bluuebunny/crossref_metadata_2025 huggingface dataset:
Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein
Authors: Tetzlaff Michael T. , Yu Wei , Li Mamie , Zhang Pumin , Finegold Milton , Mahon Kathleen , Harper J. Wade , Schwartz Robert J. , Elledge Stephen J.
Published: March 2004

The mammalian F-box protein Fbw7 and its
Caenorhabditis elegans
counterpart Sel-10 have been implicated in the ubiquitin-mediated turnover of cyclin E as well as the Notch/Lin-12 family of transcriptional activators. Both unregulated Notch and cyclin E promote tumorigenesis, and inactivating mutations in human
Fbw7
suggest that it may be a tumor suppressor. To generate an
in vivo
system to assess the consequences of such unregulated signaling, we generated mice deficient for
Fbw7. Fbw7
-null mice die around 10.5 days post coitus because of a combination of deficiencies in hematopoietic and vascular development and heart chamber maturation. The absence of Fbw7 results in elevated levels of cyclin E, concurrent with inappropriate DNA replication in placental giant trophoblast cells. Moreover, the levels of both Notch 1 and Notch 4 intracellular domains were elevated, leading to stimulation of downstream transcriptional pathways involving Hes1, Herp1, and Herp2. These data suggest essential functions for Fbw7 in controlling cyclin E and Notch signaling pathways in the mouse.
The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world
Authors: Egloff Marie-Pierre , Ferron François , Campanacci Valérie , Longhi Sonia , Rancurel Corinne , Dutartre Hélène , Snijder Eric J. , Gorbalenya Alexander E. , Cambillau Christian , Canard Bruno
Published: March 2004

The recently identified etiological agent of the severe acute respiratory syndrome (SARS) belongs to
Coronaviridae
(CoV), a family of viruses replicating by a poorly understood mechanism. Here, we report the crystal structure at 2.7-Å resolution of nsp9, a hitherto uncharacterized subunit of the SARS-CoV replicative polyproteins. We show that SARS-CoV nsp9 is a single-stranded RNA-binding protein displaying a previously unreported, oligosaccharide/oligonucleotide fold-like fold. The presence of this type of protein has not been detected in the replicative complexes of RNA viruses, and its presence may reflect the unique and complex CoV viral replication/transcription machinery.
Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase
Authors: Evans John C. , Huddler Donald P. , Hilgers Mark T. , Romanchuk Gail , Matthews Rowena G. , Ludwig Martha L.
Published: March 2004

B
12
-dependent methionine synthase (MetH) is a large modular enzyme that utilizes the cobalamin cofactor as a methyl donor or acceptor in three separate reactions. Each methyl transfer occurs at a different substrate-binding domain and requires a different arrangement of modules. In the catalytic cycle, the cobalamin-binding domain carries methylcobalamin to the homocysteine (Hcy) domain to form methionine and returns cob(I)alamin to the folate (Fol) domain for remethylation by methyltetrahydrofolate (CH
3
-H
4
folate). Here, we describe crystal structures of a fragment of MetH from
Thermotoga maritima
comprising the domains that bind Hcy and CH
3
-H
4
folate. These substrate-binding domains are (βα)
8
barrels packed tightly against one another with their barrel axes perpendicular. The properties of the domain interface suggest that the two barrels remain associated during catalysis. The Hcy and CH
3
-H
4
folate substrates are bound at the C termini of their respective barrels in orientations that position them for reaction with cobalamin, but the two active sites are separated by ≈50 Å. To complete the catalytic cycle, the cobalamin-binding domain must travel back and forth between these distant active sites.
Timing of brain-derived neurotrophic factor exposure affects life expectancy of new neurons
Authors: Alvarez-Borda Benjamín , Haripal Bhagwattie , Nottebohm Fernando
Published: March 2004

The high vocal center (HVC) of adult male canaries,
Serinus canaria
, is necessary for the production of learned song. New neurons are added to HVC every day, where they replace older neurons that have died, but the length of their survival depends on the time of year when they are born. A great number of HVC neurons born in the fall, when adult canaries learn a new song, are still present 8 mo later, when this song is used during the breeding season. By contrast, most of the neurons born in HVC in the spring, when little song learning takes place, disappear much sooner. Here we show that infusion of brain-derived neurotrophic factor into HVC during days 14-20 after new HVC neurons are born in the spring confers on them a life expectancy comparable to that of fall-born neurons; this extension on life is not seen when infusion occurs 10 days earlier or later. We suggest that there is, in the adult HVC, a subset of neurons whose life expectancy is determined by brain-derived neurotrophic factor during a sensitive period soon after these neurons reach destination and start forming connections.
Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling
Authors: Blondeau Francois , Ritter Brigitte , Allaire Patrick D. , Wasiak Sylwia , Girard Martine , Hussain Natasha K. , Angers Annie , Legendre-Guillemin Valerie , Roy Line , Boismenu Daniel , Kearney Robert E. , Bell Alexander W. , Bergeron John J. M. , McPherson Peter S.
Published: March 2004

