bluuebunny/crossref_metadata_2025 huggingface dataset:
Human ASIC3 channel dynamically adapts its activity to sense the extracellular pH in both acidic and alkaline directions
Authors: Delaunay Anne , Gasull Xavier , Salinas Miguel , Noël Jacques , Friend Valérie , Lingueglia Eric , Deval Emmanuel
Published: July 2012

In rodent sensory neurons, acid-sensing ion channel 3 (ASIC3) has recently emerged as a particularly important sensor of nonadaptive pain associated with tissue acidosis. However, little is known about the human ASIC3 channel, which includes three splice variants differing in their C-terminal domain (hASIC3a, hASIC3b, and hASIC3c). hASIC3a transcripts represent the main mRNAs expressed in both peripheral and central neuronal tissues (dorsal root ganglia [DRG], spinal cord, and brain), where a small proportion of hASIC3c transcripts is also detected. We show that hASIC3 channels (hASIC3a, hASIC3b, or hASIC3c) are able to directly sense extracellular pH changes not only during acidification (up to pH 5.0), but also during alkalization (up to pH 8.0), an original and inducible property yet unknown. When the external pH decreases, hASIC3 display a transient acid mode with brief activation that is relevant to the classical ASIC currents, as previously described. On the other hand, an external pH increase activates a sustained alkaline mode leading to a constitutive activity at resting pH. Both modes are inhibited by the APETx2 toxin, an ASIC3-type channel inhibitor. The alkaline sensitivity of hASIC3 is an intrinsic property of the channel, which is supported by the extracellular loop and involves two arginines (R68 and R83) only present in the human clone. hASIC3 is thus able to sense the extracellular pH in both directions and therefore to dynamically adapt its activity between pH 5.0 and 8.0, a property likely to participate in the fine tuning of neuronal membrane potential and to neuron sensitization in various pH environments.
Profibrogenic chemokines and viral evolution predict rapid progression of hepatitis C to cirrhosis
Authors: Farci Patrizia , Wollenberg Kurt , Diaz Giacomo , Engle Ronald E. , Lai Maria Eliana , Klenerman Paul , Purcell Robert H. , Pybus Oliver G. , Alter Harvey J.
Published: July 2012

Chronic hepatitis C may follow a mild and stable disease course or progress rapidly to cirrhosis and liver-related death. The mechanisms underlying the different rates of disease progression are unknown. Using serial, prospectively collected samples from cases of transfusion-associated hepatitis C, we identified outcome-specific features that predict long-term disease severity. Slowly progressing disease correlated with an early alanine aminotransferase peak and antibody seroconversion, transient control of viremia, and significant induction of IFN-γ and MIP-1β, all indicative of an effective, albeit insufficient, adaptive immune response. By contrast, rapidly progressive disease correlated with persistent and significant elevations of alanine aminotransferase and the profibrogenic chemokine MCP-1 (CCL-2), greater viral diversity and divergence, and a higher rate of synonymous substitution. This study suggests that the long-term course of chronic hepatitis C is determined early in infection and that disease severity is predicted by the evolutionary dynamics of hepatitis C virus and the level of MCP-1, a chemokine that appears critical to the induction of progressive fibrogenesis and, ultimately, the ominous complications of cirrhosis.
Photophysics and optical switching in green fluorescent protein mutants
Authors: Creemers T. M. H. , Lock A. J. , Subramaniam V. , Jovin T. M. , Völker S.
Published: July 2012

We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable “one-color” systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in “color” on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species.
Tcl1 enhances Akt kinase activity and mediates its nuclear translocation
Authors: Pekarsky Yuri , Koval Anatoliy , Hallas Cora , Bichi Roberta , Tresini Maria , Malstrom Scott , Russo Giandomenico , Tsichlis Philip , Croce Carlo M.
Published: July 2012

The
TCL1
oncogene at 14q32.1 is involved in the development of human mature T-cell leukemia. The mechanism of action of Tcl1 is unknown. Because the virus containing the
v-akt
oncogene causes T-cell lymphoma in mice and Akt is a key player in transduction of antiapoptotic and proliferative signals in T-cells, we investigated whether Akt and Tcl1 function in the same pathway. Coimmunoprecipitation experiments showed that endogenous Akt1 and Tcl1 physically interact in the T-cell leukemia cell line SupT11; both proteins also interact when cotransfected into 293 cells. Using several
AKT1
constructs in cotransfection experiments, we determined that this interaction occurs through the pleckstrin homology domain of the Akt1 protein. We further demonstrated that, in 293 cells transfected with
TCL1
, the endogenous Akt1 bound to Tcl1 is 5–10 times more active compared with Akt1 not bound to Tcl1. The intracellular localization of Tcl1 and Akt1 in mouse fibroblasts was investigated by immunofluorescence. When transfected alone, Akt1 was found only in cytoplasm whereas Tcl1 was localized in the cytoplasm and in the nucleus. Interestingly, Akt1 was also found in the nucleus when
AKT1
was cotransfected with
TCL1
, suggesting that Tcl1 promotes the transport of Akt1 to the nucleus. These findings were supported by the intracellular localization of Akt1 or Tcl1 when Tcl1 or Akt1, respectively, were confined to the specific cellular compartments. Thus, we demonstrate that Tcl1 is a cofactor of Akt1 that enhances Akt1 kinase activity and promotes its nuclear transport.
Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity
Authors: Edelman Gerald M. , Meech Robyn , Owens Geoffrey C. , Jones Frederick S.
Published: July 2012

