bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular dissection of the mouse interleukin-4 promoter.
Authors: Bruhn K W , Nelms K , Boulay J L , Paul W E , Lenardo M J
Published: May 2006

Understanding the molecular mechanisms regulating the expression of interleukin 4 (IL-4) may shed light on the differentiation of lymphokine-producing phenotypes of CD4+ T cells. We have identified two DNA segments that are necessary for full phorbol 12-myristate 13-acetate (PMA)-induced activity of the IL-4 promoter region in the thymoma cell line EL4. Through deletion and mutation analyses, one of these segments (-57 through -47) was shown to be indispensable for promoter function. We designated this sequence consensus sequence 1 (CS1), as it shares homology with a sequence (ATTTTCCNNTG) that appears five times in the proximal 302-base-pair (bp) region 5' of the gene. We examined CS1 in further detail, as well as a second consensus sequence, CS2, located at nucleotides -75 through -65; both are within a minimal 83-bp construct that expresses full promoter activity. CS1- and CS2-spanning oligonucleotides bound apparently distinct PMA-inducible, sequence-specific factors in mobility-shift assays. Multimer constructs linking CS1- or CS2-spanning oligonucleotides to a heterologous promotor revealed that the CS1 construct had the greater enhancer activity in EL4 cells. Mutating the CS1 sequence within the context of the 302-bp promoter abolished all activity of the promoter, while mutating the CS2 sequence alone had little effect. Furthermore, a CS1 multimer could drive a heterologous promoter in an IL-4-producing [helper T-cell type 2 (TH2-type)] T-cell clone but not in a non-IL-4-producing (TH1-type) clone, suggesting a mechanism by which IL-4 production could be differentially regulated in TH subsets.
Molecular cloning of the human and monkey sperm surface protein PH-20.
Authors: Lin Y , Kimmel L H , Myles D G , Primakoff P
Published: May 2006

The guinea pig sperm surface protein PH-20 has an essential function in sperm adhesion to the zona pellucida of guinea pig eggs. Fully effective contraception has been achieved by immunizing either male or female guinea pigs with purified guinea pig PH-20. Here we report the isolation of human and cynomolgus monkey PH-20 cDNAs as a key step toward testing the function of primate PH-20 and the contraceptive efficacy of PH-20 immunization in primates. The deduced amino acid sequence of human PH-20 has 509 residues and is 59% identical with guinea pig PH-20, suggesting they may have a conserved function and immunogenicity. Southern blots show that there is a single PH-20 gene in the human genome and Northern blots of human testis poly(A)+ RNA show a 2.4-kb message. Northern blots of tissues other than testis are negative for PH-20, indicating that human PH-20 is testis-specific.
Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon gamma production and diminishes interleukin 4 inhibition of such priming.
Authors: Seder R A , Gazzinelli R , Sher A , Paul W E
Published: May 2006

Naive CD4+ T cells produce interleukin 2 (IL-2) but little IL-4 or interferon gamma (IFN-gamma). In vitro, they develop into IL-4 or IFN-gamma producers depending on the conditions of the priming culture. Using T-cell receptor transgenic CD4+ T cells, the role of IL-12 and IL-4 in antigen-specific priming was examined. IL-12 substantially enhanced the ability of naive CD4+ T cells to develop into cells that produced IFN-gamma upon restimulation. However, it was not essential since anti-IL-12 antibodies failed to block the priming for IFN-gamma observed in the absence of exogenous IL-12. When both IL-12 and IL-4 were present in the priming culture, IL-12 did not inhibit priming for IL-4 production. In contrast, IL-4 diminished but did not abolish priming for IFN-gamma production. In an accessory cell-independent priming system, IL-12 strikingly augmented priming for IFN-gamma production, indicating that it acts directly on T cells. IFN-gamma itself did not enhance priming for IFN-gamma production in either accessory cell-dependent or independent systems. In an accessory cell-dependent system, the IL-12-mediated enhancement was not blocked by adding neutralizing anti-IFN-gamma monoclonal antibody. However, in an accessory cell-independent system, anti-IFN-gamma antibody did inhibit priming for IFN-gamma production leaving open a role for IFN-gamma in the priming process. These data indicate that IL-12 has a major effect on the inductive phase of T-cell priming by enhancing commitment to IFN-gamma production and thus can profoundly influence the state of immunity that develops.
On the role of macrophages in anthrax.
Authors: Hanna P C , Acosta D , Collier R J
Published: May 2006

