bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental regulation and the role of insulin and insulin receptor in metanephrogenesis
Authors: Liu Zheng Z. , Kumar Anil , Ota Kosuke , Wallner Elisabeth I. , Kanwar Yashpal S.
Published: July 2002

The insulin family of peptides and their receptors influence cellular growth in very early preimplantation embryos. In this study their expression and role in renal organogenesis was investigated. By immunofluorescence microscopy and
in situ
hybridization, insulin receptor (IR) expression was seen in the ureteric bud branches and early nephron precursors in mouse metanephroi harvested at day 13 of gestation. The expression gradually decreased in successive stages of gestation, and it was confined mainly to renal tubules in 1-week-old mice. Similar developmental regulation of the IR and insulin was observed by reverse transcriptase–polymerase chain reaction (RT-PCR) analyses. Addition of insulin into the culture medium at low concentrations, ranging from 40 to 400 ng/ml, induced trophic changes and increased [
3
H]thymidine incorporation in the embryonic renal explants, and inclusion of IR β-subunit-specific antisense oligodeoxynucleotide caused marked dysmorphogenesis and growth retardation of the metanephroi. Specificity of the antisense effect was reflected by immunoprecipitation experiments in which translational blockade of the β subunit of the IR was observed. RT-PCR analyses revealed that the α subunit of the IR was unaffected by the antisense treatment of metanephric explants. Concomitantly,
de novo
synthesis of morphogenetic regulatory extracellular matrix proteins, especially the proteoglycans, was decreased. Gel-shift analyses indicated a failure in the activation of c-
fos
promoter region binding protein(s) by insulin in the antisense oligodeoxynucleotide-treated explants. These studies suggest that insulin and its putative receptor are developmentally regulated in the murine embryonic metanephros, and they play a role in renal organogenesis, possibly by affecting other modulators of morphogenesis—i.e., extracellular matrix proteins and protooncogenes.
Continuous
in vitro
propagation of the malaria parasite
Plasmodium vivax
Authors: Golenda Claudia F. , Li Jun , Rosenberg Ronald
Published: July 2002

The difficulty in controlling
Plasmodium vivax
, the most common cause of human malaria, has been complicated by growing drug resistance. We have established a method to cycle parasite generations in continuous culture using human blood cells. Chesson strain parasites were passaged from owl monkey erythrocytes to human reticulocytes in McCoy’s 5A medium modified with L-glutamine with 25 mM Hepes buffer supplemented with 20% AB
+
human serum. Reticulocytes were separated by differential centrifugation in homologous plasma from the peripheral blood of a hemochromatosis patient. Parasites were grown during each 48-hr cycle in a static candle jar environment until the beginning of schizogony, at about 36–40 hr, when reticulocytes were added and cultures transferred to a shaker for 10–12 hr. The addition of a concentration of 10% reticulocytes resulted in stabilizing parasite densities between 0.28 and 0.57 after cycle 3 and increasing the total number of parasites at least 2-fold with each generational cycle. Cultured parasites successfully infected an owl monkey. The morphology of cultured parasites was typical of
P. vivax
, with highly ameboid trophozoites evident; however, infected erythrocytes were enlarged and distorted on thin film preparations. The species identity of cultivated parasites was confirmed by analysis of the A and C 18S rRNA genes from genomic DNA and expression of only the A gene during erythrocytic asexual growth. The ability to culture
P. vivax
opens new opportunities to develop vaccines, test drugs, and clone parasites for genome sequencing.
The p53 tumor suppressor targets a novel regulator of G protein signaling
Authors: Buckbinder Leonard , Velasco-Miguel Susana , Chen Yan , Xu Ningzhi , Talbott Randy , Gelbert Larry , Gao Jizong , Seizinger Bernd R. , Gutkind J. Silvio , Kley Nikolai
Published: July 2002

Heterotrimeric G proteins transduce multiple growth-factor-receptor-initiated and intracellular signals that may lead to activation of the mitogen-activated or stress-activated protein kinases. Herein we report on the identification of a novel p53 target gene (A28-RGS14) that is induced in response to genotoxic stress and encodes a novel member of a family of regulators of G protein signaling (RGS) proteins with proposed GTPase-activating protein activity. Overexpression of A28-RGS14p protein inhibits both Gi- and Gq-coupled growth-factor-receptor-mediated activation of the mitogen-activated protein kinase signaling pathway in mammalian cells. Thus, through the induction of A28-RGS14, p53 may regulate cellular sensitivity to growth and/or survival factors acting through G protein-coupled receptor pathways.
Integrality of Tate-cycles
Authors: Faltings Gerd
Published: July 2002

We explain a technical result about
p
-adic
cohomology and apply it to the study of Shimura varieties. The
technical result applies to algebraic varieties with torsion-free
cohomology, but for simplicity we only treat abelian varieties.
Feature integration in pattern perception
Authors: Levi Dennis M. , Sharma Vineeta , Klein Stanley A.
Published: July 2002

