP directly modulates a “positive-acting” receptor.
Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor
Authors: Fry David W. , Bridges Alexander J. , Denny William A. , Doherty Annette , Greis Kenneth D. , Hicks James L. , Hook Kenneth E. , Keller Paul R. , Leopold Wilbur R. , Loo Joseph A. , McNamara Dennis J. , Nelson James M. , Sherwood Veronika , Smaill Jeff B. , Trumpp-Kallmeyer Susanne , Dobrusin Ellen M.
Published: July 2002

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and
14
C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior
in vivo
antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.
Low frequency vibrational modes in proteins: Changes induced by point-mutations in the protein-cofactor matrix of bacterial reaction centers
Authors: Rischel Christian , Spiedel Diane , Ridge Justin P. , Jones Michael R. , Breton Jacques , Lambry Jean-Christophe , Martin Jean-Louis , Vos Marten H.
Published: July 2002

As a step toward understanding their functional role, the low frequency vibrational motions (<300 cm
−1
) that are coupled to optical excitation of the primary donor bacteriochlorophyll cofactors in the reaction center from
Rhodobacter sphaeroides
were investigated. The pattern of hydrogen-bonding interaction between these bacteriochlorophylls and the surrounding protein was altered in several ways by mutation of single amino acids. The spectrum of low frequency vibrational modes identified by femtosecond coherence spectroscopy varied strongly between the different reaction center complexes, including between different mutants where the pattern of hydrogen bonds was the same. It is argued that these variations are primarily due to changes in the nature of the individual modes, rather than to changes in the charge distribution in the electronic states involved in the optical excitation. Pronounced effects of point mutations on the low frequency vibrational modes active in a protein-cofactor system have not been reported previously. The changes in frequency observed indicate a strong involvement of the protein in these nuclear motions and demonstrate that the protein matrix can increase or decrease the fluctuations of the cofactor along specific directions.
Novel role of phosphorylation in Fe–S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1
Authors: Brown Nina M. , Anderson Sheila A. , Steffen Daniel W. , Carpenter Tami B. , Kennedy M. Claire , Walden William E. , Eisenstein Richard S.
Published: July 2002

Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5′ or 3′ untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe–4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe–4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe–S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe–4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.
Evaluating multiple alternative hypotheses for the origin of Bilateria: An analysis of 18S rRNA molecular evidence
Authors: Collins Allen G.
Published: July 2002

Six alternative hypotheses for the phylogenetic origin of Bilateria are evaluated by using complete 18S rRNA gene sequences for 52 taxa. These data suggest that there is little support for three of these hypotheses. Bilateria is not likely to be the sister group of Radiata or Ctenophora, nor is it likely that Bilateria gave rise to Cnidaria or Ctenophora. Instead, these data reveal a close relationship between bilaterians, placozoans, and cnidarians. From this, several inferences can be drawn. Morphological features that previously have been identified as synapomorphies of Bilateria and Ctenophora, e.g., mesoderm, more likely evolved independently in each clade. The endomesodermal muscles of bilaterians may be homologous to the endodermal muscles of cnidarians, implying that the original bilaterian mesodermal muscles were myoepithelial. Placozoans should have a gastrulation stage during development. Of the three hypotheses that cannot be falsified with the 18S rRNA data, one is most strongly supported. This hypothesis states that Bilateria and Placozoa share a more recent common ancestor than either does to Cnidaria. If true, the simplicity of placozoan body architecture is secondarily derived from a more complex ancestor. This simplification may have occurred in association with a planula-type larva becoming reproductive before metamorphosis. If this simplification took place during the common history that placozoans share with bilaterians, then placozoan genes that contain a homeobox, such as
Trox2
, should be explored, for they may include the gene or genes most closely related to
Hox
genes of bilaterians.
Fatty acids suppress voltage-gated Na
+
currents in HEK293t cells transfected with the α-subunit of the human cardiac Na
+
channel
Authors: Xiao Yong-Fu , Wright Sterling N. , Wang Ging Kuo , Morgan James P. , Leaf Alexander
Published: July 2002