Bacillus anthracis, the causative agent of anthrax, produces systemic shock and death in susceptible animals, primarily through the action of its lethal toxin. This toxin, at high concentrations, induces lysis of macrophages in vitro but shows little or no effect on other cells. We found that when mice were specifically depleted of macrophages by silica injections, they became resistant to the toxin. Sensitivity could be restored by coinjection of toxin-sensitive cultured macrophages (RAW 264.7 cells) but not by coinjection of other cell lines tested. These results implied that macrophages mediate the action of lethal toxin in vivo and led us to investigate their role in death of the mammalian host. Sublytic concentrations of lethal toxin, orders of magnitude lower than those required to induce lysis of RAW 264.7 cells, were found to induce these cells to express interleukin 1 (IL-1) and tumor necrosis factor in vitro. Passive immunization against IL-1 or injection of an IL-1 receptor antagonist protected mice from toxin challenge, whereas anti-tumor necrosis factor provided little, if any, protection. These results imply that systemic shock and death from anthrax result primarily from the effects of high levels of cytokines, principally IL-1, produced by macrophages that have been stimulated by the anthrax lethal toxin.
mu opiate receptor: cDNA cloning and expression.
Authors: Wang J B , Imai Y , Eppler C M , Gregor P , Spivak C E , Uhl G R
Published: May 2006

mu opiate receptors recognize morphine with high affinity. A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described delta and kappa opiate receptor sequences was identified through polymerase chain reaction and cDNA homology approaches. This cDNA recognizes a 10.5-kb mRNA that is expressed in thalamic neurons. COS-cell expression confers naloxonazine-, Na(+)-, and GTP-sensitive binding of mu but not delta or kappa opioid ligands. Expressing cells bind morphine, [D-Ala2,N-methyl-Phe4,glyol5]enkephalin (DAMGO), and [D-Ala2,D-Leu5]enkephalin (DADLE) with nanomolar or subnanomolar affinities, defining a mu opiate receptor that avidly recognizes analgesic and euphoric opiate drugs and opioid peptides.
G-protein alpha o subunit: mutation of conserved cysteines identifies a subunitcontact surface and alters GDP affinity.
Authors: Thomas T C , Schmidt C J , Neer E J
Published: May 2006

The reversible association of alpha and beta gamma subunits of GTP-binding
proteins is important for signal transmission from a variety of cell-surface receptors to intracellular
effectors. Previous work showed that 1,6-bis(maleimido)hexane, which crosslinks cysteine residues, crosslinks
alpha o and alpha i-1 to beta gamma. These crosslinks are likely to form through a conserved cysteine
because 1,6-bis(maleimido)hexane can also crosslink alpha i-2, alpha 1, alpha s and Drosophila alpha
1 to give products of the same apparent molecular weight as crosslinked alpha o beta gamma and alpha
i-1 beta gamma. These proteins have only two cysteines in common. Therefore, we mutated each of the two
conserved cysteines of alpha o to alanines. Mutation of Cys215 prevents crosslinking to beta gamma, but
does not affect binding of guanosine 5'-[gamma-thio]triphosphate or the ability of the mutated alpha
subunit to bind beta gamma. In models of the alpha subunit based on the crystal structure of p21ras,
Cys215 is located on the face opposite to the GTP-binding site and near an area that changes conformation
depending on the nucleotide bound. This surface on the alpha subunit overlaps a putative effector binding
region, raising important questions about the spatial organization of the proteins as they form ternary
complexes. Mutation of Cys325 has no effect on crosslinking but, surprisingly, decreases by a factor
of 10 the affinity of the mutated protein for GDP, relative to wild type, without changing the affinity
for guanosine 5'-[gamma-thio]triphosphate. This mutation falls within a region thought to contact receptors
and may represent a site through which receptors enhance the release of GDP.
An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a subfamily of aldo-keto reductases.
Authors: Ellis E M , Judah D J , Neal G E , Hayes J D
Published: May 2006

Protection of liver against the toxic and carcinogenic effects of aflatoxin B1 (AFB1) can be achieved through the induction of detoxification enzymes by chemoprotectors such as the phenolic antioxidant ethoxyquin. We have cloned and sequenced a cDNA encoding an aldehyde reductase (AFB1-AR), which is expressed in rat liver in response to dietary ethoxyquin. Expression of the cDNA in Escherichia coli and purification of the recombinant enzyme reveals that the protein exhibits aldehyde reductase activity and is capable of converting the protein-binding dialdehyde form of AFB1-dihydrodiol to the nonbinding dialcohol metabolite. We show that the mRNA encoding this enzyme is markedly elevated in the liver of rats fed an ethoxyquin-containing diet, correlating with acquisition of resistance to AFB1. AFB1-AR represents the only carcinogen-metabolizing aldehyde reductase identified to date that is induced by a chemoprotector. Alignment of the amino acid sequence of AFB1-AR with other known and putative aldehyde reductases shows that it defines a subfamily within the aldo-keto reductase superfamily.
Molecular cloning and characterization of the human A3 adenosine receptor.
Authors: Salvatore C A , Jacobson M A , Taylor H E , Linden J , Johnson R G
Published: May 2006