The human visual system is able to effortlessly integrate local features to form our rich perception of patterns, despite the fact that visual information is discretely sampled by the retina and cortex. By using a novel perturbation technique, we show that the mechanisms by which features are integrated into coherent percepts are scale-invariant and nonlinear (phase and contrast polarity independent). They appear to operate by assigning position labels or “place tags” to each feature. Specifically, in the first series of experiments, we show that the positional tolerance of these place tags in foveal, and peripheral vision is about half the separation of the features, suggesting that the neural mechanisms that bind features into forms are quite robust to topographical jitter. In the second series of experiment, we asked how many stimulus samples are required for pattern identification by human and ideal observers. In human foveal vision, only about half the features are needed for reliable pattern interpolation. In this regard, human vision is quite efficient (ratio of ideal to real ≈ 0.75). Peripheral vision, on the other hand is rather inefficient, requiring more features, suggesting that the stimulus may be relatively underrepresented at the stage of feature integration.
The crystal structure of human interferon β at 2.2-Å resolution
Authors: Karpusas Michael , Nolte Matthias , Benton Christopher B. , Meier Werner , Lipscomb William N. , Goelz Susan
Published: July 2002

Type I interferons (IFNs) are helical cytokines that have diverse biological activities despite the fact that they appear to interact with the same receptor system. To achieve a better understanding of the structural basis for the different activities of α and β IFNs, we have determined the crystal structure of glycosylated human IFN-β at 2.2-Å resolution by molecular replacement. The molecule adopts a fold similar to that of the previously determined structures of murine IFN-β and human IFN-α
2b
but displays several distinct structural features. Like human IFN-α
2b
, human IFN-β contains a zinc-binding site at the interface of the two molecules in the asymmetric unit, raising the question of functional relevance for IFN-β dimers. However, unlike the human IFN-α
2b
dimer, in which homologous surfaces form the interface, human IFN-β dimerizes with contact surfaces from opposite sides of the molecule. The relevance of the structure to the effects of point mutations in IFN-β at specific exposed residues is discussed. A potential role of ligand–ligand interactions in the conformational assembly of IFN receptor components is discussed.
Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: Evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion
Authors: Mehta Anand , Lu Xuanyong , Block Timothy M. , Blumberg Baruch S. , Dwek Raymond A.
Published: July 2002

The role of N-linked glycosylation and glycan trimming in the function of glycoproteins remains a central question in biology. Hepatitis B virus specifies three glycoproteins (L, M, and S) that are derived from alternate translation of the same ORF. All three glycoproteins contain a common N-glycosylation site in the S domain while M possesses an additional N-glycosylation site at its amino terminus. In the presence of
N
-butyl-deoxnojirimycin (an inhibitor of α-glucosidase) virions and the M protein are surprisingly retained. Preliminary evidence suggests that the retained M protein is hyperglucosylated and localized to lysosomal vesicles. In contrast, the S and L proteins are secreted, and their glycosylation state is unaffected by the presence of the inhibitor. Site-directed mutagenesis provides evidence that virion secretion requires the glycosylation sequon in the pre-S2 domain of M. This highlights the potential role of the M protein oligosaccharide as a therapeutic target.
Identification of components of a phosphoinositide signaling pathway in retinal rod outer segments
Authors: Peng Y.-W. , Rhee S. G. , Yu W.-P. , Ho Y.-K. , Schoen T. , Chader G. J. , Yau K.-W.
Published: July 2002

Phototransduction in retinal rods involves a G protein-coupled signaling cascade that leads to cGMP hydrolysis and the closure of cGMP-gated cation channels that are open in darkness, producing a membrane hyperpolarization as the light response. For many years there have also been reports of the presence of a phosphoinositide pathway in the rod outer segment, though its functions and the molecular identities of its components are still unclear. Using immunocytochemistry with antibodies against various phosphoinositide-specific phospholipase C (PLC) isozymes (β1–4, γ1–2, and δ1–2), we have found PLCβ4-like immunoreactivity in rod outer segments. Similar experiments with antibodies against the α-subunits of the G
q
family of G proteins, which are known to activate PLCβ4, have also demonstrated G
α11
-like immunoreactivity in this location. Immunoblots of total proteins from whole retina or partially purified rod outer segments with anti-PLCβ4 and anti-G
α11
antibodies gave, respectively, a single protein band of the expected molecular mass, suggesting specific labelings. The retinal locations of the two proteins were also supported by
in situ
hybridization experiments on mouse retina with probes specific for the corresponding mouse genes. These two proteins, or immunologically identical isoforms, therefore likely mediate the phosphoinositide signaling pathway in the rod outer segment. At present, G
α11
or a G
α11
-like protein represents the only G protein besides transducin (which mediates phototransduction) identified so far in the rod outer segment. Although absent in the outer segment layer, other PLC isoforms as well as G
αq
(another G
q
family member), are present elsewhere in the retina.
Replication synchrony–PCR: A sampling-time-independent assay for replication synchrony in human tissues and tumors
in situ
Authors: De Francesco Laura , Klevecz Robert R.
Published: July 2002