Eukaryotic transcriptional regulation in different cells involves large numbers and arrangements of cis and trans elements. To survey the number of cis regulatory elements that are active in different contexts, we have devised a high-throughput selection procedure permitting synthesis of active cis motifs that enhance the activity of a minimal promoter. This synthetic promoter construction method (SPCM) was used to identify >100 DNA sequences that showed increased promoter activity in the neuroblastoma cell line Neuro2A. After determining DNA sequences of selected synthetic promoters, database searches for known elements revealed a predominance of eight motifs: AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1/MAZ. The most active of the selected synthetic promoters contain composites of a number of these motifs. Assays of DNA binding and promoter activity of three exemplary motifs (ETS, CREB, and SP1/MAZ) were used to prove the effectiveness of SPCM in uncovering active sequences. Up to 10% of 133 selected active sequences had no match in currently available databases, raising the possibility that new motifs and transcriptional regulatory proteins to which they bind may be revealed by SPCM. The method may find uses in constructing databases of active cis motifs, in diagnostics, and in gene therapy.
Heterologous expression in
Escherichia coli
of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic
Authors: Trauger John W. , Walsh Christopher T.
Published: July 2012

The gene cluster from
Amycolotopsis orientalis
responsible for biosynthesis of the vancomycin-type glycopeptide antibiotic chloroeremomycin was recently sequenced, indicating that this antibiotic derives from a seven-residue peptide synthesized by a three-subunit (CepA, CepB, and CepC) modular nonribosomal peptide synthetase. Expression of all or parts of the peptide synthetase in
Escherichia coli
would facilitate biochemical characterization of its substrate specificity, an important step toward the development of more potent glycopeptides by combinatorial biosynthesis. To determine whether CepA, a three-module 3,158-residue peptide synthetase expected to assemble the first three residues of the heptapeptide precursor, could be heterologously expressed in
E. coli
and converted to active, holo form by posttranslational priming with a phosphopantetheinyltransferase, we expressed two CepA fragments (CepA1-575 and CepA1-1596) as well as full-length CepA (CepA1-3158). All three constructs were expressed in soluble form. We find that the CepA1-575 fragment, containing adenylation and peptidyl carrier protein domains (A1-PCP1), specifically adenylates
l
-leucine and
d
-leucine in a 6:1 ratio, and it can be converted to holo form by the phosphopantetheinyltransferase Sfp; also, we find that holo-CepA1-575 can be covalently aminoacylated with
l
-leucine on the peptidyl carrier protein 1 domain. However, no amino acid-dependent adenylation or aminoacylation activity was detected for the larger CepA constructs with
l
-leucine or other expected amino acid substrates, suggesting severe folding problems in the multidomain proteins.
Functional overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis
Authors: Salghetti Simone E. , Muratani Masafumi , Wijnen Herman , Futcher Bruce , Tansey William P.
Published: July 2012

Many transcription factors, particularly those involved in the control of cell growth, are unstable proteins destroyed by ubiquitin-mediated proteolysis. In a previous study of sequences targeting the transcription factor Myc for destruction, we observed that the region in Myc signaling ubiquitin-mediated proteolysis overlaps closely with the region in Myc that activates transcription. Here, we present evidence that the overlap of these two activities is not unique to Myc, but reflects a more general phenomenon. We show that a similar overlap of activation domains and destruction elements occurs in other unstable transcription factors and report a close correlation between the ability of an acidic activation domain to activate transcription and to signal proteolysis. We also show that destruction elements from yeast cyclins, when tethered to a DNA-binding domain, activate transcription. The intimate overlap of activation domains and destruction elements reveals an unexpected convergence of two very different processes and suggests that transcription factors may be destroyed because of their ability to activate transcription.
Large exons encoding multiple ectodomains are a characteristic feature of protocadherin genes
Authors: Wu Qiang , Maniatis Tom
Published: July 2012