The human A3 adenosine receptor was cloned from a striatal cDNA library using a probe derived from the homologous rat sequence. The cDNA encodes a protein of 318 amino acids and exhibits 72% and 85% overall identity with the rat and sheep A3 adenosine receptor sequences, respectively. Specific and saturable binding of the adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine [125I]ABA was measured on the human A3 receptor stably expressed in Chinese hamster ovary cells with a Kd = 10 nM. The potency order for adenosine receptor agonists was N-ethylcarboxamidoadenosine (NECA) > or = (R)-N6-phenyl-2-propyladenosine [(R)-PIA] > N6-cyclopentyladenosine (CPA) > (S)-N6-phenyl-2-propyladenosine [(S)-PIA]. The human receptor was blocked by xanthine antagonists, most potently by 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propylxanthine (I-ABOPX) with a potency order of I-ABOPX > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amino congener >> 1,3-dipropyl-8-cyclopentylxanthine. Adenosine, NECA, (R)- and (S)-PIA, and CPA inhibited forskolin-stimulated cAMP accumulation by 30-40% in stably transfected cells; I-ABA is a partial agonist. When measured in the presence of antagonists, the dose-response curves of NECA-induced inhibition of forskolin-stimulated cAMP accumulation were right-shifted. Antagonist potencies determined by Schild analyses correlated well with those established by competition for radioligand binding. The A3 adenosine receptor transcript is widespread and, in contrast to the A1, A2a, and A2b transcripts, the most abundant expression is found in the lung and liver. The tissue distribution of A3 mRNA is more similar to the widespread profile found in sheep than to the restricted profile found in the rat. This raises the possibility that numerous physiological effects of adenosine may be mediated by A3 adenosine receptors.
Formation of layer-specific fiber projections to the hippocampus in vitro.
Authors: Frotscher M , Heimrich B
Published: May 2006

The factors determining the layer-specific termination of hippocampal afferents are not known. Previous studies have suggested that the laminated termination of afferent fiber systems is caused by their sequential ingrowth during development. Here we have tested this temporal hypothesis of fiber segregation by an in vitro confrontation system in which the sequential arrival of entorhinal and commissural fibers was reversed. However, despite the temporal reversal of ingrowth, both fiber systems terminated in their normal positions. We conclude that the sequence of fiber ingrowth does not determine the lamination of hippocampal afferents.
The intelligent camera: images of computer vision.
Authors: Grimson W E
Published: May 2006

The field of computer vision is concerned with the problem of deducing properties of object surfaces and the position and orientation of objects in scenes. I illustrate the fundamental difficulty of the problem, describe common approaches taken in the field, and provide representative examples of current solutions.
D-chiro-inositol metabolism in diabetes mellitus.
Authors: Ostlund R E , McGill J B , Herskowitz I , Kipnis D M , Santiago J V , Sherman W R
Published: May 2006

D-chiro-inositol is a rare inositol isomer present in inositol phosphoglycans which are proposed mediators of insulin action. To study D-chiro-inositol metabolism in diabetes mellitus, a sensitive and specific assay was developed using negative-ion chemical ionization gas chromatography/mass spectrometry. Median urinary D-chiro-inositol excretion, which was 2.1 mumol/day in nondiabetics, was substantially increased to 12 mumol/day in non-insulin-dependent diabetes (P < 0.0001) and to 74 mumol/day in insulin-dependent diabetes (P < 0.0001). Urinary D-chiro-inositol was strongly correlated with fasting plasma glucose (r = 0.568, P < 0.0001), glycated hemoglobin (r = 0.529, P < 0.0001), and urinary glucose (r = 0.368, P = 0.01). The renal clearance of D-chiro-inositol was selectively elevated in both non-insulin-dependent and insulin-dependent diabetes when compared with the clearances of L-chiro-inositol or myo-inositol and exceeded the glomerular filtration rate in 71% of the diabetics but in none of the nondiabetics. In poorly controlled diabetic patients insulin treatment reduced urinary D-chiro-inositol losses by 63% and increased plasma levels by 8.8-fold. The metabolism of D-chiro-inositol is abnormal in diabetes and appears to be influenced by short- and long-term metabolic control.
Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages.
Authors: Nunoshiba T , deRojas-Walker T , Wishnok J S , Tannenbaum S R , Demple B
Published: May 2006

Nitric oxide is a free radical (NO) formed biologically through the oxidation of L-arginine by nitric oxide synthases. NO is produced transiently in mammalian cells for intercellular signaling and in copious quantities to cause cytostasis and cytotoxicity. In the latter situation, NO is a deliberate cytotoxic product of activated macrophages, along with other reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide (O2-). Escherichia coli has a complex set of responses to H2O2 and O2- that involves approximately 80 inducible proteins; we wondered whether these bacteria might induce analogous defenses against nitric oxide. We show here that a multigene system controlled by the redox-sensitive transcriptional regulator SoxR is activated by NO in vivo. This induction confers bacterial resistance to activated murine macrophages with kinetics that parallel the production of NO by these cells. Elimination of specific SoxR-regulated genes diminishes the resistance of these bacteria to the cytotoxic macrophages. The required functions include manganese-containing superoxide dismutase, endonuclease IV (a DNA-repair enzyme for oxidative damage), and micF, an antisense regulator of the outer membrane porin OmpF. These results demonstrate that SoxR is a sensor for cellular exposure to NO, and that the soxRS response system may contribute to bacterial virulence.
Uroguanylin: structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase.
Authors: Hamra F K , Forte L R , Eber S L , Pidhorodeckyj N V , Krause W J , Freeman R H , Chin D T , Tompkins J A , Fok K F , Smith C E
Published: May 2006

