bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease
Authors: Cutler Roy G. , Kelly Jeremiah , Storie Kristin , Pedersen Ward A. , Tammara Anita , Hatanpaa Kimmo , Troncoso Juan C. , Mattson Mark P.
Published: February 2004

Alzheimer's disease (AD) is an age-related disorder characterized by deposition of amyloid β-peptide (Aβ) and degeneration of neurons in brain regions such as the hippocampus, resulting in progressive cognitive dysfunction. The pathogenesis of AD is tightly linked to Aβ deposition and oxidative stress, but it remains unclear as to how these factors result in neuronal dysfunction and death. We report alterations in sphingolipid and cholesterol metabolism during normal brain aging and in the brains of AD patients that result in accumulation of long-chain ceramides and cholesterol. Membrane-associated oxidative stress occurs in association with the lipid alterations, and exposure of hippocampal neurons to Aβ induces membrane oxidative stress and the accumulation of ceramide species and cholesterol. Treatment of neurons with α-tocopherol or an inhibitor of sphingomyelin synthesis prevents accumulation of ceramides and cholesterol and protects them against death induced by Aβ. Our findings suggest a sequence of events in the pathogenesis of AD in which Aβ induces membrane-associated oxidative stress, resulting in perturbed ceramide and cholesterol metabolism which, in turn, triggers a neurodegenerative cascade that leads to clinical disease.
Chemical-genetic inhibition of a sensitized mutant myosin Vb demonstrates a role in peripheral-pericentriolar membrane traffic
Authors: Provance D. William , Gourley Christopher R. , Silan Colleen M. , Cameron L. C. , Shokat Kevan M. , Goldenring James R. , Shah Kavita , Gillespie Peter G. , Mercer John A.
Published: February 2004

Selective,
in situ
inhibition of individual unconventional myosins is a powerful approach to determine their specific physiological functions. Here, we report the engineering of a myosin Vb mutant that still hydrolyzes ATP, yet is selectively sensitized to an
N
6
-substituted ADP analog that inhibits its activity, causing it to remain tightly bound to actin. Inhibition of the sensitized mutant causes inhibition of accumulation of transferrin in the cytoplasm and increases levels of plasma-membrane transferrin receptor, suggesting that myosin Vb functions in traffic between peripheral and pericentrosomal compartments.
Inducible, reversible, and stable RNA interference in mammalian cells
Authors: Gupta Sunita , Schoer Rebecca A. , Egan James E. , Hannon Gregory J. , Mittal Vivek
Published: February 2004

RNA interference is a powerful genetic approach for efficiently silencing target genes. The existing method of gene suppression by the constitutive expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes but limits the analysis of genes essential for cell survival, cell cycle regulation, and cell development. We have developed an inducible U6 promoter for synthesis of shRNAs in both human and murine cells. Cells containing stably integrated shRNA expression constructs demonstrate stringent dosage- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer ecdysone. Inducible suppression of human p53 in glioblastoma cells shows striking morphological changes and defects in cell cycle arrest caused by DNA damage, as expected. Remarkably, the inducibility is reversible after withdrawal of the inducer, as observed by reappearance of the protein and a restoration of the original cell phenotype. Inducible and reversible regulation of RNA interference has broad applications in the areas of mammalian genetics and molecular therapeutics.
Computational design of water-soluble analogues of the potassium channel KcsA
Authors: Slovic Avram M. , Kono Hidetoshi , Lear James D. , Saven Jeffery G. , DeGrado William F.
Published: February 2004

Although the interiors of membrane and water-soluble proteins are similar in their physicochemical properties, membrane proteins differ in having larger fractions of hydrophobic residues on their exteriors. Thus, it should be possible to water-solubilize membrane proteins by mutating their lipid-contacting side chains to more polar groups. Here, a computational approach was used to generate water-soluble variants of the potassium channel KcsA. As a probe of the correctness of the fold, the proteins contain an agitoxin2 binding site from a mammalian homologue of the channel. The resulting proteins express in high yield in
Escherichia coli
and share the intended functional and structural properties with KcsA, including secondary structure, tetrameric quaternary structure, and tight specific binding to both agitoxin2 and a small molecule channel blocker.
A capsid protein of nonenveloped Bluetongue virus exhibits membrane fusion activity
Authors: Forzan Mario , Wirblich Christoph , Roy Polly
Published: February 2004

The outer capsid layer of Bluetongue virus, a member of the nonenveloped
Reoviridae
family, is composed of two proteins, a receptor-binding protein, VP2, and a second protein, VP5, which shares structural features with class I fusion proteins of enveloped viruses. In the replication cycle of Bluetongue virus VP5 acts as a membrane permeabilization protein that mediates release of viral particles from endosomal compartments into the cytoplasm. Here, we show that VP5 can also act as a fusion protein and induce syncytium formation when it is fused to a transmembrane anchor and expressed on the cell surface. Fusion activity is strictly pH-dependent and is triggered by short exposure to low pH. No cell-cell fusion is observed at neutral pH. Deletion of the first 40 amino acids, which can fold into two amphipathic helices, abolishes fusion activity. Syncytium formation by VP5 is inhibited in the presence of VP2 when it is expressed in a membrane-anchored form. The data indicate an interaction between the outer capsid protein VP2 and VP5 and show that VP5 undergoes pH-dependent conformational changes that render it capable of interacting with cellular membranes. More importantly, our data show that a membrane permeabilization protein of a nonenveloped virus can evolve into a fusion protein by the addition of an appropriate transmembrane anchor. The results strongly suggest that the mechanism of membrane permeabilization by VP5 and membrane fusion by viral fusion proteins require similar structural features and conformational changes.
DDB2 gene disruption leads to skin tumors and resistance to apoptosis after exposure to ultraviolet light but not a chemical carcinogen
Authors: Itoh Toshiki , Cado Dragana , Kamide Ryoichi , Linn Stuart
Published: February 2004