Replication synchrony within a cell population can be demonstrated by pulse-labeling followed by PCR amplification of immunoprecipitated 5-iodo-2′-deoxyuridine (IdUrd)-labeled DNA from cells of otherwise indeterminant kinetic stages. This replication synchrony–PCR approach may be valuable in understanding the dynamics of human normal tissue or solid tumor replication
in situ
where access for repeated sampling is severely limited. IdUrd labeling provides a sampling-time-independent method for assessing the replicative status of a cell population at the time when the label was presented. Using genes whose time of replication in S phase is already known, the presence of a cell in early or late S phase can be determined and a qualitative measure made of replication synchrony in the population. This approach was evaluated in synchronous and random cultures of Ej cells using the early replicating
PGK-1
gene to identify cells in early S phase at the time of labeling and the late replicating factor IX gene to identify cells that were in late S phase. To test the feasibility of clinical application of this technique, human tumor cells from patients with advanced cancers, given IdUrd therapeutically at specified times of the day, were evaluated. In some patients, replication synchrony–PCR provided evidence of parasynchronous DNA replication in tumor cells. This technique could be appended to existing clinical studies in which BrdUrd or IdUrd is being given to patients either diagnostically or therapeutically.
Target-independent cholinergic differentiation in the rat sympathetic nervous system
Authors: Schäfer Martin K.-H. , Schütz Burkhard , Weihe Eberhard , Eiden Lee E.
Published: July 2002

Chemical coding in the sympathetic nervous system involves both noradrenergic and, for a minority of neurons, cholinergic neurotransmission. The expression of the cholinergic phenotype in the developing sympathetic nervous system was examined to determine if coding for cholinergic transmission occurs before or after innervation of peripheral target organs. The vesicular acetylcholine transporter (VAChT) and choline acetyltransferase, the products of the “cholinergic gene locus” determining the cholinergic phenotype, were expressed in principal cells of the paravertebral, but only rarely in prevertebral, sympathetic chains as early as embryonic day 14. A subpopulation of VAChT- and choline acetyltransferase-positive sympathetic ganglion cells persisted throughout development of the stellate and more caudal paravertebral ganglia into anatomically distinct cell groups, and into adulthood. The forepaw eccrine sweat glands, innervated exclusively by the stellate ganglion, received VAChT-positive nerve terminals at least as early as postembryonic day 4, coincident with the development of the sweat glands themselves. These terminals, like the VAChT-positive cell bodies of the developing stellate ganglion, have some noradrenergic traits including expression of tyrosine hydroxylase, but did not express the vesicular monoamine transporter, and are therefore not functionally noradrenergic. Development of the cholinergic phenotype in principal cells of the sympathetic paravertebral ganglia apparently occurs via receipt of instructive cues, or selection, within the sympathetic chain itself or perhaps even during migration of the cells of the neural crest from which the paravertebral ganglia arise.
Insertional editing of nascent mitochondrial RNAs in
Physarum
Authors: Visomirski-Robic Linda M. , Gott Jonatha M.
Published: July 2002

Maturation of
Physarum
mitochondrial RNA involves the highly specific insertion of nonencoded nucleotides at multiple locations. To investigate the mechanism(s) by which this occurs, we previously developed an isolated mitochondrial system in which run-on transcripts are accurately and efficiently edited by nucleotide insertion. Here we show that under limiting concentrations of exogenous nucleotides the mitochondrial RNA polymerases stall, generating a population of nascent RNAs that can be extended upon addition of limiting nucleotide. Several of these RNA species have been characterized and were found to be fully edited, indicating that nascent RNA is a substrate for nucleotide insertion in isolated
Physarum
mitochondria. Remarkably, these RNAs are edited at positions located within 14–22 nucleotides of the polymerase active site, suggesting that insertional editing may be physically or functionally associated with transcription. The absence of unedited RNA in these experiments indicates that large tracts of RNA downstream of editing sites are not required for nucleotide addition, and argues that insertional editing in
Physarum
occurs with a 5′ to 3′ polarity. These data also provide strong evidence that insertional editing in
Physarum
is mechanistically distinct from editing in kinetoplastid systems.
DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA
Authors: Sugiyama Tomohiko , New James H. , Kowalczykowski Stephen C.
Published: July 2002