The intestinal hormone guanylin and bacterial heat-stable enterotoxins (STs) are members of a peptide family that activates intestinal membrane guanylate cyclase. Two different peptides that activate the human intestinal T84 cell guanylate cyclase have been purified from urine and intestinal mucosa of opossums (Didelphis virginiana). The highly acidic peptide, QEDCELCINVACTGC, was named uroguanylin because it was isolated from urine and shares 53% identity with guanylin. A second peptide, SHTCEICAFAACAGC, was purified from urine and intestinal mucosa. This alanine-rich peptide was 47% identical to uroguanylin and 73% identical to human guanylin, suggesting that it may be an opossum homologue of guanylin. Synthetic uroguanylin-(2-15) (i.e., EDCELCINVACTGC) was 10-fold more potent than synthetic rat guanylin, but both peptides were less potent than Escherichia coli ST in the T84 cell cGMP bioassay. Uroguanylin-(2-15) and guanylin inhibited 125I-ST binding to T84 cell receptors in competitive radioligand binding assays. Transepithelial Cl- secretion was stimulated by 1 microM uroguanylin, indicated by an increase in the short circuit current of T84 cells. Thus, uroguanylin is another paracrine hormone in the emerging peptide family that activates intestinal membrane guanylate cyclase. The second peptide may be the opossum form of guanylin, or perhaps, it is still another member of this peptide family. The presence of uroguanylin and guanylin in urine and receptors in proximal tubules suggests that these peptides may also originate from renal tissue and may regulate kidney function.
Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence.
Authors: Jones J I , Gockerman A , Busby W H , Wright G , Clemmons D R
Published: May 2006

Insulin-like growth factor (IGF)-binding protein 1 (IGFBP-1) contains an Arg-Gly-Asp (RGD) integrin recognition sequence. In vitro mutagenesis was used to alter this RGD sequence to Trp-Gly-Asp (WGD). Migration of Chinese hamster ovary (CHO) cells expressing the wild-type protein was more than 3-fold greater in 48 hr compared with cells expressing the WGD mutant form of IGFBP-1. Similarly, wild-type IGFBP-1 added to the media of control CHO cells stimulated migration 2-fold compared with the WGD protein. A synthetic RGD-containing peptide, when added to the medium with wild-type IGFBP-1, blocked the effect of IGFBP-1 on cell migration. The addition of IGF-I to the culture medium had no effect on the migration of cells expressing IGFBP-1 or vector alone. Affinity chromatography of 125I-labeled CHO cell membrane proteins, using IGFBP-1 coupled to agarose, identified the alpha 5 beta 1 integrin (fibronectin receptor) as the only cell surface molecule capable of binding IGFBP-1 in an RGD-dependent manner. Furthermore, wild-type IGFBP-1, but not the WGD mutant form, could be coprecipitated from CHO cells with an antibody directed against the alpha 5 integrin subunit. These studies demonstrate that IGFBP-1 stimulates CHO cell migration and binds to the alpha 5 beta 1 integrin receptor, both by an RGD-dependent mechanism. The effect of IGFBP-1 on migration is independent of IGF-I and is probably mediated through the alpha 5 beta 1 integrin.
Mechanistic aspects of genome-wide demethylation in the preimplantation mouse embryo.
Authors: Kafri T , Gao X , Razin A
Published: May 2006

Gene-specific methylation patterns in mammals play a role in a variety of biological processes in the embryo and adult tissues. These patterns are established during embryo development by a process that involves genome-wide demethylation in the morula and de novo methylation in the pregastrula. To elucidate the mechanism of demethylation in the early mouse embryo, we have injected mouse zygotes with gene sequences that were methylated in vitro by Hpa II methylase and analyzed the methylation status of specific sites in blastocyst DNA. Because it had been propagated in Escherichia coli, the DNA used for these injections was also methylated at adenine residues in GATC sites. This allowed us to eliminate fully methylated, unintegrated DNA by Dpn I digestion and fully unmethylated, integrated DNA that underwent several rounds of replication by Mbo I digestion. The integrated, originally injected DNA strands were in a hemimethylated state and survived this treatment. The methylation status of Hpa II sites in these molecules was analyzed by Hpa II digestion of the genomic DNA isolated from blastocysts, followed by PCR amplification using appropriate primers. The results demonstrate that demethylation is achieved by an active mechanism and that specific sites in imprinted genes escape demethylation, maintaining a methylated state throughout preimplantation development.
Ectopic expression of wild-type or a dominant-negative mutant of transcription factor NTF-1 disrupts normal Drosophila development.
Authors: Attardi L D , Von Seggern D , Tjian R
Published: May 2006

