bluuebunny/crossref_metadata_2025 huggingface dataset:
Signal transduction in macrophages by glycosylphosphatidylinositols ofPlasmodium,Trypanosoma, andLeishmania: Activation of protein tyrosine kinases and protein kinase C by inositolglycan and diacylglycerol moieties
Authors: Tachado Souvenir D. , Gerold Peter , Schwarz Ralph , Novakovic Suzanna , McConville Malcolm , Schofield Louis
Published: July 2002

The perturbation of various glycosylphosphatidylinositol (GPI)-anchored surface proteins imparts profound regulatory signals to macrophages, lymphocytes and other cell types. The specific contribution of the GPI moieties to these events however is unclear. This study demonstrates that purified GPIs ofPlasmodium falciparum,Trypanosoma brucei, andLeishmania mexicanaorigin are sufficient to initiate signal transduction when added alone to host cells as chemically defined agonists. GPIs (10 nM–1 μM) induce rapid activation of the protein tyrosine kinase (PTK) p59hckin macrophages. The minimal structural requirement for PTK activation is the evolutionarily conserved core glycan sequence Manα1-2Manα1-6Manα1-4GlcN1-6myo-inositol. GPI-associated diacylglycerols independently activate the calcium-independent ɛ isoform of protein kinase C. Both signals collaborate in regulating the downstream NF-κB/rel-dependent gene expression of interleukin 1α, tumor necrosis factor (TNF) α, and inducible NO synthase. The alkylacylglycerol-containing iM4 GIPL ofL. mexicana, however, is unable to activate protein kinase C and inhibits TNF expression in response to other agonists, establishing signaling specificity among structurally distinct GPIs. GPI alone appears sufficient to mimic the activities of malaria parasite extracts in the signaling pathway leading to TNF expression. A mAb to GPI blocks TNF induction by parasite extracts indicating that GPI is a necessary agent in this response. As protozoal GPIs are closely related to their mammalian counterparts, the data indicate that GPIs do indeed constitute a novel outside-in signaling system, acting as both agonists and second messenger substrates, and imparting at least two separate signals through the structurally distinct glycan and fatty acid domains. These activities may underlie aspects of pathology and immune regulation in protozoal infections.
Gp91

phox

is the heme binding subunit of the superoxide-generating NADPH oxidase
Authors: Yu Lixin , Quinn Mark T. , Cross Andrew R. , Dinauer Mary C.
Published: July 2002

The phagocyte NADPH oxidase flavocytochrome
b
558
is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91

phox

, and a nonglycosylated subunit, p22

phox

. It contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O
2
), resulting in the generation of superoxide ion (O
2
−
). However, the location of the hemes within the flavocytochrome heterodimer remains controversial. In this study, we have used transgenic COS7 cell lines expressing gp91

phox

, p22

phox

, or both polypeptides to examine the relative role of each flavocytochrome
b
558
subunit in heme binding and O
2
−
formation. A similar membrane localization was observed when gp91

phox

and p22

phox

were either expressed individually or coexpressed, as analyzed by confocal microscopy and immunoblotting of subcellular fractions. Spectral analysis of membranes prepared from COS7 cell lines expressing either gp91

phox

or both gp91

phox

and p22

phox

showed a
b
-type cytochrome with spectral characteristics identical to those of human neutrophil flavocytochrome
b
558
. In contrast, no heme spectrum was detected in wild-type COS7 membranes or those containing only p22

phox

. Furthermore, redox titration studies suggested that two heme groups were contained in gp91

phox

expressed in COS7 membranes, with midpoint potentials of −264 and −233 mV that were very similar to those obtained for neutrophil flavocytochrome
b
558
. These results provide strong support for the hypothesis that gp91

phox

is the sole heme binding subunit of flavocytochrome
b
558
. However, coexpression of gp91

phox

and p22

phox

in COS7 membranes was required to support O
2
−
production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome
b
558
.
A mutant cell line defective in response to double-stranded RNA and in regulating basal expression of interferon-stimulated genes
Authors: Leaman Douglas W. , Salvekar Anupama , Patel Rekha , Sen Ganes C. , Stark George R.
Published: July 2002