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na
+
current (I
Na,α
) in human embryonic kidney (HEK293t) cells transfected with the α-subunit of the human cardiac Na
+
channel (hH1
α
). Extracellular application of 0.01 to 30 μM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I
Na,α
with an IC
50
of 0.51 ± 0.06 μM. The EPA-induced suppression of I
Na,α
was concentration- and voltage-dependent. EPA at 5 μM significantly shifted the steady-state inactivation relationship by −27.8 ± 1.2 mV (
n
= 6,
P
< 0.0001) at the V
1/2
point. In addition, EPA blocked I
Na,α
with a higher “binding affinity” to hH1
α
channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 μM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 μM EPA, from 2.1 ± 0.8 ms for control to 34.8 ± 2.1 ms (
n
= 5,
P
< 0.001). The effects of EPA on I
Na,α
were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), α-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 μM and all-
trans
-retinoic acid at 10 μM had similar effects on I
Na,α
as EPA. Even 5 μM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited I
Na,α
. In contrast, 5 μM EPA ethyl ester did not alter I
Na,α
(8 ± 4%,
n
= 8,
P
> 0.05). The present data demonstrate that free fatty acids suppress I
Na,α
with high “binding affinity” to hH1
α
channels in the inactivated state and prolong the duration of recovery from inactivation.
Maternal care during infancy regulates the development of neural systems mediating the expression of fearfulness in the rat
Authors: Caldji Christian , Tannenbaum Beth , Sharma Shakti , Francis Darlene , Plotsky Paul M. , Meaney Michael J.
Published: July 2002

The mothers of infant rats show individual differences in the frequency of licking/grooming and arched-back nursing (LG-ABN) of pups that contribute to the development of individual differences in behavioral responses to stress. As adults, the offspring of mothers that exhibited high levels of LG-ABN showed substantially reduced behavioral fearfulness in response to novelty compared with the offspring of low LG-ABN mothers. In addition, the adult offspring of the high LG-ABN mothers showed significantly (
i
) increased central benzodiazepine receptor density in the central, lateral, and basolateral nuclei of the amygdala as well as in the locus ceruleus, (
ii
) increased α
2
adrenoreceptor density in the locus ceruleus, and (
iii
) decreased corticotropin-releasing hormone (CRH) receptor density in the locus ceruleus. The expression of fear and anxiety is regulated by a neural circuitry that includes the activation of ascending noradrenergic projections from the locus ceruleus to the forebrain structures. Considering the importance of the amygdala, notably the anxiogenic influence of CRH projections from the amygdala to the locus ceruleus, as well as the anxiolytic actions of benzodiazepines, for the expression of behavioral responses to stress, these findings suggest that maternal care during infancy serves to “program” behavioral responses to stress in the offspring by altering the development of the neural systems that mediate fearfulness.
Quantitative analysis of hepatitis C virus-specific CD8+T cells in peripheral blood and liver using peptide-MHC tetramers
Authors: He Xiao-Song , Rehermann Barbara , López-Labrador F. Xavier , Boisvert Judie , Cheung Ramsey , Mumm John , Wedemeyer Heiner , Berenguer Marina , Wright Teresa L. , Davis Mark M. , Greenberg Harry B.
Published: July 2002

