bluuebunny/crossref_metadata_2025 huggingface dataset:
Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway
Authors: Shoham Nitza G. , Centola Michael , Mansfield Elizabeth , Hull Keith M. , Wood Geryl , Wise Carol A. , Kastner Daniel L.
Published: November 2003

Pyrin, the familial Mediterranean fever protein, is found in association with the cytoskeleton in myeloid/monocytic cells and modulates IL-1β processing, NF-κB activation, and apoptosis. These effects are mediated in part through cognate interactions with the adaptor protein ASC, which shares an N-terminal motif with pyrin. We sought additional upstream regulators of inflammation by using pyrin as the bait in yeast two-hybrid assays. We now show that proline serine threonine phosphatase-interacting protein [PSTPIP1, or CD2-binding protein 1 (CD2BP1)], a tyrosine-phosphorylated protein involved in cytoskeletal organization, also interacts with pyrin. Recently, PSTPIP1/CD2BP1 mutations were shown to cause the syndrome of pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA), a dominantly inherited autoinflammatory disorder mediated predominantly by granulocytes. Endogenous PSTPIP1/CD2BP1 and pyrin are coexpressed in monocytes and granulocytes and can be coimmunoprecipitated from THP-1 cells. The B box segment of pyrin was necessary and the B box/coiled-coil segment sufficient for this interaction, whereas the SH3 and coiled-coil domains of PSTPIP1/CD2BP1 were both necessary, but neither was sufficient, for pyrin binding. The Y344F PSTPIP1/CD2BP1 mutation, which blocks tyrosine phosphorylation, was associated with a marked reduction in pyrin binding in pervanadate-treated cells. PAPA-associated A230T and E250Q PSTPIP1/CD2BP1 mutations markedly increased pyrin binding as assayed by immunoprecipitation and, relative to WT, these mutants were hyperphosphorylated when coexpressed with c-Abl kinase. Consistent with the hypothesis that these mutations exert a dominant-negative effect on the previously reported activity of pyrin, we found increased IL-1β production by peripheral blood leukocytes from a clinically active PAPA patient with the A230T PSTPIP1/CD2BP1 mutation and in cell lines transfected with both PAPA-associated mutants.
The proteome of Saccharomyces cerevisiae mitochondria
Authors: Sickmann Albert , Reinders Jörg , Wagner Yvonne , Joppich Cornelia , Zahedi René , Meyer Helmut E. , Schönfisch Birgit , Perschil Inge , Chacinska Agnieszka , Guiard Bernard , Rehling Peter , Pfanner Nikolaus , Meisinger Chris
Published: November 2003

We performed a comprehensive approach to determine the proteome of
Saccharomyces cerevisiae
mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent ≈90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.
Id-1
as a molecular target in therapy for breast cancer cell invasion and metastasis
Authors: Fong Sylvia , Itahana Yoko , Sumida Tomoki , Singh Jarnail , Coppe Jean-Philippe , Liu Yong , Richards Peter C. , Bennington James L. , Lee Nancy M. , Debs Robert J. , Desprez Pierre-Yves
Published: November 2003

Mammary epithelial cells constitutively expressing Id-1 protein are unable to differentiate, acquire the ability to proliferate, and invade the extracellular matrix. In addition, Id-1 is aberrantly over-expressed in aggressive and metastatic breast cancer cells, as well as in human breast tumor biopsies from infiltrating carcinomas, suggesting Id-1 might be an important regulator of breast cancer progression. We show that human metastatic breast cancer cells become significantly less invasive
in vitro
and less metastatic
in vivo
when
Id-1
is down-regulated by stable transduction with antisense
Id-1
. Expression of the matrix metalloproteinase MT1-MMP is decreased in proportion to the decrease in Id-1 protein levels, representing a potential mechanism for the reduction of invasiveness. Further, to more accurately recapitulate the biology of and potential therapeutic approaches to tumor metastasis, we targeted
Id-1
expression systemically in tumor-bearing mice by using a nonviral approach. We demonstrate significant reduction of both Id-1 and MT1-MMP expressions as well as the metastatic spread of 4T1 breast cancer cells in syngeneic BALB/c mice. In conclusion, our studies have identified Id-1 as a critical regulator of breast cancer progression and suggest the feasibility of developing novel therapeutic approaches to target
Id-1
expression to reduce breast cancer metastasis in humans.
Critical roles for IFN-β in lymphoid development, myelopoiesis, and tumor development: Links to tumor necrosis factor α
Authors: Deonarain Raj , Verma Amit , Porter Andrew C. G. , Gewert Dirk R. , Platanias Leonidas C. , Fish Eleanor N.
Published: November 2003

We have generated mice null for IFN-β and report the diverse consequences of IFN-β for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood, thymus, and spleen of IFN-β-/-mice, activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor α production, relative to IFN-β+/+mice. Notably, constitutive and induced expression of tumor necrosis factor α is reduced in the spleen and bone marrow (BM) macrophages, respectively, of IFN-β-/-mice. We also observe an altered splenic architecture in IFN-β-/-mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-β-/-mice, associated with a decrease in B220+ve/high/CD43-veBM-derived cells and a reduction in BP-1, IgM, and CD23 expression. Circulating IgM-, Mac-1-, and Gr-1-positive cells are also substantially decreased in IFN-β-/-mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice, shown by the reduction of colony-forming units, granulocyte-macrophage. We proceeded to evaluate thein vivogrowth of malignant cells in the IFN-β-/-background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-β-/-mice. Taken altogether, our data suggest that, in addition to the direct growth-inhibitory effects on tumor cells, IFN-β is required during different stages of maturation in the development of the immune system.
De novodesigned cyclic-peptide heme complexes
Authors: Rosenblatt Michael M. , Wang Jiangyun , Suslick Kenneth S.
Published: November 2003