Homologous recombination in
Saccharomyces cerevisiae
depends critically on
RAD52
function.
In vitro
, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither
Escherichia coli
ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.
Autoregulation of the Raf-1 serine/threonine kinase
Authors: Cutler Richard E. , Stephens Robert M. , Saracino Misty R. , Morrison Deborah K.
Published: July 2002

The Raf-1 serine/threonine kinase is a key protein involved in the transmission of many growth and developmental signals. In this report, we show that autoinhibition mediated by the noncatalytic, N-terminal regulatory region of Raf-1 is an important mechanism regulating Raf-1 function. The inhibition of the regulatory region occurs, at least in part, through binding interactions involving the cysteine-rich domain. Events that disrupt this autoinhibition, such as mutation of the cysteine-rich domain or a mutation mimicking an activating phosphorylation event (Y340D), alleviate the repression of the regulatory region and increase Raf-1 activity. Based on the striking similarites between the autoregulation of the serine/threonine kinases protein kinase C, Byr2, and Raf-1, we propose that relief of autorepression and activation at the plasma membrane is an evolutionarily conserved mechanism of kinase regulation.
Molecular dynamics study displays near in-line attack conformations in the hammerhead ribozyme self-cleavage reaction
Authors: Torres Rhonda A. , Bruice Thomas C.
Published: July 2002

We have performed molecular dynamics (MD) calculations by using one of the recently solved crystal structures of a hammerhead ribozyme. By rotating the α, β, γ, δ, ɛ, and ζ torsion angles of the phosphate linkage of residue 17, the nucleobase at the cleavage site was slightly rotated out of the active site toward the solution. Unconstrained MD simulations exceeding 1 ns were performed on this starting structure solvated in water with explicit counter ions and two Mg
2+
ions at the active site. Our results reveal that near attack conformations consistently were formed in the simulation. These near attack conformations are characterized by assumption of the 2′-hydroxyl to a near in-line position for attack on the -O-(PO
2
−
)-O- phosphorous. Also during the time course of the MD study, one Mg
2+
moved immediately to associate with a
pro
-R phosphate oxygen in the conserved core region, and the second Mg
2+
remained associated with the
pro
-R oxygen on the phosphate linkage undergoing hydrolysis. These results are in accord with a one-metal ion mechanism of catalysis and give insight into the possible roles of many of the conserved residues in the ribozyme.
Thermostabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA
Authors: Carninci Piero , Nishiyama Yoko , Westover Arthur , Itoh Masayoshi , Nagaoka Sumiharu , Sasaki Nobuya , Okazaki Yasushi , Muramatsu Masami , Hayashizaki Yoshihide
Published: July 2002

The advent of thermostable enzymes has led to great advances in molecular biology, such as the development of PCR and ligase chain reaction. However, isolation of naturally thermostable enzymes has been restricted to those existing in thermophylic bacteria. Here, we show that the disaccharide trehalose enables enzymes to maintain their normal activity (thermostabilization) or even to increase activity at high temperatures (thermoactivation) at which they are normally inactive. We also demonstrate how enzyme thermoactivation can improve the reverse transcriptase reaction. In fact, thermoactivated reverse transcriptase, which displays full activity even at 60°C, was powerful enough to synthesize full length cDNA without the early termination usually induced by stable secondary structures of mRNA.
Requirement of G
M2
ganglioside activator for phospholipase D activation
Authors: Nakamura Shun-ichi , Akisue Toshihiro , Jinnai Hitoshi , Hitomi Tomohiro , Sarkar Sukumar , Miwa Noriko , Okada Taro , Yoshida Kimihisa , Kuroda Shun’ichi , Kikkawa Ushio , Nishizuka Yasutomi
Published: July 2002

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known G
M2
ganglioside activator whose deficiency results in the AB-variant of G
M2
gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of G
M2
to G
M3
ganglioside that is catalyzed by β-hexosaminidase A. Inversely, G
M2
ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987)
Methods Enzymol.
138, 792–815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.
Inhibition of interferon γ induced interleukin 12 production: A potential mechanism for the anti-inflammatory activities of tumor necrosis factor
Authors: Hodge-Dufour Jennifer , Marino Michael W. , Horton Maureen R. , Jungbluth Achim , Burdick Marie D. , Strieter Robert M. , Noble Paul W. , Hunter Christopher A. , Puré Ellen
Published: July 2002

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) γ production. IFN-γ, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-γ inhibits production of chemokines (macrophage inflammatory proteins MIP-1α and MIP-1β). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-γ priming for production of high levels of IL-12 by macrophagesin vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-γ-induced IL-12 production contributed to the resolution of an inflammatory responsein vivo, the response of TNF+/+and TNF−/−mice injected withCorynebacterium parvumwere compared. TNF−/−mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF+/+mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold inC. parvum-treated TNF−/−mice compared with TNF+/+mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response toC. parvumin TNF−/−mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responsesin vivoinvolves its capacity to regulate macrophage IL-12 production. IFN-γ inhibition of chemokine production and inhibition of IFN-γ-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).
An artificial cell-cycle inhibitor isolated from a combinatorial library
Authors: Cohen Barak A. , Colas Pierre , Brent Roger
Published: July 2002

Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21
Cip1
, which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G
1
phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G
1
-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.
Point mutations in segment I-S6 render voltage-gated Na
+
channels resistant to batrachotoxin
Authors: Wang Sho-Ya , Wang Ging Kuo
Published: July 2002

Batrachotoxin (BTX) is a steroidal alkaloid that causes Na
+
channels to open persistently. This toxin has been used widely as a tool for studying Na
+
channel gating processes and for estimating Na
+
channel density. In this report we used point mutations to identify critical residues involved in BTX binding and to examine if such mutations affect channel gating. We show that a single asparagine → lysine substitution of the rat muscle Na
+
channel α-subunit, μ1-N434K, renders the channel completely insensitive to 5 μM BTX when expressed in mammalian cells. This mutant channel nonetheless displays normal current kinetics with minimal changes in gating properties. Another substitution, μ1-N434A, yields a partial BTX-sensitive mutant. Unlike wild-type currents, the BTX-modified μ1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional. This finding implies that the μ1-N434 residue upon binding with BTX is critical for subsequent changes on gating; alanine at the μ1–434 position apparently diminishes the efficacy of BTX on eliminating Na
+
channel inactivation. Mutants of two adjacent residues, μ1-I433K and μ1-L437K, also were found to exhibit the identical BTX-resistant phenotype. We propose that the μ1-I433, μ1-N434, and μ1-L437 residues in transmembrane segment I-S6 probably form a part of the BTX receptor.
Conditional expression of mitogen-activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-induced apoptosis
Authors: Franklin Christopher C. , Srikanth Sampathkumar , Kraft Andrew S.
Published: July 2002

UV irradiation induces apoptosis in U937 human leukemic cells that is accompanied by the activation of both the stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK) signal transduction pathways. The MAPK phosphatase, MKP-1, is capable of inactivating both SAPK and p38 MAPK
in vivo
. To determine whether MKP-1-mediated inhibition of SAPK and/or p38 MAPK activity provided cytoprotection against UV-induced apoptosis, a U937 cell line conditionally expressing MKP-1 from the human metallothionein IIa promoter was established. Conditional expression of MKP-1 was found to abolish UV-induced SAPK and p38 MAPK activity, and inhibit UV-induced apoptosis as judged by both morphological criteria and DNA fragmentation. MKP-1 was also found to inhibit other biochemical events associated with apoptosis, including activation of caspase-3 and the proteolytic cleavage of the caspase-3 substrate, poly(ADP ribose) polymerase. These findings demonstrate that MKP-1 acts at a site upstream of caspase activation within the apoptotic program. The cytoprotective properties of MKP-1 do not appear to be mediated by its ability to inhibit p38 MAPK because the p38 MAPK specific inhibitor SB203580 had no effect on UV-induced apoptosis in U937 cells. Furthermore, by titrating the level of MKP-1 expression it was found that MKP-1 inhibited UV-induced SAPK activity, DNA fragmentation, and caspase-3 activation in a similar dose-dependent manner. The dual-specificity phosphatase, PAC1, which does not inhibit UV-induced activation of SAPK, did not provide a similar cytoprotection against UV-induced apoptosis. These results are consistent with a model whereby MKP-1 provides cytoprotection against UV-induced apoptosis by inhibiting UV-induced SAPK activity.
Depudecin induces morphological reversion of transformed fibroblasts via the inhibition of histone deacetylase
Authors: Kwon Ho Jeong , Owa Takashi , Hassig Christian A. , Shimada Junichi , Schreiber Stuart L.
Published: July 2002

Depudecin is a fungal metabolite that reverts the rounded phenotype of NIH 3T3 fibroblasts transformed with v-
ras
and v-
src
oncogenes to the flattened phenotype of the nontransformed parental cells. The mechanism of detransformation induced by this agent had not been determined. Here, we demonstrate that depudecin inhibits histone deacetylase (HDAC) activity effectively both
in vivo
and
in vitro
. Depudecin induces similar morphological reversion in v-
ras
transformed NIH 3T3 cells as do other naturally occurring HDAC inhibitors such as trichostatin A or trapoxin. It competitively inhibits the binding of [
3
H]trapoxin
in vitro
and the nuclear binding of a trapoxin–coumarin fluorophore
in vivo
, suggesting that depudecin shares a nuclear binding protein and site on that protein with trapoxin. Furthermore, depudecin induces hyperacetylation of histones in a dose-dependent manner and at concentrations comparable with that required for detransformation. An
in vitro
histone deacetylase assay, using purified recombinant HDAC1, reveals that depudecin inhibits 50% of the enzyme activity at a concentration of 4.7 μM. These results demonstrate that depudecin is a novel HDAC inhibitor and suggest that its ability to induce morphological reversion of transformed cells is the result of its HDAC inhibitory activity.
The Hochschild cohomology problem for von Neumann algebras
Authors: Sinclair Allan M. , Smith Roger R.
Published: July 2002