Although much progress has been made in identifying the signaling pathways that mediate the initial responses to interferons (IFNs), much less is known about how IFN-stimulated genes (ISGs) are kept quiescent in untreated cells, how the response is sustained after the initial induction, and how ISG expression is down-regulated, even in the continued presence of IFN. We have used the cell sorter to isolate mutant cells with constitutively high ISG expression. A recessive mutant, P2.1, has higher constitutive ISG levels than the parental U4C cells, which do not respond to any IFN. Unexpectedly, P2.1 cells also are deficient in the expression of ISGs in response to double-stranded RNA (dsRNA). Electrophoretic mobility-shift assays revealed that the defect is upstream of the activation of the transcription factors NFκB and IFN regulatory factor 1. Analysis of the pivotal dsRNA-dependent serine/threonine kinase PKR revealed that the wild-type kinase is present and is activated normally in response to dsRNA in P2.1 cells. Together, these data suggest that the defect in P2.1 cells is either downstream of PKR or in a component of a distinct pathway that is involved both in activating multiple transcription factors in response to dsRNA and in regulating the basal expression of ISGs.
Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin
Authors: van de Wal Yvonne , Kooy Yvonne M. C. , van Veelen Peter A. , Peña Salvador A. , Mearin Luisa M. , Molberg Øyvind , Lundin Knut E. A. , Sollid Ludvig M. , Mutis Tuna , Benckhuijsen Willemien E. , Drijfhout Jan Wouter , Koning Frits
Published: July 2002

Celiac disease is a common severe intestinal disease resulting from intolerance to dietary wheat gluten and related proteins. The large majority of patients expresses the HLA-DQ2 and/or DQ8 molecules, and gluten-specific HLA-DQ-restricted T cells have been found at the site of the lesion in the gut. The nature of peptides that are recognized by such T cells, however, has been unclear so far. We now report the identification of a gliadin-derived epitope that dominantly is recognized by intestinal gluten-specific HLA-DQ8-restricted T cells. The characterization of such epitopes is a key step toward the development of strategies to interfere in mechanisms involved in the pathogenesis of celiac disease.
Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A
Authors: Schulman Brenda A. , Lindstrom Derek L. , Harlow Ed
Published: July 2002

An important question in the cell cycle field is how cyclin-dependent kinases (cdks) target their substrates. We have studied the role of a conserved hydrophobic patch on the surface of cyclin A in substrate recognition by cyclin A-cdk2. This hydrophobic patch is ≈35Å away from the active site of cdk2 and contains the MRAIL sequence conserved among a number of mammalian cyclins. In the x-ray structure of cyclin A-cdk2-p27, this hydrophobic patch contacts the RNLFG sequence in p27 that is common to a number of substrates and inhibitors of mammalian cdks. We find that mutation of this hydrophobic patch on cyclin A eliminates binding to proteins containing RXL motifs without affecting binding to cdk2. This docking site is critical for cyclin A-cdk2 phosphorylation of substrates containing RXL motifs, but not for phosphorylation of histone H1. Impaired substrate binding by the cyclin is the cause of the defect in RXL substrate phosphorylation, because phosphorylation can be rescued by restoring a cyclin A–substrate interaction in a heterologous manner. In addition, the conserved hydrophobic patch is important for cyclin A function in cells, contributing to cyclin A’s ability to drive cells out of the G
1
phase of the cell cycle. Thus, we define a mechanism by which cyclins can recruit substrates to cdks, and our results support the notion that a high local concentration of substrate provided by a protein–protein interaction distant from the active site is critical for phosphorylation by cdks.
A modifier screen in the eye reveals control genes for
Krüppel
activity in the
Drosophila
embryo
Authors: Carrera Pilar , Abrell Sarah , Kerber Birgit , Walldorf Uwe , Preiss Anette , Hoch Michael , Jäckle Herbert
Published: July 2002

Irregular facets
(
If
) is a dominant mutation of
Drosophila
that results in small eyes with fused ommatidia. Previous results showed that the gene
Krüppel
(
Kr
), which is best known for its early segmentation function, is expressed ectopically in
If
mutant eye discs. However, it was not known whether ectopic
Kr
activity is either the cause or the result of the
If
mutation. Here, we show that
If
is a gain-of-function allele of
Kr
. We then used the
If
mutation in a genetic screen to identify dominant enhancers and suppressors of
Kr
activity on the third chromosome. Of 30 identified
Kr
-interacting loci, two were cloned, and we examined whether they also represent components of a natural
Kr
-dependent developmental pathway of the embryo. We show that the two genes,
eyelid
(
eld
) and
extramacrochaetae
(
emc
), which encode a Bright family-type DNA binding protein and a helix-loop-helix factor, respectively, are necessary to achieve the singling-out of a unique
Kr
-expressing cell during the development of the Malpighian tubules, the excretory organs of the fly. The results indicate that the
Kr
gain-of-function mutation
If
provides a tool to identify genes that are active during eye development and that a number of them function also in the control of
Kr
-dependent developmental processes.
Active summation of excitatory postsynaptic potentials in hippocampal CA3 pyramidal neurons
Authors: Urban Nathaniel N. , Barrionuevo German
Published: July 2002