Recent studies revealed a striking difference in the genomic organization of classic cadherin genes and one family of “nonclassic cadherin” genes designated protocadherins. Specifically, the DNA sequences encoding the ectodomain repeats of classic cadherins are interrupted by multiple introns. By contrast, all of the encoded ectodomains of each member of the protocadherin gene clusters are present in one large exon. To determine whether large ectodomain exons are a general feature of protocadherin genes we have investigated the genomic organization of several additional human protocadherin genes by using DNA sequence information in GenBank. These genes include
protocadherin 12
(
Pcdh12
), an ortholog of the mouse vascular endothelial cadherin-2 gene;
hFmi1
and
hFmi2
, homologs of the
Drosophila
planar cell polarity gene,
flamingo
;
hFat2
, a homolog of the
Drosophila
tumor suppressor gene
fat
; and the
Drosophila DN-cadherin
and
DE-cadherin
genes. Each of these genes was found to be a member of the protocadherin subfamily, based on amino acid sequence comparisons of their ectodomains. Remarkably, all of these protocadherin genes share a common feature: most of the genomic DNA sequences encoding their ectodomains are not interrupted by an intron. We conclude that the presence of unusually large exons is a characteristic feature of protocadherin genes.
BRCA1 interaction with RNA polymerase II reveals a role for hRPB2 and hRPB10α in activated transcription
Authors: Schlegel Brian P. , Green Victoria J. , Ladias John A. A. , Parvin Jeffrey D.
Published: July 2012

The functions of most of the 12 subunits of the RNA polymerase II (Pol II) enzyme are unknown. In this study, we demonstrate that two of the subunits, hRPB2 and hRPB10α, mediate the regulated stimulation of transcription. We find that the transcriptional coactivator BRCA1 interacts directly with the core Pol II complex
in vitro
. We tested whether single subunits from Pol II would compete with the intact Pol II complex to inhibit transcription stimulated by BRCA1. Excess purified Pol II subunits hRPB2 or hRPB10α blocked BRCA1- and VP16-dependent transcriptional activation
in vitro
with minimal effect on basal transcription. No other Pol II subunits tested inhibited activated transcription in these assays. Furthermore, hRPB10α, but not hRPB2, blocked Sp1-dependent activation.
Protonation of the β-lactam nitrogen is the trigger event in the catalytic action of class A β-lactamases
Authors: Atanasov Boris P. , Mustafi Devkumar , Makinen Marvin W.
Published: July 2012

The pH dependence of the pK
a
values of all ionizable groups and of the electrostatic potential at grid points corresponding to catalytically important atoms in the active site of TEM-1 β-lactamase has been calculated by a mean-field approach for reaction intermediates modeled on the basis of energy minimized x-ray crystallographic coordinates. By estimating electrostatic contributions to the free energy changes accompanying the conversion of the free enzyme into the acylenzyme reaction intermediate, we found that acid-catalyzed protonation of the β-lactam nitrogen is energetically favored as the initiating event, followed by base-catalyzed nucleophilic attack on the carbonyl carbon of the β-lactam group. N-protonation is catalyzed through a hydrogen-bonded cluster involving the 2-carboxylate group of the substrate, the side chains of S130 and K234, and a solvent molecule. Nucleophilic attack on the carbonyl carbon is carried out by the side chain of S70 with proton abstraction catalyzed by a water molecule hydrogen-bonded to the side chain of E166. Stabilization of ion pairs in the active site through interactions with distant clusters of charged residues in the enzyme was concluded to be an important driving force of the catalytic mechanism.
Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells
Authors: Persad Sujata , Attwell Sarah , Gray Virginia , Delcommenne Marc , Troussard Armelle , Sanghera Jasbinder , Dedhar Shoukat
Published: July 2012

PTEN
is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. Somatic mutations of
PTEN
are found in a number of human malignancies, and loss of expression, or mutational inactivation of
PTEN,
leads to the constitutive activation of protein kinase B (PKB)/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We recently have demonstrated that the integrin-linked kinase (ILK) can phosphorylate PKB/Akt on Ser-473 in a phosphoinositide phospholipid-dependent manner. We now demonstrate that the activity of ILK is constitutively elevated in a serum- and anchorage-independent manner in PTEN-mutant cells, and transfection of wild-type (WT) PTEN into these cells inhibits ILK activity. Transfection of a kinase-deficient, dominant-negative form of ILK or exposure to a small molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473, but not on Thr-308, in the PTEN-mutant prostate carcinoma cell lines PC-3 and LNCaP. Transfection of dominant-negative ILK and WT PTEN into these cells also results in the inhibition of PKB/Akt kinase activity. Furthermore, dominant-negative ILK or WT PTEN induces G
1
phase cycle arrest and enhanced apoptosis. Together, these data demonstrate a critical role for ILK in PTEN-dependent cell cycle regulation and survival and indicate that inhibition of ILK may be of significant value in PTEN-mutant tumor therapy.
Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow
Authors: Colter David C. , Class Reiner , DiGirolamo Carla M. , Prockop Darwin J.
Published: July 2012

Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm
2
) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 10
9
-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation.
Aneuploidy vs. gene mutation hypothesis of cancer: Recent study claims mutation but is found to support aneuploidy
Authors: Li Ruhong , Sonik Arvind , Stindl Reinhard , Rasnick David , Duesberg Peter
Published: July 2012