Mutations in the human DDB2 gene give rise to xeroderma pigmentosum group E, a disease characterized by increased skin tumorigenesis in response to UV-irradiation. Cell strains derived from xeroderma pigmentosum group E individuals also have enhanced resistance to UV-irradiation due to decreased p53-mediated apoptosis. To further address the precise function(s) of DDB2 and the consequence of non-naturally occurring DDB2 mutations, we generated mice with a disruption of the gene. The mice exhibited significantly enhanced skin carcinogenesis in response to UV-irradiation, and cells from the DDB2
–/–
mice were abnormally
resistant
to killing by the radiation and had diminished UV-induced, p53-mediated apoptosis. Notably, the cancer-prone phenotype and the resistance to cellular killing were not observed after exposure to the chemical carcinogen, 7,12-dimethylbenz[
a
]anthracene (DMBA), to which mice carrying defective nucleotide excision repair genes respond with enhanced tumors and cell killing. Although cells from heterozygous DDB2
+/–
mice appeared normal, these mice had enhanced skin carcinogenesis after UV-irradiation, so that XP-E heterozygotes might be at risk for carcinogenesis. In sum, these results demonstrate that DDB2 is well conserved between humans and mice and functions as a tumor suppressor, at least in part, by controlling p53-mediated apoptosis after UV-irradiation.
Mice with cardiomyocyte-specific disruption of the endothelin-1 gene are resistant to hyperthyroid cardiac hypertrophy
Authors: Shohet Ralph V. , Kisanuki Yaz Y. , Zhao Xiao-Song , Siddiquee Zakir , Franco Fatima , Yanagisawa Masashi
Published: February 2004

Endothelin 1 (ET-1), a potent vasoconstrictor peptide expressed by endothelium, is also produced in the heart in response to a variety of stresses. It induces hypertrophy in cultured cardiac myocytes but only at concentrations far greater than those found in plasma. We tested whether ET-1 generated by cardiac myocytes
in vivo
is a local signal for cardiac hypertrophy. To avoid the perinatal lethality seen in systemic
ET-1
-null mice, we used the Cre/loxP system to generate mice with cardiac myocyte-specific disruption of the
ET-1
gene. We used the α-myosin heavy chain promoter to drive expression of Cre and were able to obtain 75% reduction in
ET-1
mRNA in cardiac myocytes isolated from these mice at baseline and after stimulation,
in vivo
, for 24 h with tri-iodothyronine (T3). Necropsy measurements of cardiac mass indexed for body weight showed a 57% reduction in cardiac hypertrophy in response to 16 days of exogenous T3 in mice homozygous for the disrupted
ET-1
allele compared to siblings with an intact
ET-1
gene. Moreover,
in vivo
MRI showed only a 3% increase in left ventricular mass indexed for body weight in mice with the disrupted allele after 3 weeks of T3 treatment versus a 27% increase in mice with an intact
ET-1
gene. A reduced hypertrophic response was confirmed by planimetry of cardiac myocytes. We conclude that ET-1, produced locally by cardiac myocytes, and acting in a paracrine/autocrine manner, is an important signal for myocardial hypertrophy that facilitates the response to thyroid hormone.
The elements of draping
Authors: Cerda E. , Mahadevan L. , Pasini J. M.
Published: February 2004

We consider the gravity-induced draping of a 3D object with a naturally flat, isotropic elastic sheet. As the size of the sheet increases, we observe the appearance of new folded structures of increasing complexity that arise because of the competition between elasticity and gravity. We analyze some of the simpler 3D structures by determining their shape and analyzing their response and stability and show that these structures can easily switch between a number of metastable configurations. For more complex draperies, we derive scaling laws for the appearance and disappearance of new length scales. Our results are consistent with commonplace observations of drapes and complement large-scale computations of draping by providing benchmarks. They also yield a qualitative guide to fashion design and virtual reality animation.
IL-15 enhances the
in vivo
antitumor activity of tumor-reactive CD8
+
T Cells
Authors: Klebanoff Christopher A. , Finkelstein Steven E. , Surman Deborah R. , Lichtman Michael K. , Gattinoni Luca , Theoret Marc R. , Grewal Navrose , Spiess Paul J. , Antony Paul A. , Palmer Douglas C. , Tagaya Yutaka , Rosenberg Steven A. , Waldmann Thomas A. , Restifo Nicholas P.
Published: February 2004