The manner in which the thousands of synaptic inputs received by a pyramidal neuron are summed is critical both to our understanding of the computations that may be performed by single neurons and of the codes used by neurons to transmit information. Recent work on pyramidal cell dendrites has shown that subthreshold synaptic inputs are modulated by voltage-dependent channels, raising the possibility that summation of synaptic responses is influenced by the active properties of dendrites. Here, we use somatic and dendritic whole-cell recordings to show that pyramidal cells in hippocampal area CA3 sum distal and proximal excitatory postsynaptic potentials sublinearly and actively, that the degree of nonlinearity depends on the magnitude and timing of the excitatory postsynaptic potentials, and that blockade of transient potassium channels linearizes summation. Nonlinear summation of synaptic inputs could have important implications for the computations performed by single neurons and also for the role of the mossy fiber and perforant path inputs to hippocampal area CA3.
ExoY, an adenylate cyclase secreted by thePseudomonas aeruginosatype III system
Authors: Yahr Timothy L. , Vallis Amy J. , Hancock Michael K. , Barbieri Joseph T. , Frank Dara W.
Published: July 2002

The exoenzyme S regulon is a set of coordinately regulated virulence genes ofPseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases ofBordetella pertussis(CyaA) andBacillus anthracis(EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains ofP. aeruginosaresulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.
Stretched and overwound DNA forms a Pauling-like structure with exposed bases
Authors: Allemand J. F. , Bensimon D. , Lavery R. , Croquette V.
Published: July 2002

We investigate structural transitions within a single stretched and supercoiled DNA molecule. With negative supercoiling, for a stretching force >0.3 pN, we observe the coexistence of B-DNA and denatured DNA from
σ
≈ −0.015 down to
σ
= −1. Surprisingly, for positively supercoiled DNA (
σ
> +0.037) stretched by 3 pN, we observe a similar coexistence of B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that this structure has ≈2.62 bases per turn and an extension 75% larger than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in common with Pauling’s early DNA structure [Pauling, L. & Corey, R. B. (1953),
Proc. Natl. Acad. Sci. USA
39, 84–97] and an unusual structure proposed for the Pf1 bacteriophage [Liu, D. J. & Day, L. A. (1994)
Science
265, 671–674].
Connexins regulate calcium signaling by controlling ATP release
Authors: Cotrina Maria Luisa , Lin Jane H.-C. , Alves-Rodrigues Alexandra , Liu Shujun , Li Jiang , Azmi-Ghadimi Hooman , Kang Jian , Naus Christian C. G. , Nedergaard Maiken
Published: July 2002

Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca
2+
mobilization and was inhibited by Cl
−
channel blockers. Calcium wave propagation also was reduced by purinergic receptor antagonists and by Cl
−
channel blockers but insensitive to gap junction inhibitors. These observations suggest that cell-to-cell signaling associated with connexin expression results from enhanced ATP release and not, as previously believed, from an increase in intercellular coupling.
Expression and regulation of GFRα3, a glial cell line-derived neurotrophic factor family receptor
Authors: Naveilhan Philippe , Baudet Christel , Mikaels Åsa , Shen Liya , Westphal Heiner , Ernfors Patrik
Published: July 2002