The Drosophila melanogaster tissue-specific transcription factor NTF-1 was originally identified in vitro as a protein that could bind to and activate transcription from the Dopa decarboxylase (Ddc) gene. A structure-function analysis of NTF-1 led to the identification of a discrete amino-terminal activation domain. Here, we report that an NTF-1 mutant lacking the activation domain acts as a trans-dominant inhibitor of NTF-1 activation in tissue culture cells by forming inactive heterodimers with the full-length protein. Ectopically expressing this dominant-negative protein or the full-length protein in developing Drosophila embryos leads to dire developmental consequences. Overexpressing the trans-dominant NTF-1 leads to lethality, while overexpressing full-length NTF-1 results in both lethality and morphogenetic defects. Our results suggest that both the activity and the regulation of NTF-1 are critical for viability and proper development of the fly.
Generation and screening of an oligonucleotide-encoded synthetic peptide library.
Authors: Needels M C , Jones D G , Tate E H , Heinkel G L , Kochersperger L M , Dower W J , Barrett R W , Gallop M A
Published: May 2006

We have prepared a library of approximately 10(6) different peptide sequences on small, spherical (10-microns diameter) beads by the combinatorial chemical coupling of both L- and D-amino acid building blocks. To each bead is covalently attached many copies of a single peptide sequence and, additionally, copies of a unique single-stranded oligonucleotide that codes for that peptide sequence. The oligonucleotide tags are synthesized through a parallel combinatorial procedure that effectively records the process by which the encoded peptide sequence is assembled. The collection of beads was screened for binding to a fluorescently labeled anti-peptide antibody using a fluorescence-activated cell sorting instrument. Those beads to which the antibody bound tightly were isolated by fluorescence-activated sorting, and the oligonucleotide identifiers attached to individual sorted beads were amplified by the PCR. Sequences of the amplified DNAs were determined to reveal the identity of peptide sequences that bound to the antibody with high affinity. By combining the capacity for information storage in an oligonucleotide code with the tremendous level of amplification possible through the PCR, we have devised a means for specifying the identity of each member of a vast library of molecules synthesized from both natural and unnatural chemical building blocks. In addition, we have shown that the use of flow cytometry instrumentation permits facile isolation of individual beads that bear high-affinity ligands for biological receptors.
Cellular epigenetics: effects of passage history on competence of cells for "spontaneous" transformation.
Authors: Rubin H
Published: May 2006

"Spontaneous" neoplastic transformation of cells in culture has been shown to be an adaptive response to moderate physiological constraints such as contact inhibition and lowered concentrations of serum. These are the same constraints that promote normal differentiation of a variety of cell types. Because both normal and neoplastic development represent enduring changes in response to constraint, spontaneous transformation can be considered a form of epigenesis. This phenomenon permits the use of established cell lines in developing general epigenetic principles with all of the advantages of manipulation, efficient cloning, and quantification afforded by such systems. Being environmentally sensitive, however, the responsive capacity of cell lines is subject to fluctuation and long-term modification. I therefore set out to define the conditions used in maintaining cells that influence their competence to undergo transformation with the aim of controlling that competence. It proved to be sensitive to both the population density and frequency of three different repetitive passage regimens. Both the saturation density of the cells and their capacity to produce transformed foci varied initially in different ways within the same regimen in two consecutive passage series starting from the same frozen stock, but both parameters eventually settled down to values characteristic of each of the three regimens. As a result, three sublines were developed of high, intermediate, and low competence for transformation. Each of the sublines has special advantages for studying different aspects of epigenetic change. In the process of developing the sublines, a number of observations were made that reinforce the epigenetic nature of neoplastic transformation and are consistent with the concept of progressive state selection as the basis of the change. That concept can be considered a first step in the formal analysis of epigenesis.
Expression of a Pim-1 transgene accelerates lymphoproliferation and inhibits apoptosis in lpr/lpr mice.
Authors: Möröy T , Grzeschiczek A , Petzold S , Hartmann K U
Published: May 2006

Transgenic mice expressing the Pim-1 kinase are predisposed to develop T-cell lymphomas with a long latency period of about 7-9 months. However, the exact functional basis of the oncogenic activity of Pim-1 remains obscure. C57BL/6 mice homozygous for the lpr mutation develop a well-described lymphoproliferative syndrome at about 26-30 weeks of age. This syndrome is characterized mainly by the accumulation of abnormal T cells in lymph nodes because of the lack of Fas receptor-induced apoptosis. We find that backcross of E mu-Pim-1 transgenics (mice with a transgene that carries the mouse Pim-1 gene under the transcriptional control of the immunoglobulin heavy chain gene enhancer E mu) into lpr/lpr mice results in strong acceleration of lymphoproliferation and dramatic enlargement of lymph nodes. In addition, we show here that cultured lymph node cells from E mu-Pim-1 lpr/lpr mice are rescued from rapid apoptosis that normally occurs in nontransgenic lpr cells in vitro. We also present evidence that CD4+/CD8+ double-positive thymocytes from lpr/lpr mice are sensitive to dexamethasone-induced apoptosis, although lpr/lpr mice lack the Fas receptor. In contrast, E mu-Pim-1 lpr/lpr animals show considerable protection from dexamethasone-induced apoptosis. These results show that Pim-1 can strongly accelerate lymphoproliferation through inhibition of apoptosis and thereby provide first insight into the functional basis for the oncogenic activity of Pim-1.
Role of dimerization in yeast aspartyl-tRNA synthetase and importance of the class II invariant proline.
Authors: Eriani G , Cavarelli J , Martin F , Dirheimer G , Moras D , Gangloff J
Published: May 2006