IL-15 and IL-2 possess similar properties, including the ability to induce T cell proliferation. However, whereas IL-2 can promote apoptosis and limit CD8
+
memory T cell survival and proliferation, IL-15 helps maintain a memory CD8
+
T cell population and can inhibit apoptosis. We sought to determine whether IL-15 could enhance the
in vivo
function of tumor/self-reactive CD8
+
T cells by using a T cell receptor transgenic mouse (pmel-1) whose CD8
+
T cells recognize an epitope derived from the self/melanoma antigen gp100. By removing endogenous IL-15 by using tumor-bearing IL-15 knockout hosts or supplementing IL-15 by means of exogenous administration, as a component of culture media or as a transgene expressed by adoptively transferred T cells, we demonstrate that IL-15 can improve the
in vivo
antitumor activity of adoptively transferred CD8
+
T cells. These results provide several avenues for improving adoptive immunotherapy of cancer in patients.
Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers
Authors: Wakabayashi Nobunao , Dinkova-Kostova Albena T. , Holtzclaw W. David , Kang Moon-Il , Kobayashi Akira , Yamamoto Masayuki , Kensler Thomas W. , Talalay Paul
Published: February 2004

Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The critical role of C273 and C288 was established by (
i
) their high reactivity when purified recombinant Keap1 was treated with dexamethasone mesylate and the dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and (
ii
) transfection of
keap1
and
nrf2
gene-deficient mouse embryonic fibroblasts with constructs expressing cysteine to alanine mutants of Keap1, and measurement of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter. Reaction of Keap1 with inducers results in formation of intermolecular disulfide bridges, probably between C273 of one Keap1 molecule and C288 of a second. Evidence for formation of such dimers was obtained by 2D PAGE of extracts of cells treated with inducers, and by the demonstration that whereas C273A and C288A mutants of Keap1 alone could not repress Nrf2 activation of the ARE-luciferase reporter, an equal mixture of these mutant constructs restored repressor activity.
The
4-coumarate:CoA ligase
gene family in
Arabidopsis thaliana
comprises one rare, sinapate-activating and three commonly occurring isoenzymes
Authors: Hamberger Björn , Hahlbrock Klaus
Published: February 2004

4-Coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role in the biosynthesis of plant secondary compounds at the divergence point from general phenylpropanoid metabolism to several major branch pathways. In
Arabidopsis thaliana
, we have identified a previously undetected, fourth and final member of the
At4CL
gene family. The encoded enzyme,
At
4CL4, exhibits the rare property of efficiently activating sinapate, besides the usual 4CL substrates (4-coumarate, caffeate, and ferulate), indicating a distinct metabolic function. Phylogenetic analysis suggests an early evolutionary and functional divergence of three of the four gene family members,
At4CL2-4
, whereas
At4CL1
appears to have originated much later by duplication of its structurally and functionally closest relative,
At4CL2
. Various characteristics shared by all known plant
4CL
genes, as well as by the encoded proteins, define and delimit the
At4CL
gene family and distinguish it from the closely related family of “
At4CL-like
” genes.
The opportunistic pathogen
Pseudomonas aeruginosa
carries a secretable arachidonate 15-lipoxygenase
Authors: Vance Russell E. , Hong Song , Gronert Karsten , Serhan Charles N. , Mekalanos John J.
Published: February 2004

In mammals, lipoxygenases play key roles in inflammation by initiating the transformation of arachidonic acid into potent bioactive lipid mediators such as leukotrienes and lipoxins. In general, most bacteria are believed to lack lipoxygenases and their polyunsaturated fatty acid substrates. It is therefore of interest that an ORF (PA1169) with high homology to eukaryotic lipoxygenases was discovered by analysis of the whole-genome sequence of the opportunistic bacterial pathogen
Pseudomonas aeruginosa
. Using TLC and liquid chromatography-UV-tandem mass spectrometry (LC-UV-MS-MS), we demonstrate that PA1169 encodes a bacterial lipoxygenase (LoxA) that converts arachidonic acid into 15-hydroxyeicosatetraenoic acid (15-HETE). Although mammalian lipoxygenases are cytoplasmic enzymes,
P. aeruginosa
LoxA activity is secreted. Taken together, these results suggest a mechanism by which a pathogen-secreted lipoxygenase may modulate host defense and inflammation via alteration of the biosynthesis of local chemical mediators.
Estrogen alters hippocampal dendritic spine shape and enhances synaptic protein immunoreactivity and spatial memory in female mice
Authors: Li Chenjian , Brake Wayne G. , Romeo Russell D. , Dunlop John C. , Gordon Marisa , Buzescu Rodica , Magarinos Ana Maria , Allen Patrick B. , Greengard Paul , Luine Victoria , McEwen Bruce S.
Published: February 2004

Estrogen (E) treatment induces axospinous synapses in rat hippocampus
in vivo
and in cultured hippocampal neurons
in vitro
. To better explore the molecular mechanisms underlying this phenomenon, we have established a mouse model for E action in the hippocampus by using Golgi impregnation to examine hippocampal dendritic spine morphology, radioimmunocytochemistry (RICC) and silver-enhanced immunocytochemistry to examine expression levels of synaptic protein markers, and hippocampal-dependent object-placement memory as a behavioral readout for the actions of E. In ovariectomized mice of several strains and F
1
hybrids, the total dendritic spine density on neurons in the CA1 region was not enhanced by E treatment, a finding that differs from that in the female rat. E treatment of ovariectomized C57BL/6J mice, however, caused an increase in the number of spines with mushroom shapes. By RICC and silver-enhanced immunocytochemistry, we found that the immunoreactivity of postsynaptic markers (PSD95 and spinophilin) and a presynaptic marker (syntaxin) were enhanced by E treatment throughout all fields of the dorsal hippocampus. In the object-placement tests, E treatment enhanced performance of object placement, a spatial episodic memory task. Taken together, the morphology and RICC results suggest a previously uncharacterized role of E in synaptic structural plasticity that may be interpreted as a facilitation of the spine-maturation process and may be associated with enhancement of hippocampal-dependent memory.
Identification of a human NF-κB-activating protein, TAB3
Authors: Jin Ge , Klika Alison , Callahan Michelle , Faga Ben , Danzig Joel , Jiang Zhengfan , Li Xiaoxia , Stark George R. , Harrington John , Sherf Bruce
Published: February 2004