We report the identification of an additional member of the glial cell line-derived neurotrophic factor (GDNF) family receptor, termed GFRα3, that is homologous to the previously identified GDNF and neurturin ligand binding receptors GFRα1 and GFRα2. GFRα3 is 32% and 37% identical to GFRα1 and GFRα2, respectively. RNase protection assays show that whereas gfrα1 and gfrα2 are abundant in both developing and adult brain, gfrα3 is exclusively expressed during development. All receptors are widely present in both the developing and adult peripheral nervous system and in peripheral organs. For instance,
in situ
hybridization shows that the developing liver, stomach, intestine, kidney, and sympathetic chain, which all contain ret-expressing cells, transcribe unique complementary and overlapping patterns of most or all of the GDNF family receptors and ligands. In sensory neurons of the trigeminal ganglion gfrα2 and gfrα3 are expressed in different subpopulations of neurons, whereas gfrα1 is coexpressed in some gfrα2 and gfrα3-positive neurons. We find that the gfrα1 population of trigeminal neurons is absent in GDNF null mutant mice, suggesting that GDNF signals
in vivo
by interacting with GFRα1. Thus, our results show that there are at least three members in the GDNF family of ligand binding receptors and that these receptors may be crucial in conferring ligand specificity
in vivo
. The unique complementary and overlapping expression of gfrα3 implies distinct functions in the developing and adult mouse from that of GFRα1 and GFRα2.
Angiotensin-converting enzyme and male fertility
Authors: Hagaman John R. , Moyer Jeffrey S. , Bachman Eric S. , Sibony Mathilde , Magyar Patricia L. , Welch Jeffrey E. , Smithies Oliver , Krege John H. , O’Brien Deborah A.
Published: July 2002

The angiotensin-converting enzyme (ACE; EC3.4.15.1) gene (Ace) encodes both a somatic isozyme found in blood and several other tissues, including the epididymis, and a testis-specific isozyme (testis ACE) found only in developing spermatids and mature sperm. We recently used gene targeting to disrupt the gene coding for both ACE isozymes in mice and reported that male homozygous mutants mate normally but have reduced fertility; the mutant females are fertile. Here we explore the male fertility defect. We demonstrate that ACE is important for achievingin vivofertilization and that sperm from mice lacking both ACE isozymes show defects in transport within the oviducts and in binding to zonae pellucidae. Males generated by gene targeting that lack somatic ACE but retain testis ACE are normally fertile, establishing that somatic ACE in males is not essential for their fertility. Furthermore, male and female mice lacking angiotensinogen have normal fertility, indicating that angiotensin I is not a necessary substrate for testis ACE. Males heterozygous for the mutation inactivating both ACE isozymes sire wild-type and heterozygous offspring at an indistinguishable frequency, indicating no selection against sperm carrying the mutation.
Structural requirements for 5-HT
2A
and 5-HT
1A
serotonin receptor potentiation by the biologically active lipid oleamide
Authors: Boger Dale L. , Patterson Jean E. , Jin Qing
Published: July 2002

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and that has been shown to effect serotonergic receptor responses and block gap junction communication. Herein, the potentiation of the 5-HT
1A
receptor response is disclosed, and a study of the structural features of oleamide required for potentiation of the 5-HT
2A
and 5-HT
1A
response to serotonin (5-HT) is described. Of the naturally occurring fatty acids, the primary amide of oleic acid (oleamide) is the most effective at potentiating the 5-HT
2A
receptor response. The structural features required for activity were found to be highly selective. The presence, position, and stereochemistry of the Δ
9
-
cis
double bond is required, and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides may replace the primary amide but follow a well defined relationship requiring small amide substituents, suggesting that the carboxamide serves as a hydrogen bond acceptor but not donor. Alternative modifications at the carboxamide as well as modifications of the methyl terminus or the hydrocarbon region spanning the carboxamide and double bond typically eliminate activity. A less extensive study of the 5-HT
1A
potentiation revealed that it is more tolerant and accommodates a wider range of structural modifications. An interesting set of analogs was identified that inhibit rather than potentiate the 5-HT
2A
, but not the 5-HT
1A
, receptor response, further suggesting that such analogs may permit the selective modulation of serotonin receptor subtypes and even have opposing effects on the different subtypes.
Severe cardiomyopathy in mice lacking dystrophin and MyoD
Authors: Megeney Lynn A. , Kablar Boris , Perry Robert L. S. , Ying Chuyan , May Linda , Rudnicki Michael A.
Published: July 2002

