bluuebunny/crossref_metadata_2025 huggingface dataset:
Epstein–Barr virus growth-transformed cells are converted to malignancy following transfection of a 1.3-kb CATR1 antisense construct independent of a change in the level of c-
myc
expression followed by a 8;14 chromosomal translocation
Authors: Li Dawei , Sun Xiao Li , Casto Bruce , Fang Jin , Theil Karl , Glaser Ronald , Milo George
Published: July 2002

The AGLCL Epstein–Barr virus (EBV) growth-transformed cell line is incapable of inducing tumors in nude mice. When the cells were transfected with a 1.3-kb CATR1 antisense cDNA construct, progressively growing lymphomas could be induced in nude mice. Chromosome analysis of the parental, transfected, and tumor cells revealed that a chromosomal translocation t(8;14)(q24.1;q32) had occurred in the transfected cells and was retained in cells derived from tumors. Moreover, enhanced c-
myc
expression, usually associated with this translocation, was either unchanged or under-expressed. These data suggest that the malignant transformation of the EBV growth-transformed cells was independent of c-
myc
expression and suggest that the
CATR1
gene may act synergistically with the chromosomal translocation facilitating the conversion of AGLCL cells from a growth-transformed state to a malignant phenotype.
Gardenification of tropical conserved wildlands: Multitasking, multicropping, and multiusers
Authors: Janzen Daniel
Published: July 2002

Tropical wildlands and their biodiversity will survive in perpetuity only through their integration into human society. One protocol for integration is to explicitly recognize conserved tropical wildlands as wildland gardens. A major way to facilitate the generation of goods and services by a wildland garden is to generate a public-domain Yellow Pages for its organisms. Such a Yellow Pages is part and parcel of high-quality search-and-delivery from wildland gardens. And, as they and their organisms become better understood, they become higher quality biodiversity storage devices than are large freezers. One obstacle to wildland garden survival is that specific goods and services, such as biodiversity prospecting, lack development protocols that automatically shunt the profits back to the source. Other obstacles are that environmental services contracts have the unappealing trait of asking for the payment of environmental credit card bills and implying delegation of centralized governmental authority to decentralized social structures. Many of the potential conflicts associated with wildland gardens may be reduced by recognizing two sets of social rules for perpetuating biodiversity and ecosystems, one set for the wildland garden and one set for the agroscape. In the former, maintaining wildland biodiversity and ecosystem survival in perpetuity through minimally damaging use is paramount, while in the agroscape, wild biodiversity and ecosystems are tools for a healthy and productive agroecosystem, and the loss of much of the original is acceptable.
Atrial natriuretic peptide-stimulated Ca
2+
reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II
Authors: Hoenderop Joost G. J. , Vaandrager Arie B. , Dijkink Lisette , Smolenski Albert , Gambaryan Stepan , Lohmann Suzanne M. , de Jonge Hugo R. , Willems Peter H. G. M. , Bindels René J. M.
Published: July 2002

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca
2+
reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca
2+
uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca
2+
and Na
+
reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca
2+
reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na
+
transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca
2+
transport. The Ca
2+
-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca
2+
transport was observed after expression of the cytosolic cGK Iβ or a membrane-bound cGK II/Iβ chimer. These results demonstrate that ANP- and NO-stimulated Ca
2+
reabsorption requires membrane-targeted cGK II.
Isolation and characterization of a dual-substrate phosphodiesterase gene family: PDE10A
Authors: Soderling Scott H. , Bayuga Sharon J. , Beavo Joseph A.
Published: July 2002

We report here the cloning, expression, and characterization of a dual-substrate, cAMP and cGMP, cyclic nucleotide phosphodiesterase (PDE) from mouse. This PDE contains the consensus sequence for a PDE catalytic domain, but shares <50% sequence identity with the catalytic domains of all other known PDEs and, therefore, represents a new PDE gene family, designated PDE10A. The cDNA for PDE10A is 3,370 nt in length. It includes a full ORF, contains three in-frame stop codons upstream of the first methionine, and is predicted to encode a 779-aa enzyme. At the N terminus PDE10A has two GAF domains homologous to many signaling molecules, including PDE2, PDE5, and PDE6, which likely constitute a low-affinity binding site for cGMP. PDE10A hydrolyzes cAMP with a
K
m
of 0.05 μM and cGMP with a
K
m
of 3 μM. Although PDE10A has a lower
K
m
for cAMP, the
V
max
ratio (cGMP/cAMP) is 4.7. RNA distribution studies indicate that PDE10A is expressed at highest levels in testis and brain.
Developmental shifts and species selection in gastropods
Authors: Duda Thomas F. , Palumbi Stephen R.
Published: July 2002