The NF-κB pathway plays a critical role in regulating cellular processes such as immune responses, stress responses, apoptosis, proliferation and differentiation, whereas dysfunction of this pathway has been associated with numerous cancer and immune disorders. We have applied our Random Activation of Gene Expression technology to an NF-κB reporter cell line to facilitate the discovery of positive regulators of NF-κB activation. A small protein expression library, corresponding to ≈0.1× genome coverage, was generated and screened for clones exhibiting constitutive activation of NF-κB. After isolation of cellular clones displaying the relevant phenotypes, we identified two known components of the NF-κB pathway and a hypothetical gene that we have designated the human ortholog of
Xenopus
TAK1-binding protein 3 (TAB3). Overexpression of human TAB3 was found to activate both NF-κB and AP-1 transcription factors. Furthermore, the activation of NF-κB by TAB3 was blocked by the NF-κB inhibitor, SN50, and by expression of dominant-negative forms of tumor necrosis factor α-associated factor 6 and transforming growth factor β-activated kinase. Taken together, these data demonstrate that TAB3 transforming growth factor is a constituent of the NF-κB pathway functioning upstream of tumor necrosis factor α-associated factor 6/transforming growth factor β-activated kinase. Interestingly, increased expression of TAB3 was found in some cancer tissues, and its overexpression in NIH 3T3 cells resulted in cellular transformation, thus establishing a causative link between elevated TAB3 expression, constitutive NF-κB activation, and oncogenesis.
Aberrant expansion of segmented filamentous bacteria in IgA-deficient gut
Authors: Suzuki Keiichiro , Meek Bob , Doi Yasuko , Muramatsu Masamichi , Chiba Tsutomu , Honjo Tasuku , Fagarasan Sidonia
Published: February 2004

The mechanism to maintain homeostasis of the gut microbiota remains largely unknown despite its critical role in the body defense. In the intestines of mice with deficiency of activation-induced cytidine deaminase (AID), the absence of hypermutated IgA is partially compensated for by the presence of large amounts of unmutated IgM and normal expression levels of defensins and angiogenins. We show here a predominant and persistent expansion of segmented filamentous bacteria throughout the small intestine of AID–/–mice. Reconstitution of lamina propria IgA production in AID–/–mice recovered the normal composition of gut flora and abolished the local and systemic activation of the immune system. The results indicate that secretions of IgAs rather than innate defense peptides are critical to regulation of commensal bacterial flora and that the segmented filamentous bacteria antigens are strong stimuli of the mucosal immune system.
The limits of protection by “memory” T cells in Ig
–/–
mice persistently infected with a γ-herpesvirus
Authors: Andreansky Samita , Liu Haiyan , Adler Heiko , Koszinowski Ulrich H. , Efstathiou Stacey , Doherty Peter C.
Published: February 2004

Can CD4
+
and CD8
+
“memory” T cells that are generated and maintained in the context of low-level virus persistence protect, in the absence of antibody, against a repeat challenge with the same pathogen? Although immune T cells exert effective, long-term control of a persistent γ-herpesvirus (γHV68) in Ig
–/–
μMT mice, subsequent exposure to a high dose of the same virus leads to further low-level replication in the lung. This lytic phase in the respiratory tract is dealt with effectively by the recall of memory T cells induced by a γHV68 recombinant (M3LacZ) that does not express the viral M3 chemokine binding protein. At least for the CD8
+
response, greater numbers of memory T cells confer enhanced protection in the M3LacZ-immune mice. However, neither WT γHV68 nor the minimally persistent M3LacZ primes the T cell response to the extent that a WT γHV68 challenge fails to establish latency in the μMT mice. Memory CD4
+
and CD8
+
T cells thus act together to limit γHV68 infection but are unable to provide absolute protection against a high-dose, homologous challenge.
Molecular cloning and characterization of two
Helicobacter pylori
genes coding for plasminogen-binding proteins
Authors: Jönsson Klas , Guo Betty P. , Monstein Hans-Jürg , Mekalanos John J. , Kronvall Göran
Published: February 2004

Helicobacter pylori
binds a number of host cell proteins, including the plasma protein plasminogen, which is the proenzyme of the serine protease plasmin. Two
H. pylori
plasminogen-binding proteins have been described; however, no genes were identified. Here we report the use of a phage display library to clone two genes from the
H. pylori
CCUG 17874 genome that mediate binding to plasminogen. DNA sequence analysis of one of these genes revealed 96.6% homology with
H. pylori
26695 HP0508. A subsequent database search revealed that the amino acid sequence of a lysine-rich C-terminal segment of HP0508 is identical to the C terminus of HP0863. Recombinant proteins expressed from HP0508 and HP0863 bound plasminogen specifically and in a lysine-dependent manner. We designate these genes
pgbA
and
pgbB
, respectively. These proteins are expressed by a variety of
H. pylori
strains, have surface-exposed domains, and do not inhibit plasminogen activation. These results indicate that
pgbA
and
pgbB
may allow
H. pylori
to coat its exterior with plasminogen, which subsequently can be activated to plasmin. The surface acquisition of protease activity may enhance the virulence of
H. pylori
.
Activation-induced cytidine deaminase shuttles between nucleus and cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide 1
Authors: Ito Satomi , Nagaoka Hitoshi , Shinkura Reiko , Begum Nasim , Muramatsu Masamichi , Nakata Mikiyo , Honjo Tasuku
Published: February 2004