The
mdx
mouse, a mouse model of Duchenne muscular dystrophy, carries a loss-of-function mutation in dystrophin, a component of the membrane-associated dystrophin–glycoprotein complex. Unlike humans,
mdx
mice rarely display cardiac abnormalities and exhibit dystrophic changes only in a small number of heavily used skeletal muscle groups. By contrast,
mdx:MyoD
−/−
mice lacking dystrophin and the skeletal muscle-specific bHLH transcription factor MyoD display a severe skeletal myopathy leading to widespread dystrophic changes in skeletal muscle and premature death around 1 year of age. The severely increased phenotype of
mdx:MyoD
−/−
muscle is a consequence of impaired muscle regeneration caused by enhanced satellite cell self-renewal. Here we report that
mdx:MyoD
−/−
mice developed a severe cardiac myopathy with areas of necrosis associated with hypertrophied myocytes. Moreover, heart tissue from
mdx:MyoD
−/−
mice exhibited constitutive activation of stress-activated signaling components, similar to
in vitro
models of cardiac myocyte adaptation. Taken together, these results support the hypothesis that the progression of skeletal muscle damage is a significant contributing factor leading to development of cardiomyopathy.
Decrease in telomerase activity in U-87MG human glioblastomas after treatment with an antagonist of growth hormone-releasing hormone
Authors: Kiaris Hippokratis , Schally Andrew V.
Published: July 2002

Antagonists of growth hormone-releasing hormone (GH-RH) inhibit the growth of various tumors through mechanisms that involve the suppression of the insulin-like growth factor I and/or insulin-like growth factor II levels or secretion. In the present study, we tested the hypothesis that the tumor inhibition is associated with a decrease in telomerase activity because telomerase is considered obligatory for continued tumor growth. Nude mice bearing xenografts of U-87MG human glioblastomas were treated with GH-RH antagonist MZ-5-156. Telomerase activity was assessed by the telomerase repeat amplification protocol. Treatment with MZ-5-156 reduced levels of telomerase activity as compared with controls. When U-87 glioblastomas, H-69 small cell lung carcinomas, H-23 non-small cell lung carcinomas, and MDA-MB-468 breast carcinoma cells were cultured
in vitro
, addition of 3 μM MZ-5-156 also inhibited telomerase activity. Reverse transcription–PCR analysis revealed that in U-87MG glioblastomas, the expression of the hTRT gene encoding for the telomerase catalytic subunit was significantly decreased by MZ-5-156, whereas the levels of mRNA for hTR and TP1, which encode for the telomerase RNA and telomerase-associated protein, respectively, were unaffected. The repression of the telomerase activity was not accompanied by a significant decrease of mRNA level for the c-
myc
protooncogene that regulates telomerase. Our findings suggest that tumor inhibition induced by the GH-RH antagonists in U-87MG glioblastomas is associated with the down-regulation of the
hTRT
gene, resulting in a decrease in telomerase activity. Further studies are needed to establish whether GH-RH antagonists produce telomerase inhibition in other tumors.
Inhibitory receptors sensing HLA-G1 molecules in pregnancy: Decidua-associated natural killer cells express LIR-1 and CD94/NKG2A and acquire p49, an HLA-G1-specific receptor
Authors: Ponte Marco , Cantoni Claudia , Biassoni Roberto , Tradori-Cappai Andrea , Bentivoglio Giorgio , Vitale Chiara , Bertone Stefania , Moretta Alessandro , Moretta Lorenzo , Mingari Maria Cristina
Published: July 2002

Trophoblastic cells lack classical HLA class I and class II molecules but express HLA-G1. Although this may prevent allorecognition by maternal T cells, it renders trophoblastic cells potentially susceptible to lysis by natural killer (NK) cells. As shown here, only a fraction of peripheral-blood NK cells in pregnant women express the HLA-G1-specific CD94/NKG2A and/or LIR-1 receptors. However, all NK cells isolated from maternal decidua during the first trimester expressed either one or both of these receptors. Perhaps more importantly, a fraction of cells expressed p49, an HLA-G1-specific inhibitory receptor, undetectable in peripheral-blood NK cells. p49 was expressed on virtually all NK cells isolated from placenta at term. Functional analyses revealed that the HLA class I-negative 221 lymphoblastoid cell line transfected with HLA-G1 was only partially protected from lysis by peripheral-blood NK cells isolated from pregnant women, whereas it was fully protected from decidual NK cells. As indicated by the addition of specific antibodies to cytolytic tests, all the above receptors contributed to HLA-G1 recognition by decidual NK cells, although p49 would appear to play a predominant role.
Deregulated signal transduction by the K1 gene product of Kaposi’s sarcoma-associated herpesvirus
Authors: Lagunoff Michael , Majeti Ravindra , Weiss Arthur , Ganem Don
Published: July 2002