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy.
Specific mutations of hepatitis B virus in plasma predict liver cancer development
Authors: Kuang Shuang-Yuan , Jackson Peta E. , Wang Jin-Bing , Lu Pei-Xing , Muñoz Alvaro , Qian Geng-Sun , Kensler Thomas W. , Groopman John D.
Published: March 2004

A major risk factor for hepatocellular carcinoma (HCC) is hepatitis B virus (HBV), whose pathogenesis is exacerbated by the acquisition of mutations that accelerate carcinogenesis. We examined, with mass spectrometry, the temporality of an HBV 1762T/1764Adouble mutation in plasma and tumors. Initial studies found that 52 of 70 (74.3%) tumors from patients residing in Qidong, People's Republic of China, contained this HBV mutation. Paired plasma samples were available for six of the tumor specimens; four tumors had the HBV 1762T/1764Amutation, whereas three of the paired plasma samples were also positive. The potential predictive value of this biomarker was explored by using stored plasma samples from a study of 120 residents of Qidong who had been monitored for aflatoxin exposure and HBV infection. After 10 years of passive follow-up, there were six cases of major liver disease including HCC (four cases), hepatitis (one case), and cirrhosis (one case). All six cases had detectable levels of the HBV 1762T/1764Amutation up to 8 years before diagnosis. Finally, 15 liver cancers were selected from a prospective cohort of 1,638 high-risk individuals in Qidong on the basis of available plasma samples spanning the years before and after diagnosis. The HBV 1762T/1764Amutation was detected in 8 of the 15 cases (53.3%) before cancer. The persistence of detection of this mutation was statistically significant (P= 0.022, two-tailed). We therefore found that a prediagnosis biomarker of specific HBV mutations can be measured in plasma and suggest this marker for use as an intermediate endpoint in prevention and intervention trials.
Local feature frequency profile: A method to measure structural similarity in proteins
Authors: Choi In-Geol , Kwon Jaimyoung , Kim Sung-Hou
Published: March 2004

Measures of structural similarity between known protein structures provide an objective basis for classifying protein folds and for revealing a global view of the protein structure universe. Here, we describe a rapid method to measure structural similarity based on the profiles of representative local features of Cαdistance matrices of compared protein structures. We first extract a finite number of representative local feature (LF) patterns from the distance matrices of all protein fold families by medoid analysis. Then, each Cαdistance matrix of a protein structure is encoded by labeling all its submatrices by the index of the nearest representative LF patterns. Finally, the structure is represented by the frequency distribution of these indices, which we call the LF frequency (LFF) profile of the protein. The LFF profile allows one to calculate structural similarity scores among a large number of protein structures quickly, and also to construct and update the “map” of the protein structure universe easily. The LFF profile method efficiently maps complex protein structures into a common Euclidean space without prior assignment of secondary structure information or structural alignment.
Transgene-driven expression of the Doppel protein in Purkinje cells causes Purkinje cell degeneration and motor impairment
Authors: Anderson Lucy , Rossi Daniela , Linehan Jackie , Brandner Sebastian , Weissmann Charles
Published: March 2004

The Doppel (Dpl) and Prion (PrP) proteins show 25% sequence identity and share several structural features with only minor differences. Dpl shows a PrP-like fold of its C-terminal globular domain and lacks the flexible N-terminal tail. The physiological functions of both proteins are unknown. However, ubiquitous Dpl overexpression in the brain of PrP knockout mice correlated with ataxia and Purkinje cell degeneration in the cerebellum. Interestingly, a similar phenotype was reported in transgenic mice expressing an N-terminally truncated PrP (ΔPrP) in Purkinje cells by the L7 promoter (TgL7-ΔPrP). Coexpression of full-length PrP rescued both the neurological syndromes caused by either Dpl or ΔPrP. To evaluate whether the two proteins caused cerebellar neurodegeneration by the same mechanism, we generated transgenic mice selectively expressing Dpl in Purkinje cells by the same L7 promoter. Such mice showed ataxia and Purkinje cell loss that depended on the level of Dpl expression. Interestingly, the effects of high levels of Dpl were not counterbalanced by the presence of two
Prnp
alleles. By contrast, PrP coexpression was sufficient to abrogate motor impairment and to delay the neurodegenerative process caused by moderate level of Dpl. A similar situation was reported for the corresponding TgL7-ΔPrP mice supporting the concept that Dpl and ΔPrP cause cell death, possibly by interfering with a common signaling cascade essential for cell survival.
Proteomic analysis of thioredoxin-targeted proteins in
Escherichia coli
Authors: Kumar Jaya K. , Tabor Stanley , Richardson Charles C.
Published: March 2004