It is believed that the hepatitis C virus (HCV)-specific CD8+cytotoxic T lymphocytes (CTLs) play a role in the development of liver cell injury and in the clearance of the virus. To develop a direct binding assay for HCV-specific CTLs, we generated two peptide-MHC tetramers by using the recombinant HLA A2.1 molecule and A2-restricted T cell epitopes of the HCV NS3 protein. With these reagents we are able to detect specific CD8+cells in the blood of 15 of 20 HLA-A2+, HCV-infected patients, at a frequency ranging from 0.01% to 1.2% of peripheral CD8+T cells. Phenotypic analysis of these specific cells indicated that there is a significant variation in the expression of the CD45 isoforms and CD27 in different patients. A 6-hour incubation of one patient’s blood with NS3 peptides resulted in the activation of the epitope-specific CD8+cells, as indicated by their expression of CD69 and IFN-γ. We also detected NS3-specific CD8+T cells in the intrahepatic lymphocyte population isolated from liver biopsies of two HCV-infected patients. The frequency of these specific CD8+cells in the liver was 1–2%, at least 30-fold higher than in the peripheral blood. All of the intrahepatic NS3-specific CD8+T cells were CD69+, suggesting that they were activated CTLs. Direct quantitation and characterization of HCV-specific CTLs should extend our understanding of the immunopathogenesis and the mechanism of clearance or persistence of HCV.
A tool for functional plant genomics: Chimeric RNA/DNA oligonucleotides cause
in vivo
gene-specific mutations
Authors: Beetham Peter R. , Kipp Peter B. , Sawycky Xenia L. , Arntzen Charles J. , May Gregory D.
Published: July 2002

Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (
i
) create stable, site-specific base substitutions in a nuclear gene and (
ii
) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene. An amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides. After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196. To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.
Crystal structure and mechanism of histone acetylation of the yeast GCN5 transcriptional coactivator
Authors: Trievel Raymond C. , Rojas Jeannie R. , Sterner David E. , Venkataramani Ravichandran N. , Wang Lian , Zhou Jianxin , Allis C. David , Berger Shelley L. , Marmorstein Ronen
Published: July 2002

The yeast GCN5 (yGCN5) transcriptional coactivator functions as a histone acetyltransferase (HAT) to promote transcriptional activation. Here, we present the high resolution crystal structure of the HAT domain of yGCN5 and probe the functional importance of a conserved glutamate residue. The structure reveals a central protein core associated with AcCoA binding that appears to be structurally conserved among a superfamily of
N
-acetyltransferases, including yeast histone acetyltransferase 1 and
Serratia marcescens
aminoglycoside 3-
N
-acetyltransferase. A pronounced cleft lying above this core, and flanked by N- and C-terminal regions that show no sequence conservation within
N
-acetyltransferase enzymes, is implicated by cross-species conservation and mutagenesis studies to be a site for histone substrate binding and catalysis. Located at the bottom of this cleft is a conserved glutamate residue (E173) that is in position to play an important catalytic role in histone acetylation. Functional analysis of an E173Q mutant yGCN5 protein implicates glutamate 173 to function as a general base for catalysis. Together, a correlation of the yGCN5 structure with functionally debilitating yGCN5 mutations provides a paradigm for understanding the structure/function relationships of the growing number of transcriptional regulators that function as histone acetyltransferase enzymes.
Phylogenetic relationships among cetartiodactyls based on insertions of short and long interpersed elements: Hippopotamuses are the closest extant relatives of whales
Authors: Nikaido Masato , Rooney Alejandro P. , Okada Norihiro
Published: July 2002

Insertion analysis of short and long interspersed elements is a powerful method for phylogenetic inference. In a previous study of short interspersed element data, it was found that cetaceans, hippopotamuses, and ruminants form a monophyletic group. To further resolve the relationships among these taxa, we now have isolated and characterized 10 additional loci. A phylogenetic analysis of these data was able to resolve relationships among the major cetartiodactyl groups, thereby shedding light on the origin of whales. The results indicated (i) that cetaceans are deeply nested within Artiodactyla, (ii) that cetaceans and hippopotamuses form a monophyletic group, (iii) that pigs and peccaries form a monophyletic group to the exclusion of hippopotamuses, (iv) that chevrotains diverged first among ruminants, and (v) that camels diverged first among cetartiodactyls. These findings lead us to conclude that cetaceans evolved from an immediate artiodactyl, not mesonychian, ancestor.
Tracking neuronal fiber pathways in the living human brain
Authors: Conturo Thomas E. , Lori Nicolas F. , Cull Thomas S. , Akbudak Erbil , Snyder Abraham Z. , Shimony Joshua S. , McKinstry Robert C. , Burton Harold , Raichle Marcus E.
Published: July 2002