The Kaposi’s sarcoma (KS)-associated herpesvirus is a lymphotropic virus strongly implicated in the pathogenesis of KS and several lymphoproliferative disorders. The KS-associated herpesvirus K1 gene encodes a transmembrane protein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM)-like sequence; it previously has been proposed to be important in viral tumorigenesis because its expression can trigger cell proliferation
in vitro
and
in vivo
. Here we show that expression of the full-length K1 protein can initiate calcium-dependent signal transduction in B cells; however, unlike other ITAM-based signal transduction events, K1 signaling occurs constitutively, in the absence of exogenous crosslinking ligands. This property is caused by its cysteine-rich ectodomain, which when transferred to other consensus ITAMs induces constitutive signaling. Although ITAM-based signaling by K1 involves classical syk and phospholipase C γ2 activation, both ITAM- and syk-independent signaling pathways are activated by K1 expression. These studies indicate that K1 is a deregulated signaling molecule with pleitropic effects that may explain its known growth deregulatory properties.
Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol—disulfide status
Authors: Åslund Fredrik , Zheng Ming , Beckwith Jon , Storz Gisela
Published: July 2002

The
Escherichia coli
transcription factor OxyR is activated by the formation of an intramolecular disulfide bond and subsequently is deactivated by enzymatic reduction of the disulfide bond. Here we show that OxyR can be activated by two possible pathways. In mutants defective in the cellular disulfide-reducing systems, OxyR is constitutively activated by a change in the thiol—disulfide redox status in the absence of added oxidants. In wild-type cells, OxyR is activated by hydrogen peroxide. By monitoring the presence of the OxyR disulfide bond after exposure to hydrogen peroxide
in vivo
and
in vitro
, we also show that the kinetics of OxyR oxidation by low concentrations of hydrogen peroxide is significantly faster than the kinetics of OxyR reduction, allowing for transient activation in an overall reducing environment. We propose that the activity of OxyR
in vivo
is determined by the balance between hydrogen peroxide levels and the cellular redox environment.
β-Trcp couples β-catenin phosphorylation-degradation and regulates
Xenopus
axis formation
Authors: Liu Chunming , Kato Yoichi , Zhang Zhuohua , Do Viet Minh , Yankner Bruce A. , He Xi
Published: July 2002

Regulation of β-catenin stability is essential for Wnt signal transduction during development and tumorigenesis. It is well known that serine-phosphorylation of β-catenin by the Axin–glycogen synthase kinase (GSK)–3β complex targets β-catenin for ubiquitination–degradation, and mutations at critical phosphoserine residues stabilize β-catenin and cause human cancers. How β-catenin phosphorylation results in its degradation is undefined. Here we show that phosphorylated β-catenin is specifically recognized by β-Trcp, an F-box/WD40-repeat protein that also associates with Skp1, an essential component of the ubiquitination apparatus. β-catenin harboring mutations at the critical phosphoserine residues escapes recognition by β-Trcp, thus providing a molecular explanation for why these mutations cause β-catenin accumulation that leads to cancer. Inhibition of endogenous β-Trcp function by a dominant negative mutant stabilizes β-catenin, activates Wnt/β-catenin signaling, and induces axis formation in
Xenopus
embryos. Therefore, β-Trcp plays a central role in recruiting phosphorylated β-catenin for degradation and in dorsoventral patterning of the
Xenopus
embryo.
Regulation of RpoS proteolysis in
Escherichia coli
: The response regulator RssB is a recognition factor that interacts with the turnover element in RpoS
Authors: Becker Gisela , Klauck Eberhard , Hengge-Aronis Regine
Published: July 2002

The degradation of the RpoS (σ
S
) subunit of RNA polymerase in
Escherichia coli
is a prime example of regulated proteolysis in prokaryotes. RpoS turnover depends on ClpXP protease, the response regulator RssB, and a hitherto uncharacterized “turnover element” within RpoS itself. Here we localize the turnover element to a small element (around the crucial amino acid lysine-173) directly downstream of the promoter-recognizing region 2.4 in RpoS. Its sequence as well as its location identify the turnover element as a unique proteolysis-promoting motif. This element is shown to be a site of interaction with RssB. Thus, RssB is functionally unique among response regulators as a direct recognition factor in ClpXP-dependent RpoS proteolysis. Binding of RssB to RpoS is stimulated by phosphorylation of the RssB receiver domain, suggesting that environmental stress affects RpoS proteolysis by modulating RssB affinity for RpoS. Initial evidence indicates that lysine-173 in RpoS, besides being essential of RpoS proteolysis, may play a role in promoter recognition. Thus the same region in RpoS is crucial for proteolysis as well as for activity as a transcription factor.
Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms
Authors: Osakabe Keishi , Tsao Cheng Chung , Li Laigeng , Popko Jacqueline L. , Umezawa Toshiaki , Carraway Daniel T. , Smeltzer Richard H. , Joshi Chandrashekhar P. , Chiang Vincent L.
Published: July 2002