The fossil record of marine gastropods has been used as evidence to support the operation of species selection; namely, that species with limited dispersal differentially increase in numbers because they are more likely to speciate than widely dispersing species. This conclusion is based on a tacit phylogenetic assumption that increases in species with limited dispersal are solely the result of speciation within monophyletic groups with low dispersal. To test this assumption, we reconstructed a phylogeny from nuclear sequence data for 70 species of the marine gastropod genusConusand used it to map the evolution of developmental mode. All eight species without planktonic life history phases recently and independently evolved this characteristic from ancestors with planktonic larval phases, showing that transitions in developmental mode are common in this group. A simple model of species diversification shows that such shifts can control the relative numbers of species with and without dispersing larval stages, leading to apparent species selection. Such results challenge the conclusion that increases in the number of nonplanktonic species relative to species with planktonic larvae over geologic time is necessarily a result of higher rates of speciation of nonplanktonic lineages and show that demonstration of species selection requires a phylogenetic framework.
Cytomegalovirus-encoded β chemokine promotes monocyte-associated viremia in the host
Authors: Saederup Noah , Lin Yu chun , Dairaghi Daniel J. , Schall Thomas J. , Mocarski Edward S.
Published: July 2002

Chemokine homologs are encoded by many large DNA viruses, suggesting that they contribute to control of host leukocyte transmigration and trafficking during viral infection. Murine cytomegalovirus carries a CC (β) chemokine homolog gene giving rise to two related proteins, murine cytomegalovirus chemokine 1 and 2 (MCK-1 and MCK-2). MCK-1 peptide was found to induce calcium signaling and adherence in murine peritoneal macrophages. Cells bearing human chemokine receptor CCR3 and the human macrophage THP1 cell line were responsive to MCK-1. This pattern suggested that MCK-1 might act as an agonist, promoting leukocyte trafficking during viral infection. Consistent with this prediction, MCK-1/MCK-2 mutant viruses exhibit dramatically reduced peak levels of monocyte-associated viremia in experimentally infected mice. Thus, MCK-1/MCK-2 appears to promote host leukocyte migration to initial sites of infection and may be responsible for attracting monocytes or macrophages that efficiently disseminate virus in the host.
Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes
Authors: Matthews D. A. , Dragovich P. S. , Webber S. E. , Fuhrman S. A. , Patick A. K. , Zalman L. S. , Hendrickson T. F. , Love R. A. , Prins T. J. , Marakovits J. T. , Zhou R. , Tikhe J. , Ford C. E. , Meador J. W. , Ferre R. A. , Brown E. L. , Binford S. L. , Brothers M. A. , DeLisle D. M. , Worland S. T.
Published: July 2002

Human rhinoviruses, the most important etiologic agents of the
common cold, are messenger-active single-stranded monocistronic RNA
viruses that have evolved a highly complex cascade of proteolytic
processing events to control viral gene expression and replication.
Most maturation cleavages within the precursor polyprotein are mediated
by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine
protease with a trypsin-like polypeptide fold. High-resolution crystal
structures of the enzyme from three viral serotypes have been used for
the design and elaboration of 3C protease inhibitors representing
different structural and chemical classes. Inhibitors having
α,β-unsaturated carbonyl groups combined with peptidyl-binding
elements specific for 3C protease undergo a Michael reaction mediated
by nucleophilic addition of the enzyme’s catalytic Cys-147, resulting
in covalent-bond formation and irreversible inactivation of the viral
protease. Direct inhibition of 3C proteolytic activity in virally
infected cells treated with these compounds can be inferred from
dose-dependent accumulations of viral precursor polyproteins as
determined by SDS/PAGE analysis of radiolabeled proteins.
Cocrystal-structure-assisted optimization of 3C-protease-directed
Michael acceptors has yielded molecules having extremely rapid
in vitro
inactivation of the viral protease, potent
antiviral activity against multiple rhinovirus serotypes and low
cellular toxicity. Recently, one compound in this series, AG7088, has
entered clinical trials.
Suppression or induction of apoptosis by opposing pathways downstream from calcium-activated calcineurin
Authors: Lotem Joseph , Kama Rachel , Sachs Leo
Published: July 2002