Cytoplasmic aspartyl-tRNA synthetase (AspRS; EC 6.1.1.12) from yeast is, as are most class II synthetases, an alpha 2 dimer. The only invariant amino acid in signature motif 1 of this class is Pro-273; this residue is located at the dimer interface. To understand the role of Pro-273 in the conserved dimeric configuration, we tested the effect of a Pro-273-->Gly (P273G) substitution on the catalytic properties of homo- and heterodimeric AspRS. Heterodimers of AspRS were produced in vivo by overexpression of their respective subunit variants from plasmid-encoded genes and purified to homogeneity in one HPLC step. The homodimer containing the P273G shows an 80% inactivation of the enzyme and an affinity decrease for its cognate tRNA(Asp) of one order of magnitude. The P273G-mutated subunit recovered wild-type enzymatic properties when associated with a native subunit or a monomer otherwise inactivated having an intact dimeric interface domain. These results, which can be explained by the crystal structure of the native enzyme complexed with its substrates, confirm the structural importance of Pro-273 for dimerization and clearly establish the functional interdependence of the AspRS subunits. More generally, the dimeric conformation may be a structural prerequisite for the activity of mononucleotide binding sites constructed from antiparallel beta strands.
Phosducin-like protein: an ethanol-responsive potential modulator of guanine nucleotide-binding protein function.
Authors: Miles M F , Barhite S , Sganga M , Elliott M
Published: May 2006

Acute and chronic exposure to ethanol produces specific changes in several signal transduction cascades. Such alterations in signaling are thought to be a crucial aspect of the central nervous system's adaptive response, which occurs with chronic exposure to ethanol. We have recently identified and isolated several genes whose expression is specifically induced by ethanol in neural cell cultures. The product of one of these genes has extensive sequence homology to phosducin, a phosphoprotein expressed in retina and pineal gland that modulates trimeric guanine nucleotide-binding protein (G protein) function by binding to G-protein beta gamma subunits. We identified from a rat brain cDNA library an isolate encoding the phosducin-like protein (PhLP), which has 41% identity and 65% amino acid homology to phosducin. PhLP cDNA is expressed in all tissues screened by RNA blot-hybridization analysis and shows marked evolutionary conservation on Southern hybridization. We have identified four forms of PhLP cDNA varying only in their 5' ends, probably due to alternative splicing. This 5'-end variation generates two predicted forms of PhLP protein that differ by 79 aa at the NH2 terminus. Treatment of NG108-15 cells for 24 hr with concentrations of ethanol seen in actively drinking alcoholics (25-100 mM) causes up to a 3-fold increase in PhLP mRNA levels. Induction of PhLP by ethanol could account for at least some of the widespread alterations in signal transduction and G-protein function that are known to occur with chronic exposure to ethanol.
Different perceptual tasks performed with the same visual stimulus attribute activate different regions of the human brain: a positron emission tomography study.
Authors: Dupont P , Orban G A , Vogels R , Bormans G , Nuyts J , Schiepers C , De Roo M , Mortelmans L
Published: May 2006

To investigate the processing of visual form in human cerebral cortex, we used the PET (positron emission tomography) activation technique to compare the human brain regions that are involved in a visual detection task and two orientation discrimination tasks: the temporal same-different (TSD) task, which includes a short-term memory component, and the identification (ID) task, which is without this component. As a control task we used passive viewing. Stimuli were identical in all four tasks. Subtraction of passive viewing from detection showed that the detection task activates early visual cortical regions (areas 17/18) as well as several motor brain regions, while decreasing activity in several higher order frontal, temporal, and parietal regions. Comparing the ID task to the detection task revealed no further visual cortical activation, while comparison of the TSD task to the detection task revealed an activation of several right visual cortical regions, one of which remained significant after the subtraction of ID from TSD (right area 19). These experiments demonstrate the task dependence of visual processing, even for very closely related tasks, and the localization of the temporal comparison component involved in orientation discrimination in human area 19.
Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase.
Authors: Chen R H , Abate C , Blenis J
Published: May 2006

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.
Expression and functional significance of an additional ligand for CTLA-4.
Authors: Lenschow D J , Su G H , Zuckerman L A , Nabavi N , Jellis C L , Gray G S , Miller J , Bluestone J A
Published: May 2006