A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate
O
-methyltransferase (COMT) from sweetgum (
Liquidambar styraciflua
) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in
Escherichia coli
methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route,
k
cat
/
K
m
of CAld5H for coniferyl aldehyde was ≈140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (
K
i
= 0.59 μM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with
in vivo
operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis.
Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis
Authors: Seifert Reinhard , Scholten Alexander , Gauss Renate , Mincheva Antoaneta , Lichter Peter , Kaupp U. Benjamin
Published: July 2002

Rhythmic activity of neurons and heart cells is endowed by pacemaker channels that are activated by hyperpolarization and directly regulated by cyclic nucleotides (termed HCN channels). These channels constitute a multigene family, and it is assumed that the properties of each member are adjusted to fit its particular function in the cell in which it resides. Here we report the molecular and functional characterization of a human subtype hHCN4. hHCN4 transcripts are expressed in heart, brain, and testis. Within the brain, the thalamus is the predominant area of hHCN4 expression. Heterologous expression of hHCN4 produces channels of unusually slow kinetics of activation and inactivation. The mean potential of half-maximal activation (V
1/2
) was −75.2 mV. cAMP shifted V
1/2
by 11 mV to more positive values. The hHCN4 gene was mapped to chromosome band 15q24–q25. The characteristic expression pattern and the sluggish gating suggest that hHCN4 controls the rhythmic activity in both thalamocortical neurons and pacemaker cells of the heart.
Synaptically released glutamate reduces γ-aminobutyric acid (GABA)ergic inhibition in the hippocampus via kainate receptors
Authors: Min Ming-Yuan , Melyan Zare , Kullmann Dimitri M.
Published: July 2002

Exogenous application of agonists at the kainate subtype of glutamate receptors has been shown to depress evoked monosynaptic inhibition by γ-aminobutyric acid (GABA)ergic interneurons in the hippocampus. This observation has led to the hypothesis that synaptic release of endogenous glutamate might have a disinhibitory effect on neuronal circuits, in addition to depolarizing neurons via postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, andN-methyl-d-aspartic acid (NMDA) receptors. It is not known, however, if glutamate released from excitatory neurons has the same kainate receptor-mediated effect on monosynaptic inhibitory transmission as exogenous agonist application. Indeed, the recent demonstration that excitatory synaptic signals elicited in interneurons are partly mediated by kainate receptors suggests that these receptors may have a pro- rather than disinhibitory role. Here, we examine the effect of synaptically released glutamate on monosynaptic inhibitory signaling. In the presence of antagonists to AMPA and NMDA receptors, brief bursts of activity in glutamatergic afferent fibers reduce GABAergic transmission. This depression of inhibition is reversibly abolished by blocking kainate receptors. It persists when GABABreceptors are blocked and is enhanced by blocking metabotropic glutamate receptors, possibly explained by presynaptic regulation of glutamate release from excitatory afferents by metabotropic autoreceptors. We conclude that the net kainate receptor-mediated effect of synaptically released glutamate is to reduce monosynaptic inhibition. Since this form of disinhibition may contribute to seizure initiation, kainate receptors may constitute an important target for anticonvulsant drug development.
Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux
Authors: Harris Z. Leah , Durley Alison P. , Man Tsz Kwong , Gitlin Jonathan D.
Published: July 2002