The structural characterization ofde novodesigned metalloproteins together with determination of chemical reactivity can provide a detailed understanding of the relationship between protein structure and functional properties. Toward this goal, we have prepared a series of cyclic peptides that bind to water-soluble metalloporphyrins (FeIIIand CoIII). Neutral and positively charged histidine-containing peptides bind with a high affinity, whereas anionic peptides bind only weakly to the negatively charged metalloporphyrin. Additionally, it was found that the peptide becomes helical only in the presence of the metalloporphyrin. CD experiments confirm that the metalloporphyrin binds specific cyclic peptides with high affinity and with isodichroic behavior. Thermal unfolding experiments show that the complex has ”native-like” properties. Finally, NMR spectroscopy produced well dispersed spectra and experimental restraints that provide a high-resolution solution structure of the complexed peptide.
Hyposulfatemia, growth retardation, reduced fertility, and seizures in mice lacking a functional NaS
i
-1 gene
Authors: Dawson Paul A. , Beck Laurent , Markovich Daniel
Published: November 2003

Inorganic sulfate is required for numerous functions in mammalian physiology, and its circulating levels are proposed to be maintained by the Na
+
-SO
4
2-
cotransporter, (NaS
i
-1). To determine the role of NaS
i
-1 in sulfate homeostasis and the physiological consequences in its absence, we have generated a mouse lacking a functional NaS
i
-1 gene,
Nas1
. Serum sulfate concentration was reduced by >75% in
Nas1
-/-
mice when compared with
Nas1
+/+
mice.
Nas1
-/-
mice exhibit increased urinary sulfate excretion, reduced renal and intestinal Na
+
-SO
4
2-
cotransport, and a general growth retardation.
Nas1
-/-
mouse body weight was reduced by >20% when compared with
Nas1
+/+
and
Nas1
+/-
littermates at 2 weeks of age and remained so throughout adulthood.
Nas1
-/-
females had a lowered fertility, with a 60% reduction in litter size. Spontaneous clonic seizures were observed in
Nas1
-/-
mice from 8 months of age. These data demonstrate NaS
i
-1 is essential for maintaining sulfate homeostasis, and its expression is necessary for a wide range of physiological functions.
ADP-ribosylation factor 6 regulates insulin secretion through plasma membrane phosphatidylinositol 4,5-bisphosphate
Authors: Lawrence John T. R. , Birnbaum Morris J.
Published: November 2003

ADP-ribosylation factor 6 (ARF6) is a small GTP-binding protein that regulates peripheral vesicular trafficking and actin cytoskeletal dynamics, and it has been implicated as critical to regulated secretion. Expression of a dominant-inhibitory ARF6 mutant, ARF6(T27N), impaired glucose-, depolarization-, and γ-thio-GTP-stimulated insulin secretion in the pancreatic β cell line, MIN6. In response to depolarization, MIN6 cells expressing ARF6(T27N) displayed an unaltered initial fast phase but an impaired subsequent slow phase of insulin secretion. Actin cytoskeletal disassembly with latrunculin A enhanced insulin secretion, whereas stabilization with jasplakinolide inhibited secretion, consistent with the actin cytoskeleton serving as a barrier to exocytosis in these cells. ARF6(T27N) led to a depolarization-dependent reduction in the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P
2
] with a time course that paralleled the inhibition of secretion. Moreover, blockade of PI(4,5)P
2
-dependent events by expression of a lipid-binding protein resulted in inhibition of depolarization-induced secretion in a manner identical to ARF6(T27N). These results indicate that ARF6 is required to sustain adequate levels of PI(4,5)P
2
during periods of increased PI(4,5)P
2
metabolism such as regulated secretion.
Identification of the protein kinase A regulatory R
I
α-catalytic subunit interface by amide H/
2
H exchange and protein docking
Authors: Anand Ganesh S. , Law Dennis , Mandell Jeffrey G. , Snead Aaron N. , Tsigelny Igor , Taylor Susan S. , Eyck Lynn F. Ten , Komives Elizabeth A.
Published: November 2003

An important goal after structural genomics is to build up the structures of higher-order protein–protein complexes from structures of the individual subunits. Often structures of higher order complexes are difficult to obtain by crystallography. We have used an alternative approach in which the structures of the individual catalytic (C) subunit and R
I
α regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen/deuterium (H/
2
H) exchange interface protection data. The resulting set of filtered solutions forms an ensemble of structures in which, besides the inhibitor peptide binding site, a flat interface between the C-terminal lobe of the C-subunit and the A- and B-helices of R
I
α is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.
Endosomal compartmentalization in three dimensions: Implications for membrane fusion
Authors: Murk J. L. A. N. , Humbel B. M. , Ziese U. , Griffith J. M. , Posthuma G. , Slot J. W. , Koster A. J. , Verkleij A. J. , Geuze H. J. , Kleijmeer M. J.
Published: November 2003