Effective T-cell activation requires antigen/major histocompatibility complex engagement by the T-cell receptor complex in concert with one or more costimulatory molecules. Recent studies have suggested that the B7 molecule, expressed on most antigen presenting cells, functions as a costimulatory molecule through its interaction with CD28 on T cells. Blocking the CD28/B7 interaction with CTLA4Ig inhibits T-cell activation in vitro and induces unresponsiveness. We demonstrate that another molecule(s), termed B7-2, is expressed constitutively on dendritic cells, is differentially regulated on B cells, and costimulates naive T cells responding to alloantigen. B7-2 is up-regulated by lipopolysaccharide in < 6 hr and is maximally expressed on the majority of B cells by 24 hr. In contrast, B7 is detected only on a subset of activated B cells late (48 hr) after stimulation. In addition, Con A directly induces B7-2 but not B7 expression on B cells. Finally, although both anti-B7 monoclonal antibodies and CTLA4Ig blocked T-cell proliferation to antigen-expressing B7 transfectants, only CTLA4Ig had any significant inhibitory effect on T-cell proliferation to antigens expressed on natural antigen presenting cells, such as dendritic cells. Thus, B7 is not the only costimulatory molecule capable of initiating T-cell responses since a second ligand, B7-2, can provide a necessary second signal for T-cell activation.
Drosophila retinal degeneration A gene encodes an eye-specific diacylglycerol kinase with cysteine-rich zinc-finger motifs and ankyrin repeats.
Authors: Masai I , Okazaki A , Hosoya T , Hotta Y
Published: May 2006

The Drosophila visual mutant, carrying the retinal degeneration A gene (rdgA), has photoreceptor cells that degenerate within a week after eclosion. Morphological studies suggested that this mutant harbors abnormalities in membrane turnover of the photoreceptor cells. Biochemically, the rdgA mutant lacks an eye-specific and membrane-associated diacylglycerol kinase (DGK; EC 2.7.1.107) activity in a gene-dosage-dependent manner, suggesting that rdgA gene encodes a DGK. We report the molecular cloning and characterization of a DGK gene, which maps to the rdgA locus. This gene, designated as DGK2, has a single open reading frame that encodes 1454 amino acids. Like porcine DGK, DGK2 has two cysteine-rich zinc-finger motifs as well as a DGK catalytic domain. The DGK2 protein contains four ankyrin-like repeats at the C-terminal region, suggesting that DGK2 is likely anchored to the membrane or cytoskeleton. Northern blot analysis and tissue in situ hybridization to adult sections revealed that DGK2 is expressed exclusively in the adult retina and that the amount of its mRNA is reduced in some of the rdgA mutant alleles. Furthermore, in two rdgA alleles, rdgA1 and rdgA2, nonsense and missense mutations occur within their DGK2 gene, respectively. Thus, we conclude that rdgA encodes an eye-specific DGK, the absence of which leads to rhabdomere degeneration due to defective phospholipid turnover.
Two motion systems with common and separate pathways for color and luminance.
Authors: Gorea A , Papathomas T V , Kovacs I
Published: May 2006

We present psychological experiments that reveal two motion systems, a specific and an unspecific one. The specific system prevails at medium to high temporal frequencies. It comprises at least two separate motion pathways that are selective for color and for luminance and that do not interact until after the motion signal is extracted separately in each. By contrast, the unspecific system prevails at low temporal frequencies and it combines color and luminance signals at an earlier stage, before motion extraction. The successful implementation of an efficient and accurate technique for assessing equiluminance corroborates further the main findings. These results offer a general framework for understanding the nature of interactions between color and luminance signals in motion perception and suggest that previously proposed dichotomies in motion processing may be encompassed by the specific/unspecific dichotomy proposed here.
Cloning of Nrf1, an NF-E2-related transcription factor, by genetic selection in yeast.
Authors: Chan J Y , Han X L , Kan Y W
Published: May 2006

We have devised a complementation assay in yeast to clone mammalian transcriptional activators and have used it to identify a human basic leucine-zipper transcription factor that we have designated Nrf1 for NF-E2-related factor 1. Nrf1 potentially encodes a 742-aa protein and displays marked homology to the mouse and human NF-E2 transcription factors. Nrf1 activates transcription via NF-E2 binding sites in yeast cells. The ubiquitous expression pattern of Nrf1 and the range of promoters containing the NF-E2 binding motif suggest that this gene may play a role in the regulation of heme synthesis and ferritin genes.
DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations.
Authors: Fynan E F , Webster R G , Fuller D H , Haynes J R , Santoro J C , Robinson H L
Published: May 2006

Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.
Induction of type I interferon genes and interferon-inducible genes in embryonal stem cells devoid of interferon regulatory factor 1.
Authors: Ruffner H , Reis L F , Näf D , Weissmann C
Published: May 2006