In 1967, when Kadison and Ringrose began the development of continuous cohomology theory for operator algebras, they conjectured that the cohomology groups

H
n

(M, M),
n
≥ 1, for a von Neumann algebra M, should all be zero. This conjecture, which has important structural implications for von Neumann algebras, has been solved affirmatively in the type
I
,
II
∞
, and
III
cases, leaving open only the type
II
1
case. In this paper, we describe a positive solution when M is type
II
1
and has a Cartan subalgebra and a separable predual.
Natural killer activating receptors trigger interferon γ secretion from T cells and natural killer cells
Authors: Mandelboim Ofer , Kent Sally , Davis Daniel M. , Wilson S. Brian , Okazaki Taku , Jackson Richard , Hafler David , Strominger Jack L.
Published: July 2002

Proliferation of human CD4+ αβ T cells expressing a natural killer cell activating receptor (NKAR) has been shown to be enhanced, particularly in response to low doses of antigen, if the target cells present appropriate human class I major histocompatibility complex (MHC) molecules. Here, we show that NKAR also enhance proliferation and killing of target cells by subsets of CD8+ αβ and CD8+ γδ T cells, as well as by NK cells. Strikingly, interferon γ secretion from all of these types of lymphocytes was markedly increased by interaction of the NKAR with their MHC class I ligands, independently of enhancement of proliferation. Thus, the recognition of class I MHC molecules by NKAR on both T cells and NK cells may provide a regulatory mechanism that affects immune responses through the secretion of interferon γ and possibly other cytokines. It represents a signal for cytokine secretion alternative and/or augmentative to that through the T cell receptor.
Characterization of differentially expressed genes in purified
Drosophila
follicle cells: Toward a general strategy for cell type-specific developmental analysis
Authors: Bryant Zev , Subrahmanyan Lakshman , Tworoger Michael , LaTray Leah , Liu Chun-Rong , Li Meng-Jin , van den Engh Ger , Ruohola-Baker Hannele
Published: July 2002

Axis formation in
Drosophila
depends on correct patterning of the follicular epithelium and on signaling between the germ line and soma during oogenesis. We describe a method for identifying genes expressed in the follicle cells with potential roles in axis formation. Follicle cells are purified from whole ovaries by enzymatic digestion, filtration, and fluorescence-activated cell sorting (FACS). Two strategies are used to obtain complementary cell groups. In the first strategy, spatially restricted subpopulations are marked for FACS selection using a green fluorescent protein (GFP) reporter. In the second, cells are purified from animals mutant for the epidermal growth factor receptor ligand
gurken
(
grk
) and from their wild-type siblings. cDNA from these samples of spatially restricted or genetically mutant follicle cells is used in differential expression screens employing PCR-based differential display or hybridization to a cDNA microarray. Positives are confirmed by
in situ
hybridization to whole mounts. These methods are found to be capable of identifying both spatially restricted and
grk
-dependent transcripts. Results from our pilot screens include (
i
) the identification of a homologue of the immunophilin FKBP-12 with dorsal anterior expression in egg chambers, (
ii
) the discovery that the ecdysone-inducible nuclear hormone receptor gene E78 is regulated by
grk
during oogenesis and is required for proper dorsal appendage formation, and (
iii
) the identification of a
Drosophila
homologue of the human SET-binding factor gene
SBF1
with elevated transcription in
grk
mutant egg chambers.
Spc29p is a component of the Spc110p subcomplex and is essential for spindle pole body duplication
Authors: Elliott Sarah , Knop Michael , Schlenstedt Gabriel , Schiebel Elmar
Published: July 2002

In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the γ-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p influences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p/Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of
SPC29
during SPB assembly is indicated by the SPB duplication defect of conditional lethal
spc29(ts)
cells and by the genetic interaction of
SPC29
with
CDC31
and
KAR1
, two genes that are involved in SPB duplication.
A practical method for simultaneously determining the effective burst sizes and cycle times of viruses
Authors: Spouge John L. , Layne Scott P.
Published: July 2002