For nearly a century, cancer has been blamed on somatic mutation. But it is still unclear whether this mutation is aneuploidy, an abnormal balance of chromosomes, or gene mutation. Despite enormous efforts, the currently popular gene mutation hypothesis has failed to identify cancer-specific mutations with transforming function and cannot explain why cancer occurs only many months to decades after mutation by carcinogens and why solid cancers are aneuploid, although conventional mutation does not depend on karyotype alteration. A recent high-profile publication now claims to have solved these discrepancies with a set of three synthetic mutant genes that “suffices to convert normal human cells into tumorigenic cells.” However, we show here that even this study failed to explain why it took more than “60 population doublings” from the introduction of the first of these genes, a derivative of the tumor antigen of simian virus 40 tumor virus, to generate tumor cells, why the tumor cells were clonal although gene transfer was polyclonal, and above all, why the tumor cells were aneuploid. If aneuploidy is assumed to be the somatic mutation that causes cancer, all these results can be explained. The aneuploidy hypothesis predicts the long latent periods and the clonality on the basis of the following two-stage mechanism: stage one, a carcinogen (or mutant gene) generates aneuploidy; stage two, aneuploidy destabilizes the karyotype and thus initiates an autocatalytic karyotype evolution generating preneoplastic and eventually neoplastic karyotypes. Because the odds are very low that an abnormal karyotype will surpass the viability of a normal diploid cell, the evolution of a neoplastic cell species is slow and thus clonal, which is comparable to conventional evolution of new species.
G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin
Authors: Ogimoto Goichi , Yudowski Guillermo A. , Barker Christopher J. , Köhler Martin , Katz Adrian I. , Féraille Eric , Pedemonte Carlos H. , Berggren Per-Olof , Bertorello Alejandro M.
Published: July 2012

Inhibition of Na+,K+-ATPase (NKA) activity in renal epithelial cells by activation of G protein-coupled receptors is mediated by phosphorylation of the catalytic α-subunit followed by endocytosis of active molecules. We examined whether agonists that counteract this effect do so by dephosphorylation of the α-subunit or by preventing its internalization through a direct interaction with the endocytic network. Oxymetazoline counteracted the action of dopamine on NKA activity, and this effect was achieved not by preventing α-subunit phosphorylation, but by impaired endocytosis of α-subunits into clathrin vesicles and early and late endosomes. Dopamine-induced inhibition of NKA activity and α-subunit endocytosis required the interaction of adaptor protein 2 (AP-2) with the catalytic α-subunit. Phosphorylation of the α-subunit is essential because dopamine failed to promote such interaction in cells lacking the protein kinase C phosphorylation residue (S18A). Confocal microscopy confirmed that oxymetazoline prevents incorporation of NKA molecules into clathrin vesicles by inhibiting the ability of dopamine to recruit clathrin to the plasma membrane. Dopamine decreased the basal levels of inositol hexakisphosphate (InsP6), whereas oxymetazoline prevented this effect. Similar increments (above basal) in the concentration of InsP6induced by oxymetazoline prevented AP-2 binding to the NKA α-subunit in response to dopamine. In conclusion, inhibition of NKA activity can be reversed by preventing its endocytosis without altering the state of α-subunit phosphorylation; increased InsP6in response to G protein-coupled receptor signals blocks the recruitment of AP-2 and thereby clathrin-dependent endocytosis of NKA.
Separation of phylogenetic and functional associations in biological sequences by using the parametric bootstrap
Authors: Wollenberg Kurt R. , Atchley William R.
Published: July 2012

Quantitative analyses of biological sequences generally proceed under the assumption that individual DNA or protein sequence elements vary independently. However, this assumption is not biologically realistic because sequence elements often vary in a concerted manner resulting from common ancestry and structural or functional constraints. We calculated intersite associations among aligned protein sequences by using mutual information. To discriminate associations resulting from common ancestry from those resulting from structural or functional constraints, we used a parametric bootstrap algorithm to construct replicate data sets. These data are expected to have intersite associations resulting solely from phylogeny. By comparing the distribution of our association statistic for the replicate data against that calculated for empirical data, we were able to assign a probability that two sites covaried resulting from structural or functional constraint rather than phylogeny. We tested our method by using an alignment of 237 basic helix–loop–helix (bHLH) protein domains. Comparison of our results against a solved three-dimensional structure confirmed the identification of several sites important to function and structure of the bHLH domain. This analytical procedure has broad utility as a first step in the identification of sites that are important to biological macromolecular structure and function when a solved structure is unavailable.
Reciprocal domain evolution within a transactivator in a restricted sequence space
Authors: Juárez Katy , Flores Humberto , Dávila Sonia , Olvera Leticia , González Víctor , Morett Enrique
Published: July 2012

Although the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the
Sinorhizobium meliloti
NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to
Bradyrhizobium japonicum
NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.
Abnormal development of Purkinje cells and lymphocytes in
Atm
mutant mice
Authors: Borghesani Paul R. , Alt Frederick W. , Bottaro Andrea , Davidson Laurie , Aksoy Saime , Rathbun Gary A. , Roberts Thomas M. , Swat Wojciech , Segal Rosalind A. , Gu Yansong
Published: July 2012