Overexpression of interferon regulatory factor 1 (IRF-1) can induce expression of the interferon (IFN) beta gene, at least in certain cells. A role of IRF-1 in the activation of IFN-alpha genes has also been claimed. We have generated embryonal stem cells in which both IRF-1 alleles were disrupted. In undifferentiated embryonal stem cells, virus-induced levels of IFN-alpha RNA were similar for wild-type and IRF-1%, and there was little induction of IFN-beta RNA in either cell type. In 8-day differentiated cells, the levels of virus-induced IFN-beta RNA, but not of IFN-alpha RNA, were about 10-fold higher than in undifferentiated cells and only slightly higher in wild-type than in IRF-1% cells. Thus, although IRF-1 at high levels may elicit or augment induction of IFN genes under certain circumstances, it is not essential for IFN gene induction by virus. Lack of IRF-1 had no effect on the IFN-induced expression levels of the IFN-inducible genes tested; however, there was little or no constitutive expression of (2'-5')oligoadenylate synthetase in IRF-1% embryonal stem cells, in contrast to wild-type cells.
DNA-binding properties and secondary structural model of the hepatocyte nuclear factor 3/fork head domain.
Authors: Li C , Tucker P W
Published: May 2006

An 84-amino acid segment of QRF-1 [glutamine (Q)-rich factor 1], a newly cloned, B-cell-derived DNA-binding protein, shows significant sequence homology with the DNA-binding domains of the hepatocyte nuclear factor 3/fork head family of proteins. Here we demonstrate that this 84-amino acid domain is necessary and sufficient for DNA binding. We also propose a secondary structural model for the domain. At the N-terminal portion of the model, a basic hook structure is followed by two amphipathic helices separated by a turn. Invariant amino acid residues within the two proposed helices form the hydrophobic cores. An aromatic kink and a third amphipathic helix comprise the center of the domain. At the C terminus, two variable-length loops flank a putative 7-amino acid helix followed by a short basic region.
Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen.
Authors: Corey E J , Matsuda S P , Bartel B
Published: May 2006

Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxysqualene mutase activity by thin-layer chromatography. One out of approximately 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis.
Rapid solution assays for retroviral integration reactions and their use in kinetic analyses of wild-type and mutant Rous sarcoma virus integrases.
Authors: Müller B , Jones K S , Merkel G W , Skalka A M
Published: May 2006

A rapid method for quantitating products of the oligodeoxynucleotide processing reaction in vitro has been developed to facilitate enzymatic studies of the retroviral integrases. Unlike earlier procedures, this assay does not depend on polyacrylamide gel electrphoresis but separates products by batch adsorption to PEI-cellulose. A joining assay has also been modified, to facilitate measurement of the two distinct steps in the integration reaction under parallel conditions. Since these methods allow quantitation of numerous samples in a short period of time, they are especially useful for investigation of kinetic parameters and to measure the effects of possible inhibitors of integrase. These assay systems were used to examine the enzymatic activity of wild-type Rous sarcoma virus integrase and selected mutant proteins with substitutions of single conserved amino acids. In contrast to previous studies, reactions were performed under conditions of substrate excess, and rates, rather than yields of product generated after a given period of incubation, were determined. The results showed that substitutions of several highly conserved residues in what is most likely an evolutionarily conserved catalytic domain of the integrases resulted in a 4- to 10-fold decrease in the apparent rate of processing relative to wild type, under optimized standard conditions. Changing an invariant acidic residue reduced the rate by approximately 60-fold. When joining activity was determined, the relative effects of the substitutions tested generally paralleled the results with processing. However, with both wild-type and mutant integrase proteins, the linear phase of the joining reaction was preceded by what appears to be an exponential "burst" phase.
Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.
Authors: Nielsen S , DiGiovanni S R , Christensen E I , Knepper M A , Harris H W
Published: May 2006

Vasopressin (antidiuretic hormone) regulates body water balance by controlling water permeability of the renal collecting ducts. The control mechanisms may involve alterations in the number or unit conductance of water channels in the apical plasma membrane of collecting-duct cells. How this occurs is unknown, but indirect evidence exists for the "shuttle" hypothesis, which states that vasopressin causes exocytic insertion of water channel-laden vesicles from the apical cytosol. To test key aspects of the shuttle hypothesis, we have prepared polyclonal antisera against the recently cloned collecting-duct water channel protein and used the antisera in immunolocalization studies (light and electron microscopic levels) in thin and ultrathin cryosections from rat kidney. Labeling was seen exclusively in collecting-duct principal cells and inner medullary collecting-duct cells. Apical membrane labeling was intense. There was heavy labeling of abundant small subapical vesicles and of membrane structures within multivesicular bodies. In addition, labeling of basolateral plasma membranes in inner medullary collecting ducts was present. Depriving rats of water for 24 or 48 hr markedly increased collecting-duct water-channel protein expression determined by immunoblotting and immunolabeling. These results are compatible with at least two complementary modes of water-channel regulation in collecting-duct cells: (i) control of channel distribution between the apical membrane and a reservoir in subapical vesicles (shuttle hypothesis) and (ii) regulation of the absolute level of expression of water-channel protein.