Thioredoxin, a ubiquitous and evolutionarily conserved protein, modulates the structure and activity of proteins involved in a spectrum of processes, such as gene expression, apoptosis, and the oxidative stress response. Here, we present a comprehensive analysis of the thioredoxin-linked
Escherichia coli
proteome by using tandem affinity purification and nanospray microcapillary tandem mass spectrometry. We have identified a total of 80 proteins associated with thioredoxin, implicating the involvement of thioredoxin in at least 26 distinct cellular processes that include transcription regulation, cell division, energy transduction, and several biosynthetic pathways. We also found a number of proteins associated with thioredoxin that either participate directly (SodA, HPI, and AhpC) or have key regulatory functions (Fur and AcnB) in the detoxification of the cell. Transcription factors NusG, OmpR, and RcsB, not considered to be under redox control, are also associated with thioredoxin.
Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR
Authors: Klein-Seetharaman Judith , Yanamala Naveena V. K. , Javeed Fathima , Reeves Philip J. , Getmanova Elena V. , Loewen Michele C. , Schwalbe Harald , Khorana H. Gobind
Published: March 2004

G protein-coupled receptors are cell-surface seven-helical membrane proteins that undergo conformational changes on activation. The mammalian photoreceptor, rhodopsin, is the best-studied member of this superfamily. Here, we provide the first evidence that activation in rhodopsin may involve differential dynamic properties of side-chain versus backbone atoms. High-resolution NMR studies of α-15N-labeled receptor revealed large backbone motions in the inactive dark state. In contrast, indole side-chain15N groups of tryptophans showed well resolved, equally intense NMR signals, suggesting restriction to a single specific conformation.
The E47 transcription factor negatively regulates CD5 expression during thymocyte development
Authors: Yang Yang , Contag Christopher H. , Felsher Dean , Shachaf Catherine M. , Cao Yuan , Herzenberg Leonard A. , Herzenberg Leonore A. , Tung James W.
Published: March 2004

The expression of CD5 increases progressively as thymocytes mature. We have shown that CD5 expression is controlled by a tissue-specific regulatory promoter located upstream of the CD5 translation start sites. Deletion of this regulatory promoter, which contains three potential transcription factor binding sites (CCAAT, κE2, and ets) reduces the promoter activity to basal level. Of these sites, only ets proved essential for CD5 expression in T cell lines. Here, we introduce a role for the E47 transcription factor and the CD5 promoter κE2 site in regulating CD5 expression during thymocyte development. Using T cell lines, we show that (i) mutation of the κE2 site in the CD5 regulatory promoter results in a significant elevation of CD5 promoter activity; (ii) the E47 transcription factor binds to the κE2 site; and (iii) overexpression of E47 inhibits CD5 expression. We then show, in high-dimensional fluorescence-activated cell sorting studies with primary thymocytes at successive developmental stages, that (i) intracellular E47 levels decrease as surface CD5 expression increases; (ii) E47 expression is down-regulated and CD5 expression is correspondingly up-regulated in DN3 thymocytes in RAG-2-deficient mice injected with anti-CD3 to mimic pre-T cell receptor stimulation; and (iii) E47 expression is down-regulated and CD5 expression is up-regulated when double positive thymocytes are stimulatedin vitrowith anti-CD3. Based on these data, we propose that E47 negatively regulates CD5 expression by interacting with the κE2 site in the CD5 regulatory promoter and that decreases in E47 in response to developmental signals are critical to the progressive increase in CD5 expression as thymocytes mature.
The architecture of complex weighted networks
Authors: Barrat A. , Barthélemy M. , Pastor-Satorras R. , Vespignani A.
Published: March 2004

Networked structures arise in a wide array of different contexts such as technological and transportation infrastructures, social phenomena, and biological systems. These highly interconnected systems have recently been the focus of a great deal of attention that has uncovered and characterized their topological complexity. Along with a complex topological structure, real networks display a large heterogeneity in the capacity and intensity of the connections. These features, however, have mainly not been considered in past studies where links are usually represented as binary states, i.e., either present or absent. Here, we study the scientific collaboration network and the world-wide air-transportation network, which are representative examples of social and large infrastructure systems, respectively. In both cases it is possible to assign to each edge of the graph a weight proportional to the intensity or capacity of the connections among the various elements of the network. We define appropriate metrics combining weighted and topological observables that enable us to characterize the complex statistical properties and heterogeneity of the actual strength of edges and vertices. This information allows us to investigate the correlations among weighted quantities and the underlying topological structure of the network. These results provide a better description of the hierarchies and organizational principles at the basis of the architecture of weighted networks.
Scanning electron microscopy of cells and tissues under fully hydrated conditions
Authors: Thiberge Stephan , Nechushtan Amotz , Sprinzak David , Gileadi Opher , Behar Vered , Zik Ory , Chowers Yehuda , Michaeli Shulamit , Schlessinger Joseph , Moses Elisha
Published: March 2004

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is ≈100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers.
The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists
Authors: Kalani M. Yashar S. , Vaidehi Nagarajan , Hall Spencer E. , Trabanino Rene J. , Freddolino Peter L. , Kalani Maziyar A. , Floriano Wely B. , Kam Victor Wai Tak , Goddard William A.
Published: March 2004

Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using first-principles theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.
T cell immunity using transgenic B lymphocytes
Authors: Gerloni Mara , Rizzi Marta , Castiglioni Paola , Zanetti Maurizio
Published: March 2004