Functional imaging with positron emission tomography and functional MRI has revolutionized studies of the human brain. Understanding the organization of brain systems, especially those used for cognition, remains limited, however, because no methods currently exist for noninvasive tracking of neuronal connections between functional regions [Crick, F. & Jones, E. (1993)
Nature (London)
361, 109–110]. Detailed connectivities have been studied in animals through invasive tracer techniques, but these invasive studies cannot be done in humans, and animal results cannot always be extrapolated to human systems. We have developed noninvasive neuronal fiber tracking for use in living humans, utilizing the unique ability of MRI to characterize water diffusion. We reconstructed fiber trajectories throughout the brain by tracking the direction of fastest diffusion (the fiber direction) from a grid of seed points, and then selected tracks that join anatomically or functionally (functional MRI) defined regions. We demonstrate diffusion tracking of fiber bundles in a variety of white matter classes with examples in the corpus callosum, geniculo-calcarine, and subcortical association pathways. Tracks covered long distances, navigated through divergences and tight curves, and manifested topological separations in the geniculo-calcarine tract consistent with tracer studies in animals and retinotopy studies in humans. Additionally, previously undescribed topologies were revealed in the other pathways. This approach enhances the power of modern imaging by enabling study of fiber connections among anatomically and functionally defined brain regions in individual human subjects.
Associative plasticity in striatal transplants
Authors: Brasted Peter J. , Watts Colin , Robbins Trevor W. , Dunnett Stephen B.
Published: July 2002

Striatal lesions disrupt both motor and cognitive performance in rats, many aspects of which can be restored by striatal transplants. Because the normal striatum is involved in the formation and maintenance of motor habits, it has been hypothesized that grafted animals may require explicit retraining to relearn previously established habits that have been disrupted by the lesions. We have used a lateralized-discrimination task to reproduce this “learning to use the transplant” effect, combined with a transfer-of-training paradigm to demonstrate that recovery requires relearning specific lateralized stimulus–response associations and cannot be explained simply by a generalized training-dependent improvement in motor skill. These results have clear implications for developing appropriate strategies for the rehabilitation of Huntington’s disease patients participating in clinical transplantation programs.
How the protease thrombin talks to cells
Authors: Coughlin Shaun R.
Published: July 2002

How does a protease act like a hormone to regulate cellular
functions? The coagulation protease thrombin (EC
3.4.21.5
) activates
platelets and regulates the behavior of other cells by means of G
protein-coupled protease-activated receptors (PARs). PAR1 is activated
when thrombin binds to and cleaves its amino-terminal exodomain to
unmask a new receptor amino terminus. This new amino terminus then
serves as a tethered peptide ligand, binding intramolecularly to the
body of the receptor to effect transmembrane signaling. The
irreversibility of PAR1’s proteolytic activation mechanism stands in
contrast to the reversible ligand binding that activates classical G
protein-coupled receptors and compels special mechanisms for
desensitization and resensitization. In endothelial cells and
fibroblasts, activated PAR1 rapidly internalizes and then sorts to
lysosomes rather than recycling to the plasma membrane as do classical
G protein-coupled receptors. This trafficking behavior is critical for
termination of thrombin signaling. An intracellular pool of thrombin
receptors refreshes the cell surface with naïve receptors,
thereby maintaining thrombin responsiveness. Thus cells have evolved a
trafficking solution to the signaling problem presented by PARs. Four
PARs have now been identified. PAR1, PAR3, and PAR4 can all be
activated by thrombin. PAR2 is activated by trypsin and by trypsin-like
proteases but not by thrombin. Recent studies with knockout mice,
receptor-activating peptides, and blocking antibodies are beginning to
define the role of these receptors
in vivo
.
CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose
Authors: Kurts Christian , Sutherland Robyn M. , Davey Gayle , Li Ming , Lew Andrew M. , Blanas Effrossini , Carbone Francis R. , Miller Jacques F. A. P. , Heath William R.
Published: July 2002

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8+T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8+T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8+T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8+T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8+T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8+T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.