Activation-induced cytidine deaminase (AID) is a molecule central to initiating class switch recombination, somatic hypermutation, and gene conversion of Ig genes. However, its mechanism to initiate these genetic alterations is still unclear. AID can convert cytosine to uracil on either mRNA or DNA and is involved in DNA cleavage. Although these events are expected to take place in the nucleus, overexpressed AID was found predominantly in the cytoplasm. Here, we demonstrated that AID is a nucleocytoplasmic shuttling protein with a bipartite nuclear localization signal and a nuclear export signal in its N and C termini, respectively. In addition to previously identified genetic, structural, and biochemical similarities of AID with apolipoprotein B mRNA editing catalytic polypeptide 1, an RNA editing enzyme of ApoB100 mRNA, the present finding provides another aspect to their resemblance, suggesting that both may have homologous reaction mechanisms.
DNA-dependent PK inhibits adeno-associated virus DNA integration
Authors: Song Sihong , Lu Yuanqing , Choi Young-Kook , Han Yinong , Tang Qiushi , Zhao Ge , Berns Kenneth I. , Flotte Terence R.
Published: February 2004

Recent studies have shown that recombinant adeno-associated virus (rAAV) can persist in episomal form; however, factors affecting rAAV persistence are poorly understood. DNA-dependent PK (DNA-PK) is a DNA repair enzyme, which we previously found played an important role in determining the molecular fate of the rAAV genome in mouse skeletal muscle. In the present study, we tested the effect of DNA-PK on AAV serotype 2 integration
in vitro
and
in vivo
in mouse liver. In an
in vitro
integration system, addition of DNA-PK decreased AAV integration, whereas antibody against DNA-PKcs increased integration.
In vivo
, matched doses of a recombinant AAV serotype 2 vector were injected into the portal vein of either C57BL/6 (DNA-PKcs
+/+
) or severe combined immunodeficient (DNA-PKcs
-/-
) mice. After partial hepatectomy to stimulate hepatocyte proliferation, retention of vector genomes and of transgene expression was substantially higher in severe combined immunodeficient mice, indicating that in the absence of DNA-PKcs, a greater proportion of genomes integrated into the cellular genome. In summary, we have provided evidence that DNA-PK inhibits AAV integration both
in vitro
and
in vivo
.
Molecular modulation of calcium oxalate crystallization by osteopontin and citrate
Authors: Qiu S. R. , Wierzbicki A. , Orme C. A. , Cody A. M. , Hoyer J. R. , Nancollas G. H. , Zepeda S. , De Yoreo J. J.
Published: February 2004

Calcium oxalate monohydrate (COM), which plays a functional role in plant physiology, is a source of chronic human disease, forming the major inorganic component of kidney stones. Understanding molecular mechanisms of biological control over COM crystallization is central to development of effective stone disease therapies and can help define general strategies for synthesizing biologically inspired materials. To date, research on COM modification by proteins and small molecules has not resolved the molecular-scale control mechanisms. Moreover, because proteins directing COM inhibition have been identified and sequenced, they provide a basis for general physiochemical investigations of biomineralization. Here, we report molecular-scale views of COM modulation by two urinary constituents, the protein osteopontin and citrate, a common therapeutic agent. Combining force microscopy with molecular modeling, we show that each controls growth habit and kinetics by pinning step motion on different faces through specific interactions in which both size and structure determine the effectiveness. Moreover, the results suggest potential for additive effects of simultaneous action by both modifiers to inhibit the overall growth of the crystal and demonstrate the utility of combining molecular imaging and modeling tools for understanding events underlying aberrant crystallization in disease.
Whole-genome shotgun assembly and comparison of human genome assemblies
Authors: Istrail Sorin , Sutton Granger G. , Florea Liliana , Halpern Aaron L. , Mobarry Clark M. , Lippert Ross , Walenz Brian , Shatkay Hagit , Dew Ian , Miller Jason R. , Flanigan Michael J. , Edwards Nathan J. , Bolanos Randall , Fasulo Daniel , Halldorsson Bjarni V. , Hannenhalli Sridhar , Turner Russell , Yooseph Shibu , Lu Fu , Nusskern Deborah R. , Shue Bixiong Chris , Zheng Xiangqun Holly , Zhong Fei , Delcher Arthur L. , Huson Daniel H. , Kravitz Saul A. , Mouchard Laurent , Reinert Knut , Remington Karin A. , Clark Andrew G. , Waterman Michael S. , Eichler Evan E. , Adams Mark D. , Hunkapiller Michael W. , Myers Eugene W. , Venter J. Craig
Published: February 2004