Adaptive immunity exists in all vertebrates and plays a defense role against microbial pathogens and tumors. T cell responses begin when precursor T cells recognize antigen on specialized antigen-presenting cells and differentiate into effector cells. Currently, dendritic cells are considered the only cells capable of stimulating T lymphocytes. Here, we show that mature naïve B lymphocytes can be genetically programmed by using nonviral DNA and turned into powerful antigen-presenting cells with a dual capacity of synthesis and presentation of antigen to T cellsin vivo. A single i.v. injection of transgenic lymphocytes activates T cell responses reproducibly and specifically even at very low cell doses (≈102). We also demonstrate that T cell priming can occur in the absence of dendritic cells and results in immunological memory with protective effector functions. These findings disclose aspects in the regulation of adaptive immunity and indicate possibilities for vaccination against viruses and cancer in humans.
Activation of the antioxidant enzyme 1-CYS peroxiredoxin requires glutathionylation mediated by heterodimerization with πGST
Authors: Manevich Y. , Feinstein S. I. , Fisher A. B.
Published: March 2004

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin superfamily, can protect cells against membrane oxidation through glutathione (GSH)-dependent reduction of phospholipid hydroperoxides to corresponding alcohols. However, purified native or recombinant enzyme
in vitro
generally lacks GSH peroxidase (GPx) activity because of oxidation of its single conserved cysteine. Reduction of the resultant oxidized cysteine is difficult because of its protected location within the homodimer formed by the oxidized protein monomers. Partial purification of 1-cysPrx from bovine lung revealed the presence of πGST in an active preparation, while purification to homogeneity yielded enzyme that inactivated with time. We show that heterodimerization of 1-cysPrx with GSH-saturated πGST results in glutathionylation of the oxidized cysteine in 1-cysPrx followed by subsequent spontaneous reduction of the mixed disulfide and restoration of enzymatic activity. Maximum activation of 1-cysPrx occurred with a 1:1 molar ratio of GSH-saturated πGST and a 2:1 molar ratio of GSH to 1-cysPrx. Liposome-mediated delivery of oxidized recombinant enzyme into NCI-H441 cells that lack 1-cysPrx but express πGST resulted in 1-cysPrx activation, whereas activation in MCF7 cells required co-delivery of πGST. Our data indicate a physiological mechanism for glutathionylation of the oxidized catalytic cysteine of 1-cysPrx by its heterodimerization with πGST followed by its GSH-mediated reduction and enzyme activation.
A variable dinucleotide repeat in the
CFTR
gene contributes to phenotype diversity by forming RNA secondary structures that alter splicing
Authors: Hefferon Timothy W. , Groman Joshua D. , Yurk Catherine E. , Cutting Garry R.
Published: March 2004

Dinucleotide repeats are ubiquitous features of eukaryotic genomes that are not generally considered to have functional roles in gene expression. However, the highly variable nature of dinucleotide repeats makes them particularly interesting candidates for modifiers of RNA splicing when they are found near splicing signals. An example of a variable dinucleotide repeat that affects splicing is a TG repeat located in the splice acceptor of exon 9 of the cystic fibrosis transmembrane conductance regulator (
CFTR
) gene. Higher repeat numbers result in reduced exon 9 splicing efficiency and, in some instances, the reduction in full-length transcript is sufficient to cause male infertility due to congenital bilateral absence of the vas deferens or nonclassic cystic fibrosis. Using a
CFTR
minigene system, we studied TG tract variation and observed the same correlation between dinucleotide repeat number and exon 9 splicing efficiency seen
in vivo
. Replacement of the TG dinucleotide tract in the minigene with random sequence abolished splicing of exon 9. Replacements of the TG tract with sequences that can self-base-pair suggested that the formation of an RNA secondary structure was associated with efficient splicing. However, splicing efficiency was inversely correlated with the predicted thermodynamic stability of such structures, demonstrating that intermediate stability was optimal. Finally, substitution with TA repeats of differing length confirmed that stability of the RNA secondary structure, not sequence content, correlated with splicing efficiency. Taken together, these data indicate that dinucleotide repeats can form secondary structures that have variable effects on RNA splicing efficiency and clinical phenotype.
Stress-induced structural remodeling in hippocampus: Prevention by lithium treatment
Authors: Wood Gwendolyn E. , Young L. Trevor , Reagan Lawrence P. , Chen Biao , McEwen Bruce S.
Published: March 2004