Endosomes are major sorting stations in the endocytic route that send proteins and lipids to multiple destinations in the cell, including the cell surface, Golgi complex, and lysosomes. They have an intricate architecture of internal membrane structures enclosed by an outer membrane. Recycling proteins remain on the outer membrane, whereas proteins that are destined for degradation in the lysosome are sorted to the interior. Recently, a retrograde pathway was discovered whereby molecules, like MHC class II of the immune system, return from the internal structures to the outer membrane, allowing their further transport to the cell surface for T cell activation. Whether this return involves back fusion of free vesicles with the outer membrane, or occurs via the continuity of the two membrane domains, is an unanswered question. By electron tomography of cryo-immobilized cells we now demonstrate that, in multivesicular endosomes of B-lymphocytes and dendritic cells, the inner membranes are free vesicles. Hence, protein transport from inner to outer membranes cannot occur laterally in the plane of the membrane, but requires fusion between the two membrane domains. This implies the existence of an intracellular machinery that mediates fusion between the exoplasmic leaflets of the membranes involved, which is opposite to regular intracellular fusion between cytoplasmic leaflets. In addition, our 3D reconstructions reveal the presence of clathrin-coated areas at the cytoplasmic face of the outer membrane, known to participate in protein sorting to the endosomal interior. Interestingly, profiles reminiscent of inward budding vesicles were often in close proximity to the coats.
Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance
Authors: Hirsch L. R. , Stafford R. J. , Bankson J. A. , Sershen S. R. , Rivera B. , Price R. E. , Hazle J. D. , Halas N. J. , West J. L.
Published: November 2003

Metal nanoshells are a class of nanoparticles with tunable optical resonances. In this article, an application of this technology to thermal ablative therapy for cancer is described. By tuning the nanoshells to strongly absorb light in the near infrared, where optical transmission through tissue is optimal, a distribution of nanoshells at depth in tissue can be used to deliver a therapeutic dose of heat by using moderately low exposures of extracorporeally applied near-infrared (NIR) light. Human breast carcinoma cells incubated with nanoshells
in vitro
were found to have undergone photothermally induced morbidity on exposure to NIR light (820 nm, 35 W/cm
2
), as determined by using a fluorescent viability stain. Cells without nanoshells displayed no loss in viability after the same periods and conditions of NIR illumination. Likewise,
in vivo
studies under magnetic resonance guidance revealed that exposure to low doses of NIR light (820 nm, 4 W/cm
2
) in solid tumors treated with metal nanoshells reached average maximum temperatures capable of inducing irreversible tissue damage (Δ
T
= 37.4 ± 6.6°C) within 4-6 min. Controls treated without nanoshells demonstrated significantly lower average temperatures on exposure to NIR light (Δ
T
< 10°C). These findings demonstrated good correlation with histological findings. Tissues heated above the thermal damage threshold displayed coagulation, cell shrinkage, and loss of nuclear staining, which are indicators of irreversible thermal damage. Control tissues appeared undamaged.
Cyclin-dependent kinase 5 is a mediator of dopaminergic neuron loss in a mouse model of Parkinson's disease
Authors: Smith Patrice D. , Crocker Stephen J. , Jackson-Lewis Vernice , Jordan-Sciutto Kelly L. , Hayley Shawn , Mount Matthew P. , O'Hare Michael J. , Callaghan Steven , Slack Ruth S. , Przedborski Serge , Anisman Hymie , Park David S.
Published: November 2003

Recent evidence indicates that cyclin-dependent kinases (CDKs, cdks) may be inappropriately activated in several neurodegenerative conditions. Here, we report that cdk5 expression and activity are elevated after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that damages the nigrostriatal dopaminergic pathway. Supporting the pathogenic significance of the cdk5 alterations are the findings that the general cdk inhibitor, flavopiridol, or expression of dominant-negative cdk5, and to a lesser extent dominant-negative cdk2, attenuates the loss of dopaminergic neurons caused by MPTP. In addition, CDK inhibition strategies attenuate MPTP-induced hypolocomotion and markers of striatal function independent of striatal dopamine. We propose that cdk5 is a key regulator in the degeneration of dopaminergic neurons in Parkinson's disease.
Essential role for RGS9 in opiate action
Authors: Zachariou Venetia , Georgescu Dan , Sanchez Nick , Rahman Zia , DiLeone Ralph , Berton Olivier , Neve Rachael L. , Sim-Selley Laura J. , Selley Dana E. , Gold Stephen J. , Nestler Eric J.
Published: November 2003

Regulators of G protein signaling (RGS) are a family of proteins known to accelerate termination of effector stimulation after G protein receptor activation. RGS9-2, a brain-specific splice variant of the RGS9 gene, is highly enriched in striatum and also expressed at much lower levels in periaqueductal gray and spinal cord, structures known to mediate various actions of morphine and other opiates. Morphine exerts its acute rewarding and analgesic effects by activation of inhibitory guanine nucleotide-binding regulatory protein-coupled opioid receptors, whereas chronic morphine causes addiction, tolerance to its acute analgesic effects, and profound physical dependence by sustained activation of these receptors. We show here that acute morphine administration increases expression of RGS9-2 in NAc and the other CNS regions, whereas chronic exposure decreases RGS9-2 levels. Mice lacking RGS9 show enhanced behavioral responses to acute and chronic morphine, including a dramatic increase in morphine reward, increased morphine analgesia with delayed tolerance, and exacerbated morphine physical dependence and withdrawal. These findings establish RGS9 as a potent negative modulator of opiate action
in vivo
, and suggest that opiate-induced changes in RGS9 levels contribute to the behavioral and neural plasticity associated with chronic opiate administration.
The low gonadotropin-independent constitutive production of testicular testosterone is sufficient to maintain spermatogenesis
Authors: Zhang Fu-Ping , Pakarainen Tomi , Poutanen Matti , Toppari Jorma , Huhtaniemi Ilpo
Published: November 2003