Motor incoordination, immune deficiencies, and an increased risk of cancer are the characteristic features of the hereditary disease ataxia–telangiectasia (A-T), which is caused by mutations in the
ATM
gene. Through gene targeting, we have generated a line of
Atm
mutant mice,
Atm

y/y

mice. In contrast to other
Atm
mutant mice,
Atm

y/y

mice show a lower incidence of thymic lymphoma and survive beyond a few months of age.
Atm

y/y

mice exhibit deficits in motor learning indicative of cerebellar dysfunction. Even though we found no gross cerebellar degeneration in older
Atm

y/y

animals, ectopic and abnormally differentiated Purkinje cells were apparent in mutant mice of all ages. These findings establish that some neuropathological abnormalities seen in A-T patients also are present in
Atm
mutant mice. In addition, we report a previously unrecognized effect of
Atm
deficiency on development or maintenance of CD4
+
8
+
thymocytes. We discuss these findings in the context of the hypothesis that abnormal development of Purkinje cells and lymphocytes contributes to the pathogenesis of A-T.
An imprinted transcript, antisense to
Nesp,
adds complexity to the cluster of imprinted genes at the mouse
Gnas
locus
Authors: Wroe Stephanie F. , Kelsey Gavin , Skinner Judith A. , Bodle Dorothy , Ball Simon T. , Beechey Colin V. , Peters Josephine , Williamson Christine M.
Published: July 2012

The
Gnas
locus in distal mouse chromosome (Chr) 2 is emerging as a complex genomic region. It contains three imprinted genes in the order
Nesp
-
Gnasxl
-
Gnas. Gnas
encodes a G protein α-subunit, and
Nesp
and
Gnasxl
encode proteins of unknown function expressed in neuroendocrine tissue. Together, these genes form a single transcription unit because transcripts of
Nesp
and
Gnasxl
are alternatively spliced onto exon 2 of
Gnas. Nesp
and
Gnasxl
are expressed from opposite parental alleles, with
Nesp
encoding a maternal-specific transcript and
Gnasxl
encoding a paternal-specific transcript. We now identify a further imprinted transcript in this cluster. Reverse transcription–PCR analysis of
Nesp
expression in 15.5-days-postcoitum embryos carrying only maternal or paternal copies of distal Chr 2 revealed an isoform that is exclusively paternally, rather than maternally, expressed. Strand-specific reverse transcription–PCR showed that this form is an antisense transcript. The existence of a paternally expressed antisense transcript was confirmed by Northern blot analysis. The sequence is contiguous with genomic sequence downstream of
Nesp
and encompasses
Nesp
exons 1 and 2 and an intervening intron. We propose that
Nespas
is an additional control element in the imprinting region of mouse distal Chr 2; it adds further complexity to the
Gnas
-imprinted gene cluster.
RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by
Agrobacterium
Authors: Reiss Bernd , Schubert Ingo , Köpchen Kerstin , Wendeler Edelgard , Schell Jeff , Puchta Holger
Published: July 2012

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange
in vivo
. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using
Agrobacterium
-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.
Severe B cell hyperplasia and autoimmune disease in TALL-1 transgenic mice
Authors: Khare Sanjay D. , Sarosi Ildiko , Xia Xing-Zhong , McCabe Susan , Miner Kent , Solovyev Irina , Hawkins Nessa , Kelley Michael , Chang David , Van Gwyneth , Ross Larry , Delaney John , Wang Ling , Lacey David , Boyle William J. , Hsu Hailing
Published: July 2012

TALL-1/Blys/BAFF is a member of the tumor necrosis factor (TNF) ligand superfamily that is functionally involved in B cell proliferation. Here, we describe B cell hyperplasia and autoimmune lupus-like changes in transgenic mice expressing TALL-1 under the control of a β-actin promoter. The TALL-1 transgenic mice showed severe enlargement of spleen, lymph nodes, and Peyer's patches because of an increased number of B220+ cells. The transgenic mice also had hypergammaglobulinemia contributed by elevations of serum IgM, IgG, IgA, and IgE. In addition, a phenotype similar to autoimmune lupus-like disease was also seen in TALL-1 transgenic mice, characterized by the presence of autoantibodies to nuclear antigens and immune complex deposits in the kidney. Prolonged survival and hyperactivity of transgenic B cells may contribute to the autoimmune lupus-like phenotype in these animals. Our studies further confirm TALL-1 as a stimulator of B cells that affect Ig production. Thus, TALL-1 may be a primary mediator in B cell-associated autoimmune diseases.
HIV-1 infection through the CCR5 receptor is blocked by receptor dimerization
Authors: Vila-Coro A. J. , Mellado M. , Martín de Ana A. , Lucas P. , del Real G. , Martínez-A. C. , Rodríguez-Frade J. M.
Published: July 2012