Ca2+-mobilizing compounds such as the Ca2+ionophore A23187 or the endoplasmic reticulum Ca2+ATPase inhibitor thapsigargin can suppress or induce apoptosis in the same cells. The use of different calcineurin inhibitors has shown that both suppression and induction of apoptosis by the Ca2+-mobilizing compounds were mediated by calcineurin activation. Ca2+-mobilizing compounds activated p38 and p44/42 mitogen-activated protein kinases (MAPKs). Induction of apoptosis by the Ca2+-mobilizing compounds was suppressed by an inhibitor of p38 MAPK but not by an inhibitor of p44/42 MAPK. These MAPK inhibitors did not suppress apoptosis induction by wild-type p53 or by withdrawal of IL-6 from IL-6-dependent cells that are mediated by calcineurin-independent pathways. These MAPK inhibitors also did not affect the ability of Ca2+-mobilizing compounds to suppress apoptosis. The results indicate that (i) Ca2+- mobilizing compounds activate different and opposing pathways that diverge downstream from calcineurin activation that can either suppress or induce apoptosis in the same cells; (ii) p38 MAPK but not p44/42 MAPK is involved in induction of apoptosis but not in its suppression by the Ca2+-mobilizing compounds; and (iii) neither p38 nor p44/42 MAPKs mediate induction of apoptosis by some calcineurin-independent pathways.
Increased levels of plasma lysosomal enzymes in patients with Lowe syndrome
Authors: Ungewickell Alexander J. , Majerus Philip W.
Published: July 2002

Lowe syndrome is an X-linked disorder that has a complex phenotype that includes progressive renal failure and blindness. The disease is caused by mutations in an inositol polyphosphate 5-phosphatase designated OCRL. It has been shown that the OCRL protein is found on the surface of lysosomes and that a renal tubular cell line deficient in OCRL accumulated substrate phosphatidylinositol 4,5-bisphosphate. Because this lipid is required for vesicle trafficking from lysosomes, we postulate that there is a defect in lysosomal enzyme trafficking in patients with Lowe syndrome that leads to increased extracellular lysosomal enzymes and might lead to tissue damage and contribute to the pathogenesis of the disease. We have measured seven lysosomal enzymes in the plasma of 15 patients with Lowe syndrome and 15 age-matched male controls. We find a 1.6- to 2.0-fold increase in all of the enzymes measured. When the data was analyzed by quintiles of activity for all of the enzymes, we found that 95% of values in the lowest quintile come from normal subjects whereas in the highest quintile 85% of the values are from patients with Lowe syndrome. The increased enzyme levels are not attributable to renal insufficiency because there was no difference in enzyme activity in the four patients with the highest creatinine levels compared with the six patients with the lowest creatinine values.
Association of terminal deoxynucleotidyl transferase with Ku
Authors: Mahajan Kiran N. , Gangi-Peterson Lisa , Sorscher David H. , Wang Jingsong , Gathy Karen N. , Mahajan Nupam P. , Reeves Westley H. , Mitchell Beverly S.
Published: July 2002

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein–protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.
Organization of an echinoderm
Hox
gene cluster
Authors: Martinez Pedro , Rast Jonathan P. , Arenas-Mena César , Davidson Eric H.
Published: July 2002

The
Strongylocentrotus purpuratus
genome contains a
single ten-gene
Hox
complex >0.5 megabase in length.
This complex was isolated on overlapping bacterial artificial
chromosome and P1 artificial chromosome genomic recombinants by using
probes for individual genes and by genomic walking. Echinoderm
Hox
genes of Paralog Groups (PG) 1 and 2 are reported.
The cluster includes genes representing all paralog groups of
vertebrate
Hox
clusters, except that there is a single
gene of the PG4–5 types and only three genes of the PG9–12 types. The
echinoderm
Hox
gene cluster is essentially similar to
those of the bilaterally organized chordates, despite the radically
altered pentameral body plans of these animals.
A cluster of oppositely imprinted transcripts at the
Gnas
locus in the distal imprinting region of mouse chromosome 2
Authors: Peters Jo , Wroe Stephanie F. , Wells Christine A. , Miller Howard J. , Bodle Dorothy , Beechey Colin V. , Williamson Christine M. , Kelsey Gavin
Published: July 2002