Spermatogenesis is thought to critically depend on the high intratesticular testosterone (T) levels induced by gonadotropic hormones. Strategies for hormonal male contraception are based on disruption of this regulatory mechanism through blockage of gonadotropin secretion. Although exogenous T or T plus progestin treatments efficiently block gonadotropin secretion and suppress testicular T production, only ≈60% of treated Caucasian men reach contraceptive azoospermia. We now report that in luteinizing hormone receptor knockout mice, qualitatively full spermatogenesis, up to elongated spermatids of late stages 13-16, is achieved at the age of 12 months, despite absent luteinizing hormone action and very low intratesticular T (2% of control level). However, postmeiotic spermiogenesis was blocked by the antiandrogen flutamide, indicating a crucial role of the residual low testicular T level in this process. The persistent follicle-stimulating hormone action in luteinizing hormone receptor knockout mice apparently stimulates spermatogenesis up to postmeiotic round spermatids, as observed in gonadotropin-deficient rodent models on follicle-stimulating hormone supplementation. The finding that spermatogenesis is possible without a luteinizing hormone-stimulated high level of intratesticular T contradicts the current dogma. Extrapolated to humans, it may indicate that only total abolition of testicular androgen action will result in consistent azoospermia, which is necessary for effective male contraception.
Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function
Authors: Côté Francine , Thévenot Etienne , Fligny Cécile , Fromes Yves , Darmon Michèle , Ripoche Marie-Anne , Bayard Elisa , Hanoun Naima , Saurini Françoise , Lechat Philippe , Dandolo Luisa , Hamon Michel , Mallet Jacques , Vodjdani Guilan
Published: November 2003

Serotonin (5-HT) controls a wide range of biological functions. In the brain, its implication as a neurotransmitter and in the control of behavioral traits has been largely documented. At the periphery, its modulatory role in physiological processes, such as the cardiovascular function, is still poorly understood. The rate-limiting enzyme of 5-HT synthesis, tryptophan hydroxylase (TPH), is encoded by two genes, the well characterizedtph1gene and a recently identifiedtph2gene. In this article, based on the study of a mutant mouse in which thetph1gene has been inactivated by replacement with the β-galactosidase gene, we establish that the neuronaltph2is expressed in neurons of the raphe nuclei and of the myenteric plexus, whereas the nonneuronaltph1, as detected by β-galactosidase expression, is in the pineal gland and the enterochromaffin cells. Anatomic examination of the mutant mice revealed larger heart sizes than in wild-type mice. Histological investigation indicates that the primary structure of the heart muscle is not affected. Hemodynamic analyses demonstrate abnormal cardiac activity, which ultimately leads to heart failure of the mutant animals. This report links loss oftph1gene expression, and thus of peripheral 5-HT, to a cardiac dysfunction phenotype. Thetph1-/-mutant may be valuable for investigating cardiovascular dysfunction observed in heart failure in humans.
Differential effects on bone of estrogen receptor α and androgen receptor activation in orchidectomized adult male mice
Authors: Movérare Sofia , Venken Katrien , Eriksson Anna-Lena , Andersson Niklas , Skrtic Stanko , Wergedal Jon , Mohan Subburaman , Salmon Phil , Bouillon Roger , Gustafsson Jan-Åke , Vanderschueren Dirk , Ohlsson Claes
Published: November 2003

Androgens may regulate the male skeleton either directly by stimulation of the androgen receptor (AR) or indirectly by aromatization of androgens into estrogens and, thereafter, by stimulation of the estrogen receptors (ERs). To directly compare the effect of ER activation on bone
in vivo
with the effect of AR activation, 9-month-old orchidectomized wild-type and ER-inactivated mice were treated with the nonaromatizable androgen 5α-dihydrotestosterone, 17β-estradiol, or vehicle. Both ERα and AR but not ERβ activation preserved the amount of trabecular bone. ERα activation resulted both in a preserved thickness and number of trabeculae. In contrast, AR activation exclusively preserved the number of trabeculae, whereas the thickness of the trabeculae was unaffected. Furthermore, the effects of 17β-estradiol could not be mediated by the AR, and the effects of 5α-dihydrotestosterone were increased rather than decreased in ER-inactivated mice. ERα, but not AR or ERβ, activation resulted in preserved thickness, volumetric density, and mechanical strength of the cortical bone. ERα activation increased serum levels of insulin-like growth factor I, which were positively correlated with all the cortical and trabecular bone parameters that were specifically preserved by ERα activation but not by AR activation, suggesting that insulin-like growth factor I might mediate these effects of ERα activation. Thus, the
in vivo
bone-sparing effect of ERα activation is distinct from the bone-sparing effect of AR activation in adult male mice. Because these two pathways are clearly distinct from each other, one may speculate that a combined treatment of selective ER modulators and selective AR modulators might be beneficial in the treatment of osteoporosis.
Visualizing the distribution and transport of mRNAs in living cells
Authors: Bratu Diana P. , Cha Byeong-Jik , Mhlanga Musa M. , Kramer Fred Russell , Tyagi Sanjay
Published: November 2003