The identification of the chemokine receptors as receptors for HIV-1 has boosted interest in these molecules, raising expectations for the development of new strategies to prevent HIV-1 infection. The discovery that chemokines block HIV-1 replication has focused attention on identifying their mechanism of action. Previous studies concluded that this inhibitory effect may be mediated by steric hindrance or by receptor down-regulation. We have identified a CCR5 receptor-specific mAb that neither competes with the chemokine for binding nor triggers signaling, as measured by Ca2+influx or chemotaxis. The antibody neither triggers receptor down-regulation nor interferes with the R5 JRFL viral strain gp120 binding to CCR5, but blocks HIV-1 replication in bothin vitroassays using peripheral blood mononuclear cells as HIV-1 targets, as well asin vivousing human peripheral blood mononuclear cell-reconstituted SCID (severe combined immunodeficient) mice. Our evidence shows that the anti-CCR5 mAb efficiently prevents HIV-1 infection by inducing receptor dimerization. Chemokine receptor dimerization also is induced by chemokines and is required for their anti-HIV-1 activity. In addition to providing a molecular mechanism through which chemokines block HIV-1 infection, these results illustrate the prospects for developing new tools that possess HIV-1 suppressor activity, but lack the undesired inflammatory side effects of the chemokines.
NF-κB activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-θ
Authors: Coudronniere Nolwenn , Villalba Martin , Englund Nathan , Altman Amnon
Published: July 2012

Protein kinase C-θ (PKCθ) is a Ca2+-independent member of the PKC family that is selectively expressed in skeletal muscle and T lymphocytes and plays an important role in T cell activation. However, the molecular basis for the important functions of PKCθ in T cells and the manner in which it becomes coupled to the T cell receptor-signaling machinery are unknown. We addressed the functional relationship between PKCθ and CD28 costimulation, which plays an essential role in T cell receptor-mediated IL-2 production. Here, we provide evidence that PKCθ is functionally coupled to CD28 costimulation by virtue of its selective ability to activate the CD28RE/activator protein-1 (AP-1) element in the IL-2 gene promoter. First, CD28 costimulation enhanced the membrane translocation and catalytic activation of PKCθ. Second, among several PKC isoforms, PKCθ was the only one capable of activating NF-κB or CD28RE/AP-1 reporters in T cells (but not in 293T cells). Third, wild-type PKCθ synergized with CD28/CD3 signals to activate CD28RE/AP-1. In addition, PKCθ selectively synergized with Tat to activate a CD28RE/AP-1 reporter. Fourth, CD3/CD28-induced CD28RE/AP-1 activation and NF-κB nuclear translocation were blocked by a selective PKCθ inhibitor. Last, PKCθ-mediated activation of the same reporter was inhibited by the proteasome inhibitor MG132 (which blocks IκB degradation) and was found to involve IκB-kinase β. These findings identify a unique PKCθ-mediated pathway for the costimulatory action of CD28, which involves activation of the IκB-kinase β/IκB/NF-κB-signaling cascade.
New studies on the heat resistance of hamster-adapted scrapie agent: Threshold survival after ashing at 600°C suggests an inorganic template of replication
Authors: Brown Paul , Rau Edward H. , Johnson Bruce K. , Bacote Alfred E. , Gibbs Clarence J. , Gajdusek D. Carleton
Published: July 2012

One-gram samples from a pool of crude brain tissue from hamsters
infected with the 263K strain of hamster-adapted scrapie agent were
placed in covered quartz-glass crucibles and exposed for either 5 or 15
min to dry heat at temperatures ranging from 150°C to 1,000°C.
Residual infectivity in the treated samples was assayed by the
intracerebral inoculation of dilution series into healthy weanling
hamsters, which were observed for 10 months; disease transmissions were
verified by Western blot testing for proteinase-resistant protein in
brains from clinically positive hamsters. Unheated control tissue
contained 9.9 log
10
LD
50
/g tissue; after
exposure to 150°C, titers equaled or exceeded 6
log
10
LD
50
/g, and after exposure to 300°C,
titers equaled or exceeded 4 log
10
LD
50
/g.
Exposure to 600°C completely ashed the brain samples, which, when
reconstituted with saline to their original weights, transmitted
disease to 5 of 35 inoculated hamsters. No transmissions occurred after
exposure to 1,000°C. These results suggest that an inorganic
molecular template with a decomposition point near 600°C is capable
of nucleating the biological replication of the scrapie agent.
Apolipoprotein E regulates dietary cholesterol absorption and biliary cholesterol excretion: Studies in C57BL/6 apolipoprotein E knockout mice
Authors: Sehayek Ephraim , Shefer Sarah , Nguyen Lien B. , Ono Jennie G. , Merkel Martin , Breslow Jan L.
Published: July 2012