We describe combined analytic and experimental methods for determining reproductive statistics from time-series data. Our computational methods derive four fundamental measures from laboratory experiments: (
i
) average number of viral daughters; (
ii
) mean viral cycle time; (
iii
) standard deviation of the viral cycling time; and (
iv
) viral doubling time. Taken together, these four reproductive statistics characterize “age-specific fertility,” a quantity that provides complete information on the reproduction of the average viral particle. In this paper, we emphasize applications relating to HIV and experiments for assessing cellular tropism, viral phenotypes, antiviral drugs, humoral immunity, and cytotoxic cellular immunity. Nevertheless, our method is quite flexible and applicable to the evaluation of drugs against bacterial, fungal, and parasitic infections, antineoplastic agents against cancer cells, and perturbations involving pest and wildlife releases in ecosystems.
Recruitment of cyclin T1/P-TEFb to an HIV type 1 long terminal repeat promoter proximal RNA target is both necessary and sufficient for full activation of transcription
Authors: Bieniasz Paul D. , Grdina Therese A. , Bogerd Hal P. , Cullen Bryan R.
Published: July 2002

Transcriptional activation of the HIV type 1 (HIV-1) long terminal repeat (LTR) promoter element by the viral Tat protein is an essential step in the HIV-1 life cycle. Tat function is mediated by the TAR RNA target element encoded within the LTR and is known to require the recruitment of a complex consisting of Tat and the cyclin T1 (CycT1) component of positive transcription elongation factor b (P-TEFb) to TAR. Here, we demonstrate that both TAR and Tat become entirely dispensable for activation of the HIV-1 LTR promoter when CycT1/P-TEFb is artificially recruited to a heterologous promoter proximal RNA target. The level of activation observed was indistinguishable from the level induced by Tat and was neither inhibited nor increased when Tat was expressed in trans. Activation by artificially recruited CycT1 depended on the ability to bind the CDK9 component of P-TEFb. In contrast, although binding to both Tat and TAR was essential for the ability of CycT1 to act as a Tat cofactor, these interactions became dispensable when CycT1 was directly recruited to the LTR. Importantly, activation of the LTR both by Tat and by directly recruited CycT1 was found to be at the level of transcription elongation. Together, these data demonstrate that recruitment of CycT1/P-TEFb to the HIV-1 LTR is fully sufficient to activate this promoter element and imply that the sole role of the Tat/TAR axis in viral transcription is to permit the recruitment of CycT1/P-TEFb.
Immortalization of primary human keratinocytes by the helix–loop–helix protein, Id-1
Authors: Alani Rhoda M. , Hasskarl Jens , Grace Miranda , Hernandez Maria-Clementia , Israel Mark A. , Münger Karl
Published: July 2002

Basic helix–loop–helix (bHLH) DNA-binding proteins have been demonstrated to regulate tissue-specific transcription within multiple cell lineages. The Id family of helix–loop–helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins. Overexpression of Id proteins within a variety of cell types has been shown to inhibit their ability to differentiate under appropriate conditions. We demonstrate that ectopic expression of Id-1 leads to activation of telomerase activity and immortalization of primary human keratinocytes. These immortalized cells have a decreased capacity to differentiate as well as activate phosphorylation of the retinoblastoma protein. Additionally, these cells acquire an impaired p53-mediated DNA-damage response as a late event in immortalization. We conclude that bHLH proteins play a pivotal role in regulating normal keratinocyte growth and differentiation, which can be disrupted by the immortalizing functions of Id-1 through activation of telomerase activity and inactivation of the retinoblastoma protein.
Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in
Arabidopsis thaliana
Authors: Jelesko John G. , Harper Ryan , Furuya Masaki , Gruissem Wilhelm
Published: July 2002

Small, multigene families organized in a tandem array can facilitate the rapid evolution of the gene cluster by a process of meiotic unequal crossing-over. To study this process in a multicellular organism, we created a synthetic
RBCSB
gene cluster in
Arabidopsis thaliana
and used this to measure directly the frequency of meiotic, intergenic unequal crossing-over between sister chromatids. The synthetic
RBCSB
gene cluster was composed of a silent
ΔRBCS1B
∷
LUC
chimeric gene fusion, lacking all 5′ transcription and translation signals, followed by
RBCS2B
and
RBC3B
genomic DNA. Expression of luciferase activity (luc
+
) required a homologous recombination event between the
ΔRBCS1B
∷
LUC
and the
RBCS3B
genes, yielding a novel recombinant
RBCS3B/ 1B
∷
LUC
chimeric gene whose expression was driven by
RBCS3B
5′ transcription and translation signals. Using sensitive, single-photon-imaging equipment, three luc
+
seedlings were identified in more than 1 million F2 seedlings derived from self-fertilized F1 plants hemizygous for the synthetic
RBCSB
gene cluster. The F2 luc
+
seedlings were isolated, and molecular and genetic analysis indicated that the luc
+
trait was caused by the formation of a recombinant chimeric
RBCS3B/1B
∷
LUC
gene. A predicted duplication of the
RBCS2B
gene also was present. The recombination resolution break points mapped adjacent to a region of intron I at which a disjunction in sequence similarity between
RBCS1B
and
RBCS3B
occurs; this provided evidence supporting models of gene cluster evolution by exon-shuffling processes. In contrast to most measures of meiotic unequal crossing-over that require the deletion of a gene in a gene cluster, these results directly measured the frequency of meiotic unequal crossing-over (≈3 × 10
−6
), leading to the expansion of the gene cluster and the formation of a novel recombinant gene.
Solution of the pulse width modulation problem using orthogonal polynomials and Korteweg-de Vries equations
Authors: Chudnovsky D. V. , Chudnovsky G. V.
Published: July 2002