We have visualized the movements of native mRNAs in living cells. Using nuclease-resistant molecular beacons, we imaged the transport and localization of
oskar
mRNA in
Drosophila melanogaster
oocytes. When the localization pattern was altered by genetic manipulation of the mRNA's 3′ untranslated region, or by chemical perturbation of the intracellular tubulin network, the distribution of the fluorescence signals changed accordingly. We tracked the migration of
oskar
mRNA in real time, from the nurse cells where it is produced to the posterior cortex of the oocyte where it is localized. Our observations reveal the presence of a transient, and heretofore elusive, stage in the transport of
oskar
mRNA. Direct visualization of specific mRNAs in living cells with molecular beacons will accelerate studies of intracellular RNA trafficking and localization, just as the use of green fluorescent protein has stimulated the study of specific proteins
in vivo
.
Functional constraints and frequency of deleterious mutations in noncoding DNA of rodents
Authors: Keightley Peter D. , Gaffney Daniel J.
Published: November 2003

Selection against deleterious mutations imposes a mutation load on populations because individuals die or fail to reproduce. In vertebrates, estimates of genomic rates of deleterious mutations in protein-coding genes imply the existence of a substantial mutation load, but many functionally important regions of the genome are thought to reside in noncoding DNA, and the contribution of noncoding DNA to the mutation load has been unresolved. Here, we infer the frequency of deleterious mutations in noncoding DNA of rodents by comparing rates of substitution at noncoding nucleotides with rates of substitution at the fastest evolving intronic sites of adjacent genes sampled from the whole genome sequences of mouse and rat. We show that the major elements of selectively constrained noncoding DNA are within 2,500 bp upstream and downstream of coding sequences and in first introns. Our estimate of the genomic deleterious point mutation rate for noncoding DNA (0.22 per diploid per generation) is similar to that for coding DNA. Mammalian populations therefore experience a substantial genetic load associated with selection against deleterious mutations in noncoding DNA. Deleterious mutations in noncoding DNA have predominantly quantitative effects and could be an important source of the burden of complex genetic disease variation in human populations.
Trp-cage: Folding free energy landscape in explicit water
Authors: Zhou Ruhong
Published: November 2003

Trp-cage is a 20-residue miniprotein, which is believed to be the fastest folder known so far. In this study, the folding free energy landscape of Trp-cage has been explored in explicit solvent by using an OPLSAA force field with periodic boundary condition. A highly parallel replica exchange molecular dynamics method is used for the conformation space sampling, with the help of a recently developed efficient molecular dynamics algorithm P3ME/RESPA (particle–particle particle–mesh Ewald/reference system propagator algorithm). A two-step folding mechanism is proposed that involves an intermediate state where two correctly formed partial hydrophobic cores are separated by an essential salt-bridge between residues Asp-9 and Arg-16 near the center of the peptide. This metastable intermediate state provides an explanation for the superfast folding process. The free energy landscape is found to be rugged at low temperatures, and then becomes smooth and funnel-like above 340 K. The lowest free energy structure at 300 K is only 1.50 Å C
α
-RMSD (C
α
-rms deviation) from the NMR structures. The simulated nuclear Overhauser effect pair distances are in excellent agreement with the raw NMR data. The temperature dependence of the Trp-cage population, however, is found to be significantly different from experiment, with a much higher melting transition temperature above 400 K (experimental 315 K), indicating that the current force fields, parameterized at room temperature, need to be improved to correctly predict the temperature dependence.
Pannexins, a family of gap junction proteins expressed in brain
Authors: Bruzzone Roberto , Hormuzdi Sheriar G. , Barbe Michael T. , Herb Anne , Monyer Hannah
Published: November 2003

Database search has led to the identification of a family of proteins, the pannexins, which share some structural features with the gap junction forming proteins of invertebrates and vertebrates. The function of these proteins has remained unclear so far. To test the possibility that pannexins underlie electrical communication in the brain, we have investigated their tissue distribution and functional properties. Here, we show that two of these genes, pannexin 1 (Px1) and Px2, are abundantly expressed in the CNS. In many neuronal cell populations, including hippocampus, olfactory bulb, cortex and cerebellum, there is coexpression of both pannexins, whereas in other brain regions, e.g., white matter, only Px1-positive cells were found. On expression in
Xenopus
oocytes, Px1, but not Px2 forms functional hemichannels. Coinjection of both pannexin RNAs results in hemichannels with functional properties that are different from those formed by Px1 only. In paired oocytes, Px1, alone and in combination with Px2, induces the formation of intercellular channels. The functional characteristics of homomeric Px1 versus heteromeric Px1/Px2 channels and the different expression patterns of Px1 and Px2 in the brain indicate that pannexins form cell type-specific gap junctions with distinct properties that may subserve different functions.
Premature telomeric loss in rheumatoid arthritis is genetically determined and involves both myeloid and lymphoid cell lineages
Authors: Schönland Stefan O. , Lopez Consuelo , Widmann Thomas , Zimmer Julia , Bryl Ewa , Goronzy Jörg J. , Weyand Cornelia M.
Published: November 2003