The present study examined the role of apolipoprotein E (apoE) in the regulation of dietary cholesterol absorption and biliary cholesterol excretion. Increasing dietary cholesterol from 0.02% to 0.5% in C57BL/6 wild-type mice decreased the percentage of dietary cholesterol that is absorbed by 25%, and this decrease was associated with a 2-fold increase in gallbladder biliary cholesterol concentration. In contrast, increasing dietary cholesterol from 0.02% to 0.5% in C57BL/6 apoE knockout mice produced no significant suppression of the percentage dietary cholesterol absorption and increased gallbladder biliary cholesterol concentration only 16%. Whereas in wild-type mice, the increase in dietary cholesterol increased the hepatic excretion of biliary cholesterol 4-fold, there was only a 2-fold increase in apoE knockout mice. On both the low- and the high-cholesterol diets, whole liver and isolated hepatocyte cholesterol content was higher in the apoE knockout mice. These results suggest that, in response to dietary cholesterol, apoE may play a critical role in decreasing the percentage absorption of dietary cholesterol and increasing biliary cholesterol excretion. These observations suggest a mechanism whereby the absence of apoE contributes to the propensity for tissue cholesterol deposition and accelerated atherogenesis in apoE knockout mice.
Transforming growth factor-β
1
hyperexpression in African-American hypertensives: A novel mediator of hypertension and/or target organ damage
Authors: Suthanthiran Manikkam , Li Baogui , Song Jong O. , Ding Ruchuang , Sharma Vijay K. , Schwartz Joseph E. , August Phyllis
Published: July 2012

Hypertension, a remediable risk factor for stroke, cardiovascular disease, and renal failure, affects 50 million individuals in the United States alone. African Americans (blacks) have a higher incidence and prevalence of hypertension and hypertension-associated target organ damage compared with Caucasian Americans (whites). Herein, we explored the hypotheses that transforming growth factor-β
1
(TGF-β
1
) is hyperexpressed in hypertensives compared with normotensives and that TGF-β
1
overexpression is more frequent in blacks compared with whites. These hypotheses were stimulated by our recent demonstration that TGF-β
1
is hyperexpressed in blacks with end-stage renal disease compared with white end-stage renal disease patients and by the biological attributes of TGF-β
1
, which include induction of endothelin-1 expression, stimulation of renin release, and promotion of vascular and renal disease when TGF-β
1
is produced in excess. TGF-β
1
profiles were determined in black and white hypertensive subjects and normotensive controls and included circulating protein concentrations, mRNA steady-state levels, and codon 10 genotype. Our investigation demonstrated that TGF-β
1
protein levels are highest in black hypertensives, and TGF-β
1
protein as well as TGF-β
1
mRNA levels are higher in hypertensives compared with normotensives. The proline allele at codon 10 (Pro
10
) was more frequent in blacks compared with whites, and its presence was associated with higher levels of TGF-β
1
mRNA and protein. Our findings support the idea that TGF-β
1
hyperexpression is a risk factor for hypertension and hypertensive complications and provides a mechanism for the excess burden of hypertension in blacks.
CD8
+
T cell-mediated suppressive activity inhibits HIV-1 after virus entry with kinetics indicating effects on virus gene expression
Authors: Tomaras Georgia D. , Lacey Simon F. , McDanal Charlene B. , Ferrari Guido , Weinhold Kent J. , Greenberg Michael L.
Published: July 2012

Individuals infected with HIV-1 have varying rates of progression to AIDS. Cellular immune responses, comprised of cytolytic and noncytolytic CD8
+
T cell effector functions, are considered important for controlling viremia and maintaining the clinically asymptomatic state. Although there is general agreement regarding CD8
+
T lymphocyte cytotoxic functions, considerable controversy exists over the nature of the noncytolytic antiviral activity of CD8
+
cells. The discovery that RANTES (regulated on activation, normal T cell expressed and secreted), MIP-1α, and MIP-1β (macrophage inflammatory protein 1 α and β) could inhibit HIV-1 replication by blocking viral entry processes led to the notion that these molecules are responsible for the CD8
+
cell suppressive activity. However, T tropic HIV isolates requiring the CXCR4 coreceptor for entry are insensitive to the antiviral effects of these β-chemokines. Using a CXCR4-dependent virus, we determined that the mechanism of CD8
+
T cell-mediated activity did act after viral entry into the host cell. We also define the kinetics of the HIV life cycle in primary activated human CD4
+
-enriched T cells by using an HIV-1 reporter virus system pseudotyped with the CXCR4-dependent HIV-1 envelope gene of NL4-3. Analysis of these kinetic data indicates that CD8
+
T cell-mediated suppressive activity acts at a stage in the viral life cycle after entry and independently of the HIV envelope. Additionally, we show that the antiviral activity targets stages of the virus life cycle correlating with transcription and early proviral gene expression. These findings not only provide a range of possible targets for the CD8
+
T cell-mediated activity but also support the notion that this antiviral activity is multifactorial in nature.
The dynamics of repeated elements: Applications to the epidemiology of tuberculosis
Authors: Tanaka Mark M. , Small Peter M. , Salamon Hugh , Feldman Marcus W.
Published: July 2012