Imprinted genes tend to occur in clusters. We have identified a cluster in distal mouse chromosome (Chr) 2, known from early genetic studies to contain both maternally and paternally imprinted, but unspecified, genes. Subsequently, one was identified as
Gnas
, which encodes a G protein α subunit, and there is clinical and biochemical evidence that the human homologue
GNAS1
, mutated in patients with Albright hereditary osteodystrophy, is also imprinted. We have used representational difference analysis, based on parent-of-origin methylation differences, to isolate candidate imprinted genes in distal Chr 2 and found two oppositely imprinted genes,
Gnasxl
and
Nesp. Gnasxl
determines a variant G protein α subunit associated with the trans-Golgi network and
Nesp
encodes a secreted protein of neuroendocrine tissues.
Gnasxl
is maternally methylated in genomic DNA and encodes a paternal-specific transcript, whereas
Nesp
is paternally methylated with maternal-specific expression. Their reciprocal imprinting may offer insight into the distal Chr 2 imprinting phenotypes. Remarkably,
Gnasxl
,
Nesp,
and
Gnas
are all part of the same transcription unit; transcripts for
Gnasxl
and
Nesp
are alternatively spliced onto exon 2 of
Gnas
. This demonstrates an imprinting mechanism in which two oppositely imprinted genes share the same downstream exons.
Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion
Authors: Van Goor Fredrick , Krsmanovic Lazar Z. , Catt Kevin J. , Stojilkovic Stanko S.
Published: July 2002

Elevation of cytosolic free Ca
2+
concentration ([Ca
2+
]
i
) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca
2+
influx. Increased [Ca
2+
]
i
stimulates Ca
2+
-sensitive K
+
channels (I
K-Ca
), and this, in turn, hyperpolarizes the cell and inhibits Ca
2+
influx. However, in some cells expressing I
K-Ca
the elevation in [Ca
2+
]
i
by depletion of intracellular stores facilitates voltage-gated Ca
2+
influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca
2+
)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca
2+
]
i
accompanied by transient hyperpolarization, followed by a sustained [Ca
2+
]
i
plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca
2+
]
i
and was mediated by apamin-sensitive I
K-Ca
channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca
2+
]
i
and were not mediated by inhibition of K
+
current, but by facilitation of a voltage-insensitive, Ca
2+
-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca
2+
current. This dual control of pacemaker activity facilitates voltage-gated Ca
2+
influx at elevated [Ca
2+
]
i
levels, but also protects cells from Ca
2+
overload. This process may also provide a general mechanism for the integration of voltage-gated Ca
2+
influx into receptor-controlled Ca
2+
mobilization.
NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone
Authors: Schuler Franz , Yano Takahiro , Di Bernardo Salvatore , Yagi Takao , Yankovskaya Victoria , Singer Thomas P. , Casida John E.
Published: July 2002

The proton-translocating NADH-quinone oxidoreductase (EC1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) inParacoccus denitrificansandThermus thermophilusHB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes ofP. denitrificansandT. thermophilusestablished photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.
Physical evidence for a phosphorylation-dependent conformational change in the enhancer-binding protein NtrC
Authors: Hwang Ingyu , Thorgeirsson Thorgeir , Lee Jonghui , Kustu Sydney , Shin Yeon-Kyun
Published: July 2002