In rheumatoid arthritis, peripheral blood T cells have age-inappropriate telomeric erosion. We examined whether
HLA-DRB1
*
04
alleles, the major susceptibility genes for this disease, confer risk for T cell senescence. In healthy individuals,
HLA-DRB1
*
04
alleles were associated with excessive loss of telomeres in CD4
+
T cells. Accelerated telomeric erosion occurred during the first two decades of life and was followed by reduced homeostatic T cell proliferation during adulthood. Premature telomeric loss also affected granulocytes, suggesting that the hematopoietic stem cell is the primary target. Telomeric repair mechanisms were intact in
HLA-DRB1
*
04
+
donors. We propose that
HLA-DRB1
*
04
alleles or genes in linkage disequilibrium regulate stem cell replication and contribute to the accumulation of senescent and autoreactive T cells in rheumatoid arthritis.
Murine cytomegalovirus m157 mutation and variation leads to immune evasion of natural killer cells
Authors: Voigt Valentina , Forbes Catherine A. , Tonkin Joanne N. , Degli-Esposti Mariapia A. , Smith Hamish R. C. , Yokoyama Wayne M. , Scalzo Anthony A.
Published: November 2003

Effective natural killer (NK) cell recognition of murine cytomegalovirus (MCMV)-infected cells depends on binding of the Ly49H NK cell activation receptor to the m157 viral glycoprotein. Here we addressed the immunological consequences of variation in m157 sequence and function. We found that most strains of MCMV possess forms of m157 that evade Ly49H-dependent NK cell activation. Importantly, repeated passage of MCMV through resistant Ly49H+mice resulted in the rapid emergence of m157 mutants that elude Ly49H-dependent NK cell responses. These data provide the first molecular evidence that NK cells can exert sufficient immunological pressure on a DNA virus, such that it undergoes rapid and specific mutation in an NK cell ligand enabling it to evade efficient NK cell surveillance.
Normal B-1a cell development requires B cell-intrinsic NFATc1 activity
Authors: Berland Robert , Wortis Henry H.
Published: November 2003

B-1a cells, an anatomically, phenotypically, and functionally distinct subset of B cells that produce the bulk of natural serum IgM and much of gut-associated IgA, are an important component of the early response to pathogens. Because the induced expression of CD5, a hallmark of B-1a cells, requires a nuclear factor of activated T cells (NFAT)-dependent enhancer, we examined the role of NFAT transcription factors in B-1a development. Here we show that the B-1a compartment is normal in mice lacking NFATc2 but essentially absent in mice lacking NFATc1. Loss of NFATc1 affects both peritoneal and splenic B-1a cells. Because there is a loss of B-1 cells defined by markers other than CD5, NFATc1 is not required simply for CD5 expression on B-1a cells. Using mixed-allotype chimeras and retroviral-mediated gene transduction we show that the requirement for NFATc1 is B cell-intrinsic. We also demonstrate that NFATc1 protein expression is elevated ≈5-fold in B-1a cells compared with B-2 cells. This is the first definitive demonstration of a B cell-intrinsic function for an NFAT family transcription factor.
Motility of Escherichia coli cells in clusters formed by chemotactic aggregation
Authors: Mittal Nikhil , Budrene Elena O. , Brenner Michael P. , van Oudenaarden Alexander
Published: November 2003

Cells of
Escherichia coli
under conditions of certain cellular stresses excrete attractants. Cells of chemotactic strains respond to these excreted signaling molecules by moving up their local concentration gradients and forming different types of stable multicellular structures. Multicellular clusters are the simplest among these structures. Fluorescence microscopy was used to characterize the macroscopic properties of the clusters and to track individual
E. coli
cells in the clusters in real time. A quantitative analysis reveals that the equilibrium cluster size is only weakly dependent on the total number of cells in the cluster. The tumble frequency of an individual cell strongly depends on the position of the cell within the cluster and its direction of movement. In the central region of the cluster, tumbles are strongly suppressed whereas near the edge of the cluster, the tumble frequency is restored for exiting cells, thereby preventing them from leaving the cluster, resulting in the maintenance of sharp cluster boundaries. A simulation based on a model of the sensory memory of
E. coli
reproduces the experimental data and indicates that the tumble rate and consequently the morphology of the cluster are determined by the sensory memory of cells.
Multiple oscillators regulate circadian gene expression inNeurospora
Authors: Correa Alejandro , Lewis Zachary A. , Greene Andrew V. , March Irene J. , Gomer Richard H. , Bell-Pedersen Deborah
Published: November 2003

High-density microarrays were used to profile circadian gene expression inNeurospora crassacultures grown in constant darkness. We identified 145 clock-controlled genes (ccgs). The ccgs peaked in mRNA accumulation at all phases of the day, with the majority peaking in the late night to early morning. The predicted or known functions of the ccgs demonstrate that the clock contributes to a wide range of cellular processes, including cell signaling, development, metabolism, and stress responses. Although the period of rhythm of most of the ccgs was found to depend on the well characterizedfrequency(FRQ)-based oscillator, three ccgs appeared to have a rhythm that was significantly short in the long period (29-h)frq7mutant strain. These ccgs accumulate mRNA rhythmically with a circadian period in afrq-null strain, confirming the existence of a second oscillator inN. crassa.
Evidence that the bypassing ribosome travels through the coding gap
Authors: Gallant Jonathan , Bonthuis Paul , Lindsley Dale
Published: November 2003