We report a whole-genome shotgun assembly (called WGSA) of the human genome generated at Celera in 2001. The Celera-generated shotgun data set consisted of 27 million sequencing reads organized in pairs by virtue of end-sequencing 2-kbp, 10-kbp, and 50-kbp inserts from shotgun clone libraries. The quality-trimmed reads covered the genome 5.3 times, and the inserts from which pairs of reads were obtained covered the genome 39 times. With the nearly complete human DNA sequence [National Center for Biotechnology Information (NCBI) Build 34] now available, it is possible to directly assess the quality, accuracy, and completeness of WGSA and of the first reconstructions of the human genome reported in two landmark papers in February 2001 [Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A.,et al.(2001)Science291, 1304–1351; International Human Genome Sequencing Consortium (2001)Nature409, 860–921]. The analysis of WGSA shows 97% order and orientation agreement with NCBI Build 34, where most of the 3% of sequence out of order is due to scaffold placement problems as opposed to assembly errors within the scaffolds themselves. In addition, WGSA fills some of the remaining gaps in NCBI Build 34. The early genome sequences all covered about the same amount of the genome, but they did so in different ways. The Celera results provide more order and orientation, and the consortium sequence provides better coverage of exact and nearly exact repeats.
A germ-line insertion in the Birt–Hogg–Dubé (
BHD
) gene gives rise to the Nihon rat model of inherited renal cancer
Authors: Okimoto Kazuo , Sakurai Junko , Kobayashi Toshiyuki , Mitani Hiroaki , Hirayama Youko , Nickerson Michael L. , Warren Michelle B. , Zbar Berton , Schmidt Laura S. , Hino Okio
Published: February 2004

A rat model of hereditary renal carcinoma (RC) was found in a rat colony of the Sprague–Dawley strain in Japan and named the “Nihon” rat. In heterozygotes, RCs, predominantly the clear cell type, develop from early preneoplastic lesions, which began to appear as early as 3 weeks of age, to adenocarcinomas by the age of 6 months. The Nihon rat is an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker (
Tsc2
gene mutant) rat. We have previously shown that the Nihon mutation was tightly linked to genes that are located on the distal part of rat chromosome 10. The order of the genes is the Eker (
Tsc2
gene (human 16p13.3)–
Il3
gene–Nihon gene–
Llgl1
locus–
Myhse
gene. We now describe a germ-line mutation in the Birt–Hogg–Dubé gene (
Bhd
) (human 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat, resulting in a frameshift and producing a stop codon 26 aa downstream. We found that the homozygous mutant condition was lethal at an early stage of fetal life in the rat. We detected a high frequency of loss of heterozygosity (LOH) in primary RCs (10/11) at the
Bhd
locus and found a point mutation (nonsense) in one LOH-negative case, fitting Knudson's “two-hit” model. The Nihon rat may therefore provide insights into a tumor-suppressor gene that is related to renal carcinogenesis and an animal model of human BHD syndrome.
Darwin's abominable mystery: Insights from a supertree of the angiosperms
Authors: Davies T. Jonathan , Barraclough Timothy G. , Chase Mark W. , Soltis Pamela S. , Soltis Douglas E. , Savolainen Vincent
Published: February 2004

Angiosperms are among the major terrestrial radiations of life and a model group for studying patterns and processes of diversification. As a tool for future comparative studies, we compiled a supertree of angiosperm families from published phylogenetic studies. Sequence data from the plastid
rbcL
gene were used to estimate relative timing of branching events, calibrated by using robust fossil dates. The frequency of shifts in diversification rate is largely constant among time windows but with an apparent increase in diversification rates within the more recent time frames. Analyses of species numbers among families revealed that diversification rate is a labile attribute of lineages at all levels of the tree. An examination of the top 10 major shifts in diversification rates indicates they cannot easily be attributed to the action of a few key innovations but instead are consistent with a more complex process of diversification, reflecting the interactive effects of biological traits and the environment.
Seasonal behavior in
Drosophila melanogaster
requires the photoreceptors, the circadian clock, and phospholipase C
Authors: Collins B. H. , Rosato E. , Kyriacou C. P.
Published: February 2004

Drosophila melanogaster
locomotor activity responds to different seasonal conditions by thermosensitive regulation of splicing of a 3′ intron in the
period
mRNA transcript. Here we demonstrate that the control of locomotor patterns by this mechanism is primarily light-dependent at low temperatures. At warmer temperatures, when it is vitally important for the fly to avoid midday desiccation, more stringent regulation of splicing is observed, requiring the light input received through the visual system during the day and the circadian clock at night. During the course of this study, we observed that a mutation in the

no-receptor-potential-A
P41

(

norpA
P41

) gene, which encodes phospholipase-C, generated an extremely high level of 3′ splicing. This cannot be explained simply by the mutation's effect on the visual pathway and suggests that

norpA
P41

is directly involved in thermosensitivity.
Rapid transformation of white adipocytes into fat-oxidizing machines
Authors: Orci Lelio , Cook William S. , Ravazzola Mariella , Wang May-yun , Park Byung-Hyun , Montesano Roberto , Unger Roger H.
Published: February 2004

Adenovirus-induced hyperleptinemia rapidly depletes body fat in normal rats without increasing free fatty acids and ketogenesis, implying that fat-storing adipocytes are oxidizing the fat. To analyze the ultrastructural changes of adipocytes accompanying this functional transformation, we examined the fat tissue by electron microscopy. After 14 days of hyperleptinemia, adipocytes had become shrunken, fatless, and encased in a thick basement-membrane-like matrix. They were crowded with mitochondria that were much smaller than those of brown adipocytes. Their gene expression profile revealed striking up-regulation of peroxisome proliferator-activated receptor γ coactivator 1α (an up-regulator of mitochondrial biogenesis not normally expressed in white fat), increased uncoupling proteins-1 and -2, and down-regulation of lipogenic enzymes. Phosphorylation of both acetyl CoA carboxylase and AMP-activated protein kinase was increased, thus explaining the increase in fatty acid oxidation. The ability to transform adipocytes into unique fat-burning cells may suggest novel therapeutic strategies for obesity.
Detection of multistability, bifurcations, and hysteresis in a large class of biological positive-feedback systems
Authors: Angeli David , Ferrell James E. , Sontag Eduardo D.
Published: February 2004