The mathematical underpinning of the pulse width modulation (PWM) technique lies in the attempt to represent “accurately” harmonic waveforms using only square forms of a fixed height. The accuracy can be measured using many norms, but the quality of the approximation of the analog signal (a harmonic form) by a digital one (simple pulses of a fixed high voltage level) requires the elimination of high order harmonics in the error term. The most important practical problem is in “accurate” reproduction of sine-wave using the same number of pulses as the number of high harmonics eliminated. We describe in this paper a complete solution of the PWM problem using Padé approximations, orthogonal polynomials, and solitons. The main result of the paper is the characterization of discrete pulses answering the general PWM problem in terms of the manifold of all rational solutions to Korteweg-de Vries equations.
Structure of the glycosylphosphatidylinositol anchor of an arabinogalactan protein fromPyrus communissuspension-cultured cells
Authors: Oxley David , Bacic Antony
Published: July 2002

Arabinogalactan proteins (AGPs) are proteoglycans of higher plants, which are implicated in growth and development. We recently have shown that two AGPs, NaAGP1 (fromNicotiana alatastyles) and PcAGP1 (fromPyrus communiscell suspension culture), are modified by the addition of a glycosylphosphatidylinositol (GPI) anchor. However, paradoxically, both AGPs were buffer soluble rather than membrane associated. We now show that pear suspension cultured cells also contain membrane-bound GPI-anchored AGPs. This GPI anchor has the minimal core oligosaccharide structure,d-Manα(1–2)-d-Manα(1–6)-d-Manα(1–4)-d-GlcN-inositol, which is consistent with those found in animals, protozoa, and yeast, but with a partial β(1–4)-galactosyl substitution of the 6-linked Man residue, and has a phosphoceramide lipid composed primarily of phytosphingosine and tetracosanoic acid. The secreted form of PcAGP1 contains a truncated GPI lacking the phosphoceramide moiety, suggesting that it is released from the membrane by the action of a phospholipase D. The implications of these findings are discussed in relation to the potential mechanisms by which GPI-anchored AGPs may be involved in signal transduction pathways.
Patterns of nucleotide substitution in
Drosophila
and mammalian genomes
Authors: Petrov Dmitri A. , Hartl Daniel L.
Published: July 2002

To estimate patterns of molecular evolution of unconstrained DNA
sequences, we used maximum parsimony to separate phylogenetic trees of
a non-long terminal repeat retrotransposable element into either
internal branches, representing mainly the constrained evolution of
active lineages, or into terminal branches, representing mainly
nonfunctional “dead-on-arrival” copies that are unconstrained by
selection and evolve as pseudogenes. The pattern of nucleotide
substitutions in unconstrained sequences is expected to be congruent
with the pattern of point mutation. We examined the retrotransposon
Helena
in the
Drosophila virilis
species
group (subgenus
Drosophila
) and the
Drosophila
melanogaster
species subgroup (subgenus
Sophophora
). The patterns of point mutation are
indistinguishable, suggesting considerable stability over evolutionary
time (40–60 million years). The relative frequencies of different
point mutations are unequal, but the “transition bias” results
largely from an ≈2-fold excess of G⋅C to A⋅T substitutions.
Spontaneous mutation is biased toward A⋅T base pairs, with an
expected mutational equilibrium of ≈65% A + T (quite similar to that
of long introns). These data also enable the first detailed comparison
of patterns of point mutations in
Drosophila
and
mammals. Although the patterns are different, all of the statistical
significance comes from a much greater rate of G⋅C to A⋅T
substitution in mammals, probably because of methylated cytosine
“hotspots.” When the G⋅C to A⋅T substitutions are
discounted, the remaining differences are considerably reduced and not
statistically significant.
The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation
Authors: Zhang Jian-Guo , Farley Alison , Nicholson Sandra E. , Willson Tracy A. , Zugaro Lisa M. , Simpson Richard J. , Moritz Robert L. , Cary Dale , Richardson Rachael , Hausmann George , Kile Benjamin T. , Kent Stephen B. H. , Alexander Warren S. , Metcalf Donald , Hilton Douglas J. , Nicola Nicos A. , Baca Manuel
Published: July 2002

The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.