In translational bypassing, a peptidyl-tRNA::ribosome complex skips over a number of nucleotides in a messenger sequence and resumes protein chain elongation after a “landing site” downstream of the bypassed region. The present experiments demonstrate that the complex “scans” processively through the bypassed region. This conclusion rests on three observations. (
i
) When two potential “landing sites” are present, the protein sequence of the product shows that virtually all ribosomes land at the first and virtually none at the second. (
ii
) In such a sequence with two landing sites, the presence of a terminator triplet in phase in the coding region immediately after the first landing site drastically reduces the efficiency of bypassing. (
iii
) Internally complementary sequences that can form a stable stemloop in the bypassed region significantly reduce the efficiency of bypassing. We analyze bypassing from a given “takeoff” site to “landing sites” at different distances downstream so as to derive estimates of the frequency of ribosome takeoff and of the stability of the bypassing complex.
Botulinum toxin type B micromechanosensor
Authors: Liu W. , Montana Vedrana , Chapman Edwin R. , Mohideen U. , Parpura Vladimir
Published: November 2003

Botulinum neurotoxin (BoNT) types A, B, E, and F are toxic to humans; early and rapid detection is essential for adequate medical treatment. Presently available tests for detection of BoNTs, although sensitive, require hours to days. We report a BoNT-B sensor whose properties allow detection of BoNT-B within minutes. The technique relies on the detection of an agarose bead detachment from the tip of a micromachined cantilever resulting from BoNT-B action on its substratum, the synaptic protein synaptobrevin 2, attached to the beads. The mechanical resonance frequency of the cantilever is monitored for the detection. To suspend the bead off the cantilever we use synaptobrevin's molecular interaction with another synaptic protein, syntaxin 1A, that was deposited onto the cantilever tip. Additionally, this bead detachment technique is general and can be used in any displacement reaction, such as in receptor-ligand pairs, where the introduction of one chemical leads to the displacement of another. The technique is of broad interest and will find uses outside toxicology.
Protein profiles associated with survival in lung adenocarcinoma
Authors: Chen Guoan , Gharib Tarek G , Wang Hong , Huang Chiang-Ching , Kuick Rork , Thomas Dafydd G. , Shedden Kerby A. , Misek David E. , Taylor Jeremy M. G. , Giordano Thomas J. , Kardia Sharon L. R. , Iannettoni Mark D. , Yee John , Hogg Philip J. , Orringer Mark B. , Hanash Samir M. , Beer David G.
Published: November 2003

Morphologic assessment of lung tumors is informative but insufficient to adequately predict patient outcome. We previously identified transcriptional profiles that predict patient survival, and here we identify proteins associated with patient survival in lung adenocarcinoma. A total of 682 individual protein spots were quantified in 90 lung adenocarcinomas by using quantitative two-dimensional polyacrylamide gel electrophoresis analysis. A leave-one-out cross-validation procedure using the top 20 survival-associated proteins identified by Cox modeling indicated that protein profiles as a whole can predict survival in stage I tumor patients (P= 0.01). Thirty-three of 46 survival-associated proteins were identified by using mass spectrometry. Expression of 12 candidate proteins was confirmed as tumor-derived with immunohistochemical analysis and tissue microarrays. Oligonucleotide microarray results from both the same tumors and from an independent study showed mRNAs associated with survival for 11 of 27 encoded genes. Combined analysis of protein and mRNA data revealed 11 components of the glycolysis pathway as associated with poor survival. Among these candidates, phosphoglycerate kinase 1 was associated with survival in the protein study, in both mRNA studies and in an independent validation set of 117 adenocarcinomas and squamous lung tumors using tissue microarrays. Elevated levels of phosphoglycerate kinase 1 in the serum were also significantly correlated with poor outcome in a validation set of 107 patients with lung adenocarcinomas using ELISA analysis. These studies identify new prognostic biomarkers and indicate that protein expression profiles can predict the outcome of patients with early-stage lung cancer.
Paternal and maternal genomes confer opposite effects on proliferation, cell-cycle length, senescence, and tumor formation
Authors: Hernandez Lidia , Kozlov Serguei , Piras Graziella , Stewart Colin L.
Published: November 2003

Loss of imprinting is the silencing of active imprinted genes or the activation of silent imprinted genes, and it is one of the most common epigenetic changes associated with the development of a wide variety of tumors. Here, we have analyzed the effects that global imprinted gene expression has on cell proliferation and transformation. Primary mouse embryonic fibroblasts (MEFs), whose entire genome is either exclusively paternal (androgenetic) or maternal (parthenogenetic), exhibit dramatically contrasting patterns of growth. In comparison with biparental MEFs, andro-genetic proliferation is characterized by a shorter cell cycle, increased saturation density, spontaneous transformation, and formation of tumors at low passage number. Parthenogenetic MEFs reach a lower saturation density, senesce, and die. The maternally expressed imprinted genes