The bacterial enhancer-binding protein nitrogen regulatory protein C (NtrC) activates transcription by σ
54
-containing RNA polymerase in a reaction that depends on ATP hydrolysis. Phosphorylation of an aspartate residue in the N-terminal receiver domain of NtrC induces oligomerization of the protein and activates the ATPase activity, which is a function of its central output domain. To study the role of the receiver domain of NtrC, which is known to act positively, we isolated mutant forms of the protein carrying single cysteine residues and derivatized them with a sulfhydryl-specific nitroxide reagent for electron paramagnetic resonance studies. Single cysteines were placed at four positions at which we had obtained constitutive amino acid substitutions, those that yield activity without phosphorylation. In only one case, derivatized C86 in α-helix 4 of the receiver domain, did the motion of the side chain become dramatically slower upon phosphorylation. Importantly, derivatized NtrC
D86C
(NtrC
D86C*
) activated transcription normally. Additional experiments indicated that the spectral change observed upon phosphorylation of NtrC
D86C*
was due to interdomain interactions rather than a conformational change within the N-terminal domain itself. These interactions did not appear to occur within a monomer. Although it is not clear whether the spectral change seen upon phosphorylation of NtrC
D86C*
is due to an interaction that occurs within a dimer of NtrC or requires the formation of higher-order oligomers, the change indicated that α-helix 4 of the receiver domain probably plays an important role in communication with the remainder of the protein.
Convergent adaptive radiations in Madagascan and Asian ranid frogs reveal covariation between larval and adult traits
Authors: Bossuyt Franky , Milinkovitch Michel C.
Published: July 2002

Recent studies have reported that independent adaptive radiations can lead to identical ecomorphs. Our phylogenetic analyses of nuclear and mitochondrial DNA sequences here indicate that a major radiation of ranid frogs on Madagascar produced morphological, physiological, and developmental characters that are remarkably similar to those that independently evolved on the Indian subcontinent. We demonstrate further that, in several cases, adult and larval stages each evolved sets of characters which are not only convergent between independent lineages, but also allowed both developmental stages to invade the same adaptive zone. It is likely that such covariations are produced by similar selective pressures on independent larval and adult characters rather than by genetic or functional linkage. We briefly discuss why larval/adult covariations might constitute an important evolutionary phenomenon in species for which more than one developmental stage potentially has access to multiple environmental conditions.
Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion
Authors: Steinberger Peter , Andris-Widhopf Jennifer , Bühler Bernd , Torbett Bruce E. , Barbas Carlos F.
Published: July 2002

Studies of naturally occurring polymorphisms of the CCR5 gene have shown that deletion of the functional receptor or reduced expression of the gene can have beneficial effects in preventing HIV-1 infection or delaying disease. Because these polymorphisms are found in otherwise healthy people, strategies that aim to prevent or limit expression of CCR5 should be beneficial in the treatment of HIV-1 disease. To test this approach we have developed a CCR5-specific single-chain antibody that was expressed intracellularly and retained in the endoplasmic reticulum. This CCR5-intrabody efficiently blocked surface expression of human and rhesus CCR5 and thus prevented cellular interactions with CCR5-dependent HIV-1 and simian immunodeficiency virus envelope glycoprotein. Intrabody-expressing cells were shown to be highly refractory to challenge with R5 HIV-1 viruses or infected cells. These results suggest that gene therapy approaches that deliver this intracellular antibody could be of benefit to infected individuals. Because the antibody reacts with a conserved primate epitope on CCR5 this strategy can be tested in nonhuman lentivirus models of HIV-1 disease.
Distinct but overlapping roles of histone acetylase PCAF and of the closely related PCAF-B/GCN5 in mouse embryogenesis
Authors: Yamauchi Teruo , Yamauchi Jun , Kuwata Takeshi , Tamura Tomohiko , Yamashita Tsuyoshi , Bae Nancy , Westphal Heiner , Ozato Keiko , Nakatani Yoshihiro
Published: July 2002

PCAF plays a role in transcriptional activation, cell-cycle arrest,
and cell differentiation in cultured cells. PCAF contributes to
transcriptional activation by acetylating chromatin and transcription
factors through its intrinsic histone acetylase activity. In this
report, we present evidence for the
in vivo
function of
PCAF and the closely related PCAF-B/GCN5. Mice lacking PCAF are
developmentally normal without a distinct phenotype. In
PCAF
null-zygous mice, protein levels of PCAF-B/GCN5
are drastically elevated in lung and liver, where PCAF is abundantly
expressed in wild-type mice, suggesting that PCAF-B/GCN5 functionally
compensates for PCAF. In contrast, animals lacking PCAF-B/GCN5 die
between days 9.5 and 11.5 of gestation. Normally,
PCAF-B
/
GCN5
mRNA is expressed at high
levels already by day 8, whereas
PCAF
mRNA is first
detected on day 12.5, which may explain, in part, the distinct knockout
phenotypes. These results provide evidence that PCAF and PCAF-B/GCN5
play distinct but functionally overlapping roles in embryogenesis.
RelB nuclear translocation regulates B cell MHC molecule, CD40 expression, and antigen-presenting cell function
Authors: O'Sullivan Brendan J. , MacDonald Kelli P. A. , Pettit Allison R. , Thomas Ranjeny
Published: July 2002