Chronic restraint stress, psychosocial stress, as well as systemic or oral administration of the stress-hormone corticosterone induces a morphological reorganization in the rat hippocampus, in which adrenal steroids and excitatory amino acids mediate a reversible remodeling of apical dendrites on CA3 pyramidal cell neurons of the hippocampus. This stress-induced neuronal remodeling is accompanied also by behavioral changes, some of which can be prevented with selective antidepressant and anticonvulsive drug treatments. Lithium is an effective treatment for mood disorders and has neuroprotective effects, which may contribute to its therapeutic properties. Thus, we wanted to determine whether lithium treatment could prevent the effects of chronic stress on CA3 pyramidal cell neuroarchitecture and the associated molecular and behavioral measures. Chronic lithium treatment prevented the stress-induced decrease in dendritic length, as well as the stress-induced increase in glial glutamate transporter 1 (GLT-1) mRNA expression and the phosphorylation of cAMP-response element binding in the hippocampus. Lithium treatment, however, did not prevent stress effects on behavior in the open field or the plus-maze. These data demonstrate that chronic treatment with lithium can protect the hippocampus from potentially deleterious effects of chronic stress on glutamatergic activation, which may be relevant to its therapeutic efficacy in the treatment of major depressive disorder and bipolar disorder.
The molecular structure and catalytic mechanism of a novel carboxyl peptidase from
Scytalidium lignicolum
Authors: Fujinaga Masao , Cherney Maia M. , Oyama Hiroshi , Oda Kohei , James Michael N. G.
Published: March 2004

The molecular structure of the pepstatin-insensitive carboxyl peptidase from
Scytalidium lignicolum
, formerly known as scytalidopepsin B, was solved by multiple isomorphous replacement phasing methods and refined to an
R
factor of 0.230 (
R
free
= 0.246) at 2.1-Å resolution. In addition to the structure of the unbound peptidase, the structure of a product complex of cleaved angiotensin II bound in the active site of the enzyme was also determined. We propose the name scytalidocarboxyl peptidase B (SCP-B) for this enzyme. On the basis of conserved, catalytic residues identified at the active site, we suggest the name Eqolisin for the enzyme family. The previously uninvestigated SCP-B fold is that of a β-sandwich; each sheet has seven antiparallel strands. A tripeptide product, Ala-Ile-His, bound in the active site of SCP-B has allowed for identification of the catalytic residues and the residues in subsites S1, S2, and S3, which are important for substrate binding. The most likely hydrolytic mechanism involves nucleophilic attack of a general base (Glu-136)-activated water (OH
-
) on the
si
-face of the scissile peptide carbonylcarbon atom to form a tetrahedral intermediate. Electrophilic assistance and oxyanion stabilization is provided by the side-chain amide of Gln-53. Protonation of the leaving-group nitrogen is accomplished by the general acid function of the protonated carboxyl group of Glu-136.
Acute myeloid leukemia with complex karyotypes and abnormal chromosome 21: Amplification discloses overexpression of
APP
,
ETS2
, and
ERG
genes
Authors: Baldus Claudia D. , Liyanarachchi Sandya , Mrózek Krzysztof , Auer Herbert , Tanner Stephan M. , Guimond Martin , Ruppert Amy S. , Mohamed Nehad , Davuluri Ramana V. , Caligiuri Michael A. , Bloomfield Clara D. , de la Chapelle Albert
Published: March 2004

Molecular mechanisms of leukemogenesis have been successfully unraveled by studying genes involved in simple rearrangements including balanced translocations and inversions. In contrast, little is known about genes altered in complex karyotypic abnormalities. We studied acute myeloid leukemia (AML) patients with complex karyotypes and abnormal chromosome 21. High-resolution bacterial artificial chromosome (BAC) array-based comparative genomic hybridization disclosed amplification predominantly in the 25- to 30-megabase (MB) region that harbors the
APP
gene (26.3 MB) and at position 38.7-39.1 MB that harbors the transcription factors
ERG
and
ETS2
. Using oligonucleotide arrays,
APP
was by far the most overexpressed gene (mean fold change 19.74,
P
= 0.0003) compared to a control group of AML with normal cytogenetics;
ERG
and
ETS2
also ranked among the most highly expressed chromosome 21 genes. Overexpression of
APP
and
ETS2
correlated with genomic amplification, but high
APP
expression occurred even in a subset of AML patients with normal cytogenetics (10 of 64, 16%).
APP
encodes a glycoprotein of unknown function previously implicated in Alzheimer's disease, but not in AML. We hypothesize that
APP
and the transcription factors
ERG
and
ETS2
are altered by yet unknown molecular mechanisms involved in leukemogenesis. Our results highlight the value of molecularly dissecting leukemic cells with complex karyotypes.
β2-Glycoprotein I binds factor XI and inhibits its activation by thrombin and factor XIIa: Loss of inhibition by clipped β2-glycoprotein I
Authors: Shi Tong , Iverson G. Michael , Qi Jian C. , Cockerill Keith A. , Linnik Matthew D. , Konecny Pamela , Krilis Steven A.
Published: March 2004