It is becoming increasingly clear that bistability (or, more generally, multistability) is an important recurring theme in cell signaling. Bistability may be of particular relevance to biological systems that switch between discrete states, generate oscillatory responses, or “remember” transitory stimuli. Standard mathematical methods allow the detection of bistability in some very simple feedback systems (systems with one or two proteins or genes that either activate each other or inhibit each other), but realistic depictions of signal transduction networks are invariably much more complex. Here, we show that for a class of feedback systems of arbitrary order the stability properties of the system can be deduced mathematically from how the system behaves when feedback is blocked. Provided that this open-loop, feedback-blocked system is monotone and possesses a sigmoidal characteristic, the system is guaranteed to be bistable for some range of feedback strengths. We present a simple graphical method for deducing the stability behavior and bifurcation diagrams for such systems and illustrate the method with two examples taken from recent experimental studies of bistable systems: a two-variable Cdc2/Wee1 system and a more complicated five-variable mitogen-activated protein kinase cascade.
The role of Shc and insulin-like growth factor 1 receptor in mediating the translocation of estrogen receptor α to the plasma membrane
Authors: Song Robert X. , Barnes Christopher J. , Zhang Zhenguo , Bao Yongde , Kumar Rakesh , Santen Richard J.
Published: February 2004

Our previous studies demonstrated that 17β-estradiol (E2) rapidly induces the interaction of estrogen receptor α (ERα) with the adapter protein Shc, the translocation of ERα to the cell membrane, and the formation of dynamic membrane structures in MCF-7 breast cancer cells. The present study examined how E2 causes ERα to translocate to the region of the plasma membrane and focused on mechanisms whereby Shc and the insulin-like growth factor-1 receptor (IGF-1R) mediate this process. Shc physically interacts with IGF-1R in the plasma membrane, and E2 activates IGF-1R. We reasoned that ERα, when bound to Shc, would be directed to the region of the plasma membrane by the same processes, causing membrane translocation of Shc. We confirmed that E2 rapidly induced IGF-1R phosphorylation and demonstrated that E2 induced formation of a ternary protein complex among Shc, ERα, and IGF-1R. Knock down of Shc with a specific small inhibitory RNA decreased the association of ERα with IGF-1R by 87%, suggesting that Shc is a crucial molecule in the formation of this ternary complex. Confocal microscopy studies provided further confirmation of the functional roles of Shc and the IGF-1R in the translocation of ERα to the region of the membrane. Down-regulation of Shc, ERα, or IGF-1R with specific small inhibitory RNAs all blocked E2-induced mitogen-activated protein kinase phosphorylation. Together, our results demonstrate that Shc and IGF-1R serve as key elements in the translocation of ERα to the cell membrane and in the facilitation of ERα-mediated rapid E2 action.
Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes
Authors: Kang Moon-Il , Kobayashi Akira , Wakabayashi Nobunao , Kim Sang-Geon , Yamamoto Masayuki
Published: February 2004

Transcription factor Nrf2 regulates basal and inducible expression of phase 2 proteins that protect animal cells against the toxic effects of electrophiles and oxidants. Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a multidomain, cysteinerich protein that is bound to the actin cytoskeleton. Keap1 acts both as a repressor of the Nrf2 transactivation and as a sensor of phase 2 inducers. Electrophiles and oxidants disrupt the Keap1–Nrf2 complex, resulting in nuclear accumulation of Nrf2, where it enhances the transcription of phase 2 genes via a common upstream regulatory element, the antioxidant response element. Reporter cotransfection–transactivation analyses with a series of Keap1 deletion mutants revealed that in the absence of the double glycine repeat domain Keap1 does not bind to Nrf2. In addition, deletion of either the intervening region or the C-terminal region also abolished the ability of Keap1 to sequester Nrf2, indicating that all of these domains contribute to the repressor activity of Keap1. Immunocytochemical and immunoprecipitation analyses demonstrated that Keap1 associates with actin filaments in the cytoplasm through its double glycine repeat domain. Importantly, disruption of the actin cytoskeleton promotes nuclear entry of an Nrf2 reporter protein. The actin cytoskeleton therefore provides scaffolding that is essential for the function of Keap1, which is the sensor for oxidative and electrophilic stress.
Physiological contribution of the astrocytic environment of neurons to intersynaptic crosstalk
Authors: Piet Richard , Vargová Lydia , Syková Eva , Poulain Dominique A. , Oliet Stéphane H. R.
Published: February 2004