Mice with targeted RelB mutations demonstrated an essential role for RelB in immune responses and in myeloid dendritic cell differentiation. Human studies suggested a more global transcriptional role in antigen presentation. Burkitt lymphoma cell lines were used as a model to examine the role of RelB in antigen presentation. After transient transfection of BJAB with RelB, strong nuclear expression of RelB-p50 heterodimers was associated with increased APC function and expression of CD40 and MHC class I. Antisense RelB in DG75 reduced antigen-presenting capacity and CD40-mediated up-regulation of MHC molecules. The data indicate that RelB transcriptional activity directly affects antigen presentation and CD40 synthesis. Stimulation of RelB transcriptional activity may provide a positive feedback loop for facilitating productive APC/T cell interactions.
Rational design of a global inhibitor of the virulence response in
Staphylococcus aureus
, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC
Authors: Lyon Gholson J. , Mayville Patricia , Muir Tom W. , Novick Richard P.
Published: July 2002

Two-component signaling systems involving receptor-histidine
kinases are ubiquitous in bacteria and have been found in yeast and
plants. These systems provide the major means by which bacteria
communicate with each other and the outside world. Remarkably, very
little is known concerning the extracellular ligands that presumably
bind to receptor-histidine kinases to initiate signaling. The
two-component
agr
signaling circuit in
Staphylococcus aureus
is one system where the ligands
are known in chemical detail, thus opening the door for detailed
structure–activity relationship studies. These ligands are short (8-
to 9-aa) peptides containing a thiolactone structure, in which the
α-carboxyl group of the C-terminal amino acid is linked to the
sulfhydryl group of a cysteine, which is always the fifth amino acid
from the C terminus of the peptide. One unique aspect of the
agr
system is that peptides that activate virulence
expression in one group of
S. aureus
strains also
inhibit virulence expression in other groups of
S.
aureus
strains. Herein, it is demonstrated by switching the
receptor-histidine kinase, AgrC, between strains of different
agr
specificity types, that intragroup activation and
intergroup inhibition are both mediated by the same group-specific
receptors. These results have facilitated the development of a global
inhibitor of virulence in
S. aureus
, which consists of a
truncated version of one of the naturally occurring thiolactone
peptides.
Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation
Authors: Yang Hui , Mujtaba Tahimina , Venkatraman Giri , Wu Yuan Yuan , Rao Mahendra S. , Luskin Marla B.
Published: July 2002

Spinal cord neuronal restricted progenitor (NRP) cells, when
transplanted into the neonatal anterior forebrain subventricular zone,
migrate to distinct regions throughout the forebrain including the
olfactory bulb, frontal cortex, and occipital cortex but not to the
hippocampus. Their migration pattern and differentiation potential is
distinct from anterior forebrain subventricular zone NRPs. Irrespective
of their final destination, NRP cells do not differentiate into glia.
Rather they synthesize neurotransmitters, acquire region-specific
phenotypes, and receive synapses from host neurons after
transplantation. Spinal cord NRPs express choline acetyl transferase
even in regions where host neurons do not express this marker. The
restricted distribution of transplanted spinal cord NRP cells and their
acquisition of varied region-specific phenotypes suggest that their
ultimate fate and phenotype is dictated by a combination of intrinsic
properties and extrinsic cues from the host.
Identification and localization of two brefeldin A-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors in a macromolecular complex
Authors: Yamaji Ryoichi , Adamik Ronald , Takeda Kazuyo , Togawa Akira , Pacheco-Rodriguez Gustavo , Ferrans Victor J. , Moss Joel , Vaughan Martha
Published: July 2002

Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain cytosol associated with >670-kDa macromolecular complexes. When observed by immunofluorescence in HeLa S3 and HepG2 cells, endogenous BIG1 and coexpressed BIG2 were distributed in a punctate pattern throughout the cytosol, and also concentrated in the perinuclear region, where endogenous BIG1 and BIG2 each partially colocalized with Golgi-specific 58K protein and γ-adaptin. On Western blot analysis, both BIG1 and BIG2 were clearly more abundant in the cytosol than in the microsomal fractions. After density gradient centrifugation of a microsomal fraction, BIG1 and BIG2 were recovered in the same fraction as β-COP, a marker for Golgi membranes. When cytosol from HeLa S3 cells was subjected to gel filtration and fractions were analyzed by Western blotting, the largest percentages of both BIG1 and BIG2 were detected in fractions containing proteins with a molecular mass of >670 kDa. Western blotting using anti-peptide antibodies specific for BIG1 or BIG2 demonstrated that ≈70% of BIG2 was immunoprecipitated along with 100% of BIG1 by the anti-BIG1 IgG, and ≈75% of BIG1 was coprecipitated with 100% of BIG2 by the anti-BIG2 IgG. All observations were consistent with the conclusion that significant fractions of BIG1 and BIG2 exist as components of the same macromolecular complexes in bovine brain cytosol and are similarly localized in cultured cells.
Requirement of a leucine residue for (apical) membrane expression of type IIb NaPi cotransporters
Authors: Karim-Jimenez Zoubida , Hernando Nati , Biber Jürg , Murer Heini
Published: July 2002

Type II NaPi cotransporters mediate epithelial phosphate (P
i
) reabsorption. In mammals the type IIb protein is expressed in the small intestinal apical membrane and other epithelia; it is not expressed in the renal proximal tubule where we find the type IIa isoform. To look for molecular determinant(s) involved in apical expression of type IIb cotransporters, we have made deletion mutations within the C-terminal tails of mouse IIb (mIIb) and human IIb (hIIb) transporter proteins. The constructs were fused to the enhanced green fluorescent protein and transiently transfected into intestinal CaCo2-cells. Both mIIb and hIIb were located exclusively in the apical membrane of the cells. For mIIb, the removal of a cysteine cluster or the last three amino acids (TVF) had no effect on the location of the protein. However, truncation at the level of the conserved L691/689 prevented the apical membrane expression of both mIIb and hIIb, respectively, and the mutated proteins were located in endosomal and lysosomal structures. A similar expression pattern of the mIIb and hIIb constructs was found in renal proximal tubular opossum kidney cells. Our data suggest that L691/689 is involved in mechanisms leading to an apical expression of type IIb NaPi cotransporters.
The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells
Authors: Salas Pedro J.I. , Rodriguez Marcelo L. , Viciana Ana L. , Vega-Salas Dora E. , Hauri Hans-Peter
Published: July 2002

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+– K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.
Thrombomucin, a Novel Cell Surface Protein that Defines Thrombocytes and Multipotent Hematopoietic Progenitors
Authors: McNagny Kelly M. , Pettersson Inger , Rossi Fabio , Flamme Ingo , Shevchenko Andrej , Mann Matthias , Graf Thomas
Published: July 2002

MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets–transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571–amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein–1, a rabbit cell surface glycoprotein of kidney podocytes.

Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell–specific proteins with possibly overlapping functions in early hematopoietic progenitors.
Titin Extensibility In Situ: Entropic Elasticity of Permanently Folded and Permanently Unfolded Molecular Segments
Authors: Trombitás Karoly , Greaser Marion , Labeit Siegfried , Jin Jian-Ping , Kellermayer Miklós , Helmes Michiel , Granzier Henk
Published: July 2002

Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293–296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (∼2.0 μm) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from ∼2.0 to 2.7 μm, the “contracted” tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond ∼2.7 μm, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112–1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force–SL relation that is not achievable with a single elastic segment.
Interferon Regulatory Factor-2 Is a Transcriptional Activator in Muscle Where It Regulates Expression of Vascular Cell Adhesion Molecule-1
Authors: Jesse Traci L. , LaChance Rhonda , Iademarco Michael F. , Dean Douglas C.
Published: July 2002