Activation of factor XI (FXI) by thrombinin vivoplays a role in coagulation by providing an important positive feedback mechanism for additional thrombin generation. FXI is activatedin vitroby thrombin, or FXIIa in the presence of dextran sulfate. In this report, we investigated the effect of β2-glycoprotein I (β2GPI) on the activation of FXI. β2GPI bound FXIin vitroand inhibited its activation to FXIa by thrombin and FXIIa. The affinity of the interaction between β2GPI and FXI was equivalent to the interaction between FXI and high molecular weight kininogen. Inhibition of FXI activation occurred with lower concentrations of β2GPI than found in human plasma. Proteolytic clipping of β2GPI by plasmin abolished its inhibition of FXI activation. The results suggest a mechanism of regulation whereby physiological concentrations of β2GPI may attenuate thrombin generationin vivoby inhibition of FXI activation. Plasmin cleavage of β2GPI provides a negative feedback that counteracts its inhibition of FXI activation.
Cholesterol depletion induces PKA-mediated basolateral-to-apical transcytosis of the scavenger receptor class B type I in MDCK cells
Authors: Burgos Patricia V. , Klattenhoff Carla , de la Fuente Erwin , Rigotti Attilio , González Alfonso
Published: March 2004

Cholesterol-based membrane microdomains, or lipid rafts, are believed to play important, yet poorly defined, roles in protein trafficking and signal transduction. In polarized epithelial cells, the current view is that rafts are involved in apical but not in basolateral protein transport from the trans-Golgi network (TGN). We report here that cholesterol is required in a post-TGN mechanism of basolateral regionalization. Permanently transfected Madin-Darby canine kidney cells segregated the caveolae/raft-associated high-density lipoprotein scavenger receptor class B type I (SR-BI) predominantly to the basolateral domain where it was constitutively internalized and recycled basolaterally. Acute cholesterol depletion did not significantly alter SR-BI internalization, implying a cholesterol depletion-insensitive endocytic process but instead induced its transcytosis through a protein kinase A (PKA)- and microtubule-dependent mechanism. Forskolin also elicited SR-BI transcytosis. The basolateral distribution of endogenous epidermal growth factor receptor remained unaffected. Strikingly, cholesterol depletion induced PKA activity without increasing the cAMP levels. Thus, our results are consistent with a scenario in which cholesterol-based rafts promote internalization and basolateral recycling of internalized SR-BI whereas a PKA pool sensitive to cholesterol depletion mediates SR-BI transcytosis. Regulated transcytosis of SR-BI may provide an additional mechanism to control cholesterol homeostasis. These results disclose relationships between cholesterol-based rafts and PKA activity operating in a post-TGN mechanism of regulated apical-to-basolateral cell surface protein distribution.
Interleukin 2 plays a central role in Th2 differentiation
Authors: Cote-Sierra Javier , Foucras Gilles , Guo Liying , Chiodetti Lynda , Young Howard A. , Hu-Li Jane , Zhu Jinfang , Paul William E.
Published: March 2004

Differentiation of naïve CD4 T cells into T helper (Th) 2 cells requires signaling through the T cell receptor and an appropriate cytokine environment. IL-4 is critical for such Th2 differentiation. We show that IL-2 plays a central role in this process. The effect of IL-2 on Th2 generation does not depend on its cell growth or survival effects. Stat5a-/-cells show diminished differentiation to IL-4 production, and forced expression of a constitutively active form of Stat5a replaces the need for IL-2.In vivoIL-2 neutralization inhibits IL-4 production in two models. Studies of restriction enzyme accessibility and binding of Stat5 to chromatin indicate that IL-2 mediates its effect by stabilizing the accessibility of theIl4gene. Thus, IL-2 plays a critical role in the polarization of naive CD4 T cells to the Th2 phenotype.
Identification of a specific self-reactive IgM antibody that initiates intestinal ischemia/reperfusion injury
Authors: Zhang Ming , Austen William G. , Chiu Isaac , Alicot Elisabeth M. , Hung Rachel , Ma Minghe , Verna Nicola , Xu Min , Hechtman Herbert B. , Moore Francis D. , Carroll Michael C.
Published: March 2004

Reperfusion injury of ischemic tissue represents an acute inflammatory response that can cause significant morbidity and mortality. The mechanism of injury is not fully elucidated, but recent studies indicate an important role for natural antibody and the classical pathway of complement. To test the hypothesis that injury is initiated by specific IgM, we have screened a panel of IgM-producing hybridomas prepared from peritoneal cells enriched in B-1 cells. One clone, CM22, was identified that could restore pathogenic injury in RAG-1-/-mice in an intestinal model of ischemia/reperfusion (I/R).In situactivation of the classical pathway of complement was evident by deposition of IgM, complement C4, and C3 in damaged tissue after passive transfer of CM22 IgM. Sequence analysis of CM22 Ig heavy and light chains showed germ-line configurations with high homology to a VHsequence from the B-1 repertoire and a VKof a known polyreactive natural IgM. These data provide definitive evidence that I/R injury can be initiated by clonally specific natural IgM that activates the classical pathway of complement. This finding opens an avenue for identification of I/R-specific self-antigen(s) and early prevention of injury.
Multiple origins of crassulacean acid metabolism and the epiphytic habit in the Neotropical family Bromeliaceae
Authors: Crayn Darren M. , Winter Klaus , Smith J. Andrew C.
Published: March 2004