We propose a stepwise mutation model to describe the dynamics of DNA fingerprint variation in
Mycobacterium tuberculosis
. The genome of
M. tuberculosis
carries insertion sequences (IS
6110
) that are relatively stable over time periods of months but have an observable transposition rate over longer time scales. Variability in copy number and genomic location of (IS
6110
) can be harnessed to generate a DNA fingerprint for each strain, by digesting the genome with a restriction enzyme and using a portion of the element as a probe for Southern blots. The number of bands found for a given genome approximates the number of copies of IS
6110
it carries. A large data set of such fingerprints from tuberculosis (TB) cases in San Francisco provides an observed distribution of IS
6110
copy number. Implementation of the model through deterministic and stochastic simulation indicates some general features of IS/TB dynamics. By comparing observations with outcomes of the model, we conclude that the IS/TB system is very heterogeneous and far from equilibrium. We find that the transposition parameters have a much stronger effect than the epidemic parameters on copy number distribution.
Recombinant rabies virus as potential live-viral vaccines for HIV-1
Authors: Schnell Matthias J. , Foley Heather D. , Siler Catherine A. , McGettigan James P. , Dietzschold Bernhard , Pomerantz Roger J.
Published: July 2012

Recombinant, replication-competent rabies virus (RV) vaccine strain-based vectors were developed expressing HIV type I (HIV-1) envelope glycoprotein (gp160) from both a laboratory-adapted (CXCR4-tropic) and a primary (dual-tropic) HIV-1 isolate. An additional transcription stop/start unit within the RV genome was used to express HIV-1 gp160 in addition to the other RV proteins. The HIV-1 gp160 protein was stably and functionally expressed, as indicated by fusion of human T cell lines after infection with the recombinant RVs. Inoculation of mice with the recombinant RVs expressing HIV-1 gp160 induced a strong humoral response directed against the HIV-1 envelope protein after a single boost with recombinant HIV-1 gp120 protein. Moreover, high neutralization titers up to 1:800 against HIV-1 could be detected in the mouse sera. These data indicate that a live recombinant RV, a rhabdovirus, expressing HIV-1 gp160 may serve as an effective vector for an HIV-1 vaccine.
Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of adult nonhuman primate cortex
Authors: Rodriguez Miguel A. , Pesold Christine , Liu Wen S. , Kriho Virginia , Guidotti Alessandro , Pappas George D. , Costa Erminio
Published: July 2012

The expression of telencephalic reelin (Reln) and glutamic acid decarboxylase mRNAs and their respective cognate proteins is down-regulated in postmortem brains of schizophrenia and bipolar disorder patients. To interpret the pathophysiological significance of this finding, immunoelectron microscopic experiments are required, but these cannot be carried out in postmortem human brains. As an alternative, we carried out such experiments in the cortex of rats and nonhuman primates. We found that Reln is expressed predominantly in layer I of both cortices and is localized to bitufted (double-bouquet), horizontal, and multipolar γ-aminobutyric acid-ergic interneurons, which secrete Reln into extracellular matrix. Reln secretion is mediated by a constitutive mechanism that depends on the expression of a specific signal peptide present in the Reln carboxy-terminal domain. Extracellular matrix Reln is found to aggregate in proximity of postsynaptic densities expressed in apical dendrite spines, which include also the α
3
subunit of integrin receptors. Most pyramidal neurons of various cortical layers express the mouse-disabled 1 (Dab1) protein, which, after phosphorylation by a soluble tyrosine kinase, functions as an adapter protein, probably mediating a modulation of cytoskeleton protein expression. We hypothesize that the decrease of neuropil and dendritic spine density reported to exist in the neocortex of psychiatric patients may be related to a down-regulation of Reln–integrin interactions and the consequent decrease of cytoskeleton protein turnover.
GABAρ1/GABA
A
α1 receptor chimeras to study receptor desensitization
Authors: Martínez-Torres Ataúlfo , Demuro Angelo , Miledi Ricardo
Published: July 2012

γ-Aminobutyrate type C (GABA
C
) receptors are ligand-gated ion channels that are expressed preponderantly in the vertebrate retina and are characterized, among other things, by a very low rate of desensitization and resistance to the specific GABA
A
antagonist bicuculline. To examine which structural elements determine the nondesensitizing character of the human homomeric ρ1 receptor, we used a combination of gene chimeras and electrophysiology of receptors expressed in
Xenopus
oocytes. Two chimeric genes were constructed, made up of portions of the ρ1-subunit and of the α1-subunit of the GABA
A
receptor. When expressed in
Xenopus
oocytes, one chimeric gene (ρ1/α1) formed functional homooligomeric receptors that were fully resistant to bicuculline and were blocked by the specific GABA
C
antagonist (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid and by zinc. Moreover, these chimeric receptors had a fast-desensitizing component, even faster than that of heterooligomeric GABA
A
receptors, in striking contrast to the almost nil desensitization of wild-type ρ1 (wt ρ1) receptors. To see whether the fast-desensitizing characteristic of the chimera was determined by the amino acids forming the ion channels, we replaced the second transmembrane segment (TM2) of ρ1 by that of the α1-subunit of GABA
A
. Although the α1-subunit forms fast-desensitizing receptors when coexpressed with other GABA
A
subunits, the sole transfer of the α1TM2 segment to ρ1 was not sufficient to form desensitizing receptors. All this suggests that the slow-desensitizing trait of ρ1 receptors is determined by a combination of several interacting domains along the molecule.