Previously, we have suggested that vascular cell adhesion molecule-1 (VCAM-1) and its integrin receptor α4β1 mediate cell–cell interactions important for skeletal myogenesis. Expression of the receptors subsequently subsides in muscle after birth. Here, we examine the mechanism regulating VCAM-1 gene expression in muscle. An enhancer located between the TATA box and the transcriptional start site is responsible for VCAM-1 gene expression in muscle—this element is inactive in endothelial cells where VCAM-1 expression is dependent on nuclear factor κB sites and inflammatory cytokines. We identify interferon regulatory factor-2 (IRF-2), a member of the interferon regulatory factor family, as the enhancer-binding transcription factor and show that expression of IRF-2 parallels that of VCAM-1 during mouse skeletal myogenesis. IRF-2 is not dependent upon cytokines for expression or activity, and it has been shown to act as a repressor in other nonmuscle cell types. We show that the basic repressor motif located near the COOH-terminal of IRF-2 is not active in muscle cells, but instead an acidic region in the center of the molecule functions as a transactivating domain. Although IRF-2 and VCAM-1 expression diminishes on adult muscle fiber, they are retained on myogenic stem cells (satellite cells). These satellite cells proliferate and fuse to regenerate muscle fiber after injury or disease. We present evidence that VCAM-1 on satellite cells mediates their interaction with α4β1(+) leukocytes that invade the muscle after injury or disease. We propose that VCAM-1 on endothelium generally recruits leukocytes to muscle after injury, whereas subsequent interaction with VCAM-1 on regenerating muscle cells focuses the invading leukocytes specifically to the sites of regeneration.
Intracellular Localization of Proteasomal Degradation of a Viral Antigen
Authors: Antón Luis C. , Schubert Ulrich , Bacík Igor , Princiotta Michael F. , Wearsch Pamela A. , Gibbs James , Day Patricia M. , Realini Claudio , Rechsteiner Martin C. , Bennink Jack R. , Yewdell Jonathan W.
Published: July 2002

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I–bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.
Ip-10 Inhibits Epidermal Growth Factor–Induced Motility by Decreasing Epidermal Growth Factor Receptor–Mediated Calpain Activity
Authors: Shiraha Hidenori , Glading Angela , Gupta Kiran , Wells Alan
Published: July 2002

During wound healing, fibroblasts are recruited from the surrounding tissue to accomplish repair. The requisite migration and proliferation of the fibroblasts is promoted by growth factors including those that activate the epidermal growth factor receptor (EGFR). Counterstimulatory factors in wound fluid are postulated to limit this response; among these factors is the ELR-negative CXC chemokine, interferon inducible protein-10 (IP-10). We report here that IP-10 inhibited EGF- and heparin-binding EGF-like growth factor–induced Hs68 human dermal fibroblast motility in a dose-dependent manner (to 52% and 44%, respectively, at 50 ng/ml IP-10), whereas IP-10 had no effect on either basal or EGFR-mediated mitogenesis (96 ± 15% at 50 ng/ml). These data demonstrate for the first time a counterstimulatory effect of IP-10 on a specific induced fibroblast response, EGFR-mediated motility.

To define the molecular basis of this negative transmodulation of EGFR signaling, we found that IP-10 did not adversely impact receptor or immediate postreceptor signaling as determined by tyrosyl phosphorylation of EGFR and two major downstream effectors phospholipase C-γ and erk mitogen-activated protein kinases. Morphological studies suggested which biophysical steps may be affected by demonstrating that IP-10 treatment resulted in an elongated cell morphology reminiscent of failure to detach the uropod; in support of this, IP-10 pretreatment inhibited EGF-induced cell detachment. These data suggested that calpain activity may be involved. The cell permeant agent, calpain inhibitor I, limited EGF-induced motility and de-adhesion similarly to IP-10. IP-10 also prevented EGF- induced calpain activation (reduced by 71 ± 7%). That this inhibition of EGF-induced calpain activity was secondary to IP-10 initiating a cAMP-protein kinase A-calpain cascade is supported by the following evidence: (a) the cell permeant analogue 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) prevented EGF-induced calpain activity and motility; (b) other ELR-negative CXC chemokines, monokine induced by IFN-γ and platelet factor 4 that also generate cAMP, inhibited EGF-induced cell migration and calpain activation; and (c) the protein kinase A inhibitor Rp-8-Br-cAMPS abrogated IP-10 inhibition of cell migration, cell detachment, and calpain activation. Our findings provide a model by which IP-10 suppresses EGF-induced cell motility by inhibiting EGF-induced detachment of the trailing edges of motile cells.