The large Neotropical family Bromeliaceae presents an outstanding example of adaptive radiation in plants, containing a wide range of terrestrial and epiphytic life-forms occupying many distinct habitats. Diversification in bromeliads has been linked to several key innovations, including water- and nutrient-impounding phytotelmata, absorptive epidermal trichomes, and the water-conserving mode of photosynthesis known as crassulacean acid metabolism (CAM). To clarify the origins of CAM and the epiphytic habit, we conducted a phylogenetic analysis of nucleotide sequences for 51 bromeliad taxa by using the plastid loci
matK
and the
rps16
intron, combined with a survey of photosynthetic pathway determined by carbon-isotope ratios for 1,873 species representing 65% of the family. Optimization of character-states onto the strict consensus tree indicated that the last common ancestor of Bromeliaceae was a terrestrial C
3
mesophyte, probably adapted to moist, exposed, nutrient-poor habitats. Both CAM photosynthesis and the epiphytic habit evolved a minimum of three times in the family, most likely in response to geological and climatic changes in the late Tertiary. The great majority of epiphytic forms are now found in two lineages: in subfamily Tillandsioideae, in which C
3
photosynthesis was the ancestral state and CAM developed later in the most extreme epiphytes, and in subfamily Bromelioideae, in which CAM photosynthesis predated the appearance of epiphytism. Subsequent radiation of the bromelioid line into less xeric habitats has led to reversion to C
3
photosynthesis in some taxa, showing that both gain and loss of CAM have occurred in the complex evolutionary history of this family.
Systemic delivery of human microdystrophin to regenerating mouse dystrophic muscle by muscle progenitor cells
Authors: Bachrach Estanislao , Li Sheng , Perez Antonio L. , Schienda Jaclyn , Liadaki Kalliopi , Volinski Jay , Flint Alan , Chamberlain Jeffrey , Kunkel Louis M.
Published: March 2004

Cell-based therapy for Duchenne muscular dystrophy patients and
mdx
mice has proven to be a safe but ineffective form of treatment. Recently, a group of cells called muscle side population (SP) cells have been isolated based on their ability to efflux the DNA-binding dye Hoechst. To understand the potential of skeletal muscle SP cells to serve as precursors for muscle, SP cells from the two mice strains
mdx

5cv

and C57BL/6N were isolated, transduced, and transplanted. Under coculture conditions with myogenic cells, some cells within the SP cell population can give rise to early Pax7-positive satellite cells and other later stage myogenic cells. Transduced SP cells were transplanted via the tail vein and were shown to successfully deliver enhanced GFP and human microdystrophin to the skeletal muscle of nonirradiated
mdx

5cv

mice, thus demonstrating their ability to travel through the capillaries and enter into damaged muscle. These results demonstrate that i.v. delivery of genes via SP cells is possible and that these SP cells are capable of recapitulating the myogenic lineage. Because this approach shows definitive engraftment by using autologous transplantation of noninjured recipients, our data may have substantial implications for therapy of muscular dystrophy.
Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect
Authors: Reddy Pavan , Maeda Yoshinobu , Hotary Kevin , Liu Chen , Reznikov Leonid L. , Dinarello Charles A. , Ferrara James L. M.
Published: March 2004

Acute graft-versus-host disease (GVHD) and leukemic relapse are the two major obstacles to successful outcomes after allogeneic bone marrow transplantation (BMT), an effective therapy for hematological malignancies. Several studies have demonstrated that the dysregulation of proinflammatory cytokines and the loss of gastrointestinal tract integrity contribute to GVHD, whereas the donor cytotoxic responses are critical for graft-versus-leukemia (GVL) preservation. Suberoylanilide hydroxamic acid (SAHA) is currently in clinical trials as an antitumor agent; it inhibits the activity of histone deacetylases and at low doses exhibits antiinflammatory effects by reducing the production of proinflammatory cytokines. Using two well characterized mouse models of BMT, we have studied the effects of SAHA on GVHD severity and GVL activity. Administration of SAHA from day +3 to day +7 after BMT reduced serum levels of the proinflammatory cytokines and decreased intestinal histopathology, clinical severity, and mortality from acute GVHD compared with vehicle-treated animals. However, SAHA had no effect on donor T cell proliferative and cytotoxic responses to host antigens
in vivo
or
in vitro
. When mice received lethal doses of tumor cells at the time of BMT, administration of SAHA did not impair GVL activity and resulted in significantly improved leukemia-free survival by using two different tumor and donor/recipient combinations. These findings reveal a critical role for histone deacetylase inhibition in the proinflammatory events contributing to GVHD and suggest that this class of pharmacologic agents may provide a strategy to reduce GVHD while preserving cytotoxic T cell responses to host antigens and maintaining beneficial GVL effects.
Biosynthesis
in vitro
of
Caenorhabditis elegans
phosphorylcholine oligosaccharides
Authors: Cipollo John F. , Awad Antoine , Costello Catherine E. , Robbins Phillips W. , Hirschberg Carlos B.
Published: March 2004