p57
kip2

and
M6P
/
Igf2r
retard proliferation and reduce the long-term growth of MEFs. In contrast, the paternally expressed growth factor
Igf2
is essential for the long-term proliferation of all genotypes. Increased
Igf2
expression in primary MEFs not only stimulates proliferation, but also results in their rapid conversion to malignancy with tumor formation of short latency. Our results reveal that paternally expressed imprinted genes, in the absence of maternal imprinted genes, predispose fibroblasts to rapid transformation. A potent factor in their transformation is IGF2, which on increased expression results in the rapid conversion of primary cells to malignancy. These results reveal a route by which malignant choriocarcinoma may arise from molar pregnancies. They also suggest that the derivation of stem cells from parthenogenetic embryos, for the purposes of therapeutic cloning, may be ineffective.
Inflammation is detrimental for neurogenesis in adult brain
Authors: Ekdahl Christine T. , Claasen Jan-Hendrik , Bonde Sara , Kokaia Zaal , Lindvall Olle
Published: November 2003

New hippocampal neurons are continuously generated in the adult brain. Here, we demonstrate that lipopolysaccharide-induced inflammation, which gives rise to microglia activation in the area where the new neurons are born, strongly impairs basal hippocampal neurogenesis in rats. The increased neurogenesis triggered by a brain insult is also attenuated if it is associated with microglia activation caused by tissue damage or lipopolysaccharide infusion. The impaired neurogenesis in inflammation is restored by systemic administration of minocycline, which inhibits microglia activation. Our data raise the possibility that suppression of hippocampal neurogenesis by activated microglia contributes to cognitive dysfunction in aging, dementia, epilepsy, and other conditions leading to brain inflammation.
A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis
Authors: Altan-Bonnet Nihal , Phair Robert D. , Polishchuk Roman S. , Weigert Roberto , Lippincott-Schwartz Jennifer
Published: November 2003

In mitosis, chromosome, cytoskeleton, and organelle dynamics must be coordinated for successful cell division. Here, we present evidence for a role for Arf1, a small GTPase associated with the Golgi apparatus, in the orchestration of mitotic Golgi breakdown, chromosome segregation, and cytokinesis. We show that early in mitosis Arf1 becomes inactive and dissociates from Golgi membranes. This is followed by the dispersal of numerous Arf1-dependent peripheral Golgi proteins and subsequent Golgi disassembly. If Arf1 is kept in an active state by treatment with the small molecule H89 or expression of its GTP-locked form, intact Golgi membranes with bound peripheral proteins persist throughout mitosis. These cells enter mitosis but exhibit gross defects in chromosome segregation and cytokinetic furrow ingression. These findings suggest that mitotic Golgi disassembly depends on Arf1 inactivation and is used by the cell to disperse numerous peripheral Golgi proteins for coordinating the behavior of Golgi membranes, chromosomes, and cytoskeleton during mitosis.
Exploring the origins of binding specificity through the computational redesign of calmodulin
Authors: Shifman Julia M. , Mayo Stephen L.
Published: November 2003

Calmodulin (CaM) is a second messenger protein that has evolved to bind tightly to a variety of targets and, as such, exhibits low binding specificity. We redesigned CaM by using a computational protein design algorithm to improve its binding specificity for one of its targets, smooth muscle myosin light chain kinase (smMLCK). Residues in or near the CaM/smMLCK binding interface were optimized; CaM interactions with alternative targets were not directly considered in the optimization. The predicted CaM sequences were constructed and tested for binding to a set of eight targets including smMLCK. The best CaM variant, obtained from a calculation that emphasized intermolecular interactions, showed up to a 155-fold increase in binding specificity. The increase in binding specificity was not due to improved binding to smMLCK, but due to decreased binding to the alternative targets. This finding is consistent with the fact that the sequence of wild-type CaM is nearly optimal for interactions with numerous targets.
Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane
Authors: Angevine Christine M. , Herold Kelly A. G. , Fillingame Robert H.
Published: November 2003

The role of subunit
a
in promoting proton translocation and rotary motion in the
Escherichia coli
F
1
F
o
ATP synthase is poorly understood. In the membrane-bound F
o
sector of the enzyme, H
+
binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit
c
. Protons are thought to reach Asp-61 at the center of the membrane via aqueous channels formed at least in part by one or more of the five TMHs of subunit
a
. Aqueous access pathways have previously been mapped to surfaces of
a
TMH4. Here we have substituted Cys into the second and fifth TMHs of subunit
a
and carried out chemical modification with Ag
+
and
N
-ethylmaleimide to define the aqueous accessibility of residues along these helices. Access to
c
Asp-61 at the center of the membrane may be mediated in part by Ag
+
-sensitive residues 248, 249, 251, and 252 in
a
TMH5. From the periplasmic surface, aqueous access to
c
Asp-61 may be mediated by silver-sensitive residues 115, 116, 119, 120, 122, and 126 in
a
TMH2. The Ag
+
-sensitive residues in TMH2, -4, and -5 form a continuum extending from the periplasmic to the cytoplasmic side of the membrane. In an arrangement of helices supported by second-site revertant and crosslinking analyses, these residues cluster at the interior of a four-helix bundle formed by TMH2–5. The aqueous access pathways at the interior of subunit
a
may be gated by a swiveling of helices in this bundle, alternately exposing cytoplasmic and periplasmic half channels to
c
Asp-61 during the H
+
transport cycle.