Interactions between separate synaptic inputs converging on the same target appear to contribute to the fine-tuning of information processing in the central nervous system. Intersynaptic crosstalk is made possible by transmitter spillover from the synaptic cleft and its diffusion over a distance to neighboring synapses. This is the case for glutamate, which inhibits γ-aminobutyric acid (GABA)ergic transmission in several brain regions through the activation of presynaptic receptors. Such heterosynaptic modulation depends on factors that influence diffusion in the extracellular space (ECS). Because glial cells represent a physical barrier to diffusion and, in addition, are essential for glutamate uptake, we investigated the physiological contribution of the astrocytic environment of neurons to glutamate-mediated intersynaptic communication in the brain. Here we show that the reduced astrocytic coverage of magnocellular neurons occurring in the supraoptic nucleus of lactating rats facilitates diffusion in the ECS, as revealed by tortuosity and volume fraction measurements. Under these conditions, glutamate spillover, monitored through metabotropic glutamate receptor-mediated depression of GABAergic transmission, is greatly enhanced. Conversely, impeding diffusion with dextran largely prevents crosstalk between glutamatergic and GABAergic afferent inputs. Astrocytes, therefore, by hindering diffusion in the ECS, regulate intersynaptic communication between neighboring synapses and, probably, overall volume transmission in the brain.
Type I MADS-box genes have experienced faster birth-and-death evolution than type II MADS-box genes in angiosperms
Authors: Nam Jongmin , Kim Joonyul , Lee Shinyoung , An Gynheung , Ma Hong , Nei Masatoshi
Published: February 2004

Plant MADS-box genes form a large gene family for transcription factors and are involved in various aspects of developmental processes, including flower development. They are known to be subject to birth-and-death evolution, but the detailed features of this mode of evolution remain unclear. To have a deeper insight into the evolutionary pattern of this gene family, we enumerated all available functional and nonfunctional (pseudogene) MADS-box genes from the
Arabidopsis
and rice genomes. Plant MADS-box genes can be classified into types I and II genes on the basis of phylogenetic analysis. Conducting extensive homology search and phylogenetic analysis, we found 64 presumed functional and 37 nonfunctional type I genes and 43 presumed functional and 4 nonfunctional type II genes in
Arabidopsis
. We also found 24 presumed functional and 6 nonfunctional type I genes and 47 presumed functional and 1 nonfunctional type II genes in rice. Our phylogenetic analysis indicated there were at least about four to eight type I genes and ≈15–20 type II genes in the most recent common ancestor of
Arabidopsis
and rice. It has also been suggested that type I genes have experienced a higher rate of birth-and-death evolution than type II genes in angiosperms. Furthermore, the higher rate of birth-and-death evolution in type I genes appeared partly due to a higher frequency of segmental gene duplication and weaker purifying selection in type I than in type II genes.
WNK4 regulates apical and basolateral Cl
–
flux in extrarenal epithelia
Authors: Kahle Kristopher T. , Gimenez Ignacio , Hassan Hatim , Wilson Frederick H. , Wong Robert D. , Forbush Biff , Aronson Peter S. , Lifton Richard P.
Published: February 2004

Mutations in the serine-threonine kinase
WNK4
[with no lysine (K) 4] cause pseudohypoaldosteronism type II, a Mendelian disease featuring hypertension with hyperkalemia. In the kidney, WNK4 regulates the balance between NaCl reabsorption and K
+
secretion via variable inhibition of the thiazide-sensistive NaCl cotransporter and the K
+
channel ROMK. We now demonstrate expression of WNK4 mRNA and protein outside the kidney. In extrarenal tissues, WNK4 is found almost exclusively in polarized epithelia, variably associating with tight junctions, lateral membranes, and cytoplasm. Epithelia expressing WNK4 include sweat ducts, colonic crypts, pancreatic ducts, bile ducts, and epididymis. WNK4 is also expressed in the specialized endothelium of the blood–brain barrier. These epithelia and endothelium all play important roles in Cl
–
transport. Because WNK4 is known to regulate renal Cl
–
handling, we tested WNK4's effect on the activity of mediators of epithelial Cl
–
flux whose extrarenal expression overlaps with WNK4. WNK4 proved to be a potent inhibitor of the activity of both the Na
+
-K
+
-2Cl
–
cotransporter (NKCC1) and the Cl
–
/base exchanger SLC26A6 (CFEX) (>95% inhibition of NKCC1-mediated
86
Rb influx,
P
< 0.001; >80% inhibition of CFEX-mediated [
14
C] formate uptake,
P
< 0.001), mediators of Cl
–
flux across basolateral and apical membranes, respectively. In contrast, WNK4 showed no inhibition of pendrin, a related Cl
–
/base exchanger. These findings indicate a general role for WNK4 in the regulation of electrolyte flux in diverse epithelia. Moreover, they reveal that WNK4 regulates the activities of a diverse group of structurally unrelated ion channels, cotransporters, and exchangers.
Crystal structure of HLA-DQ0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy
Authors: Siebold Christian , Hansen Bjarke E. , Wyer Jessica R. , Harlos Karl , Esnouf Robert E. , Svejgaard Arne , Bell John I. , Strominger Jack L. , Jones E. Yvonne , Fugger Lars
Published: February 2004

The MHC class II molecule DQ0602 confers strong susceptibility to narcolepsy but dominant protection against type 1 diabetes. The crystal structure of DQ0602 reveals the molecular features underlying these contrasting genetic properties. Structural comparisons to homologous DQ molecules with differential disease associations highlight a previously unrecognized interplay between the volume of the P6 pocket and the specificity of the P9 pocket, which implies that presentation of an expanded peptide repertoire is critical for dominant protection against type 1 diabetes. In narcolepsy, the volume of the P4 pocket appears central to the susceptibility, suggesting that the presentation of a specific peptide population plays a major role.