The biosynthesis
in vitro
of phosphorylcholine oligosaccharides in
Caenorhabditis elegans
has been investigated. Here we show that extracts of
C. elegans
' microsomes transfer phosphorylcholine from L-α-dipalmitoyl phosphatidylcholine to hybrid and complex type N-linked oligosaccharides containing mannose residues disubstituted with
N
-acetylglucosamine. The reaction products are consistent with structures reported for
C. elegans
as well those found in the filarial nematodes
Acanthocheilonema viteae
,
Onchocerca volvulus
, and
Brugia malayi
, strongly supporting the concept that the phosphorylcholine oligosaccharide biosynthetic enzymes are conserved in this group of organisms. Because it is thought that phosphorylcholine substitution of oligosaccharides modulates host immune response in filarial infections, this
in vitro
system may help in gaining an understanding of the basis for this response.
The long-term stability of new hippocampal place fields requires new protein synthesis
Authors: Agnihotri Naveen T. , Hawkins Robert D. , Kandel Eric R. , Kentros Clifford
Published: March 2004

The hippocampus is critical for formation of spatial memories. Hippocampal pyramidal neurons in freely behaving animals exhibit spatially selective firing patterns, which taken together form an internal representation of the environment. This representation is thought to contribute to the hippocampal spatial memory system. Behavioral long-term memories differ from short-term memories in requiring the synthesis of new proteins. Does the development of the internal hippocampal representation also require the synthesis of new proteins? We found that blocking protein synthesis in the brain of mice by 95% does not affect short-term stability of newly formed hippocampal place fields but abolishes stability in the long term. By contrast, inhibiting protein synthesis does not affect the retention and recall of previously established fields in a familiar environment, indicating that protein synthesis-dependent reconsolidation is not required for recall. Our results indicate that place fields parallel both behavioral memories and the late phase of long-term potentiation in requiring the synthesis of new proteins for consolidation.
Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation
Authors: Komeili Arash , Vali Hojatollah , Beveridge Terrance J. , Newman Dianne K.
Published: March 2004

Bacterial magnetosomes are intracellular compartments that house highly ordered magnetite crystals. By using
Magnetospirillum
sp. AMB-1 as a model system, we show that magnetosome vesicles exist in the absence of magnetite, biomineralization of magnetite proceeds simultaneously in multiple vesicles, and biomineralization proceeds from the same location in each vesicle. The magnetosome-associated protein, MamA, is required for the formation of functional magnetosome vesicles and displays a dynamic subcellular localization throughout the growth cycle of magnetotactic bacteria. Together, these results suggest that the magnetosome precisely coordinates magnetite biomineralization and can serve as a model system for the study of organelle biogenesis in noneukaryotic cells.
Chicxulub impact predates the K-T boundary mass extinction
Authors: Keller Gerta , Adatte Thierry , Stinnesbeck Wolfgang , Rebolledo-Vieyra Mario , Urrutia Fucugauchi Jaime , Kramar Utz , Stüben Doris
Published: March 2004

Since the early l990s the Chicxulub crater on Yucatan, Mexico, has been hailed as the smoking gun that proves the hypothesis that an asteroid killed the dinosaurs and caused the mass extinction of many other organisms at the Cretaceous-Tertiary (K-T) boundary 65 million years ago. Here, we report evidence from a previously uninvestigated core, Yaxcopoil-1, drilled within the Chicxulub crater, indicating that this impact predated the K-T boundary by ≈300,000 years and thus did not cause the end-Cretaceous mass extinction as commonly believed. The evidence supporting a pre-K-T age was obtained from Yaxcopoil-1 based on five independent proxies, each with characteristic signals across the K-T transition: sedimentology, biostratigraphy, magnetostratigraphy, stable isotopes, and iridium. These data are consistent with earlier evidence for a late Maastrichtian age of the microtektite deposits in northeastern Mexico.
Persistent colonization and the spread of antibiotic resistance in nosocomial pathogens: Resistance is a regional problem
Authors: Smith David L. , Dushoff Jonathan , Perencevich Eli N. , Harris Anthony D. , Levin Simon A.
Published: March 2004

Infections with antibiotic-resistant bacteria (ARB) in hospitalized patients are becoming increasingly frequent despite extensive infection-control efforts. Infections with ARB are most common in the intensive care units of tertiary-care hospitals, but the underlying cause of the increases may be a steady increase in the number of asymptomatic carriers entering hospitals. Carriers may shed ARB for years but remain undetected, transmitting ARB to others as they move among hospitals, long-term care facilities, and the community. We apply structured population models to explore the dynamics of ARB, addressing the following questions: (
i
) What is the relationship between the proportion of carriers admitted to a hospital, transmission, and the risk of infection with ARB? (
ii
) How do frequently hospitalized patients contribute to epidemics of ARB? (
iii
) How do transmission in the community, long-term care facilities, and hospitals interact to determine the proportion of the population that is carrying ARB? We offer an explanation for why ARB epidemics have fast and slow phases and why resistance may continue to increase despite infection-control efforts. To successfully manage ARB at tertiary-care hospitals, regional coordination of infection control may be necessary, including tracking asymptomatic carriers through health-care systems.