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism. Affected individuals evidence iron accumulation in tissue parenchyma in association with absent serum ceruloplasmin. Genetic studies of such patients reveal inherited mutations in the ceruloplasmin gene. To elucidate the role of ceruloplasmin in iron homeostasis, we created an animal model of aceruloplasminemia by disrupting the murine ceruloplasmin (
Cp
) gene. Although normal at birth,
Cp
−/−
mice demonstrate progressive accumulation of iron such that by one year of age all animals have a prominent elevation in serum ferritin and a 3- to 6-fold increase in the iron content of the liver and spleen. Histological analysis of affected tissues in these mice shows abundant iron stores within reticuloendothelial cells and hepatocytes. Ferrokinetic studies in
Cp
+/+
and
Cp
−/−
mice reveal equivalent rates of iron absorption and plasma iron turnover, suggesting that iron accumulation results from altered compartmentalization within the iron cycle. Consistent with this concept,
Cp
−/−
mice showed no abnormalities in cellular iron uptake but a striking impairment in the movement of iron out of reticuloendothelial cells and hepatocytes. Our findings reveal an essential physiologic role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores.
Pseudechetoxin: A peptide blocker of cyclic nucleotide-gated ion channels
Authors: Brown R. Lane , Haley Tammie L. , West Karen A. , Crabb John W.
Published: July 2002

Ion channels activated by the binding of cyclic nucleotides first were discovered in retinal rods where they generate the cell’s response to light. In other systems, however, it has been difficult to unambiguously determine whether cyclic nucleotide-dependent processes are mediated by protein kinases, their classical effector enzymes, or cyclic nucleotide-gated (CNG) ion channels. Part of this difficulty has been caused by the lack of specific pharmacological tools. Here we report the purification from the venom of the Australian King Brown snake of a peptide toxin that inhibits current through CNG channels. This toxin, which we have named Pseudechetoxin (PsTx), was purified by cation exchange and RP-HPLC and has a molecular mass of about 24 kDa. When applied to the extracellular face of membrane patches containing the α-subunit of the rat olfactory CNG channel, PsTx blocked the cGMP-dependent current with a
K
i
of 5 nM. Block was independent of voltage and required only a single molecule of toxin. PsTx also blocked CNG channels containing the bovine rod α-subunit with high affinity (100 nM), but it was less effective on the heteromeric version of the rod channel (
K
i
≈ 3 μM). We have obtained N-terminal and partial internal sequence data and the amino acid composition of PsTx. These data indicate that PsTx is a basic protein that exhibits some homology with helothermine, a toxin isolated from the venom of the Mexican beaded lizard. PsTx promises to be a valuable pharmacological tool for studies on the structure and physiology of CNG channels.
Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves
Authors: Vaquero Carmen , Sack Markus , Chandler John , Drossard Jürgen , Schuster Flora , Monecke Michael , Schillberg Stefan , Fischer Rainer
Published: July 2002

To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described
Agrobacterium
-
mediated transient expression system. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse/human chimeric antibody (cT84.66) derived from the parental murine mAb T84.66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors.
In vivo
assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant
Agrobacterium
. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS/PAGE, Western blotting, and ELISA.
The M2–2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription
Authors: Bermingham Alison , Collins Peter L.
Published: July 2002

The M2 mRNA of human respiratory syncytial virus (RSV) contains two overlapping ORFs, encoding the transcription antitermination protein (M2–1) and the 90-aa M2–2 protein of unknown function. Viable recombinant RSV was recovered in which expression of M2–2 was ablated, identifying it as an accessory factor dispensable for growth
in vitro
. Virus lacking M2–2 grew less efficiently than did the wild-type parent
in vitro
, with titers that were reduced 1,000-fold during the initial 2–5 days and 10-fold by days 7–8. Compared with wild-type virus, the intracellular accumulation of RNA by M2–2 knockout virus was reduced 3- to 4-fold or more for genomic RNA and increased 2- to 4-fold or more for mRNA. Synthesis of the F and G glycoproteins, the major RSV neutralization and protective antigens, was increased in proportion with that of mRNA. In cells infected with wild-type RSV, mRNA accumulation increased dramatically up to approximately 12–15 hr after infection and then leveled off, whereas accumulation continued to increase in cells infected with the M2–2 knockout viruses. These findings suggest that M2–2 mediates a regulatory “switch” from transcription to RNA replication, one that provides an initial high level of mRNA synthesis followed by a shift in the RNA synthetic program in favor of genomic RNA for virion assembly. With regard to vaccine development, the M2–2 knockout has a highly desirable phenotype in which virus growth is attenuated while gene expression is concomitantly increased.
Segregation-driven organization in chaotic granular flows
Authors: Hill K. M. , Khakhar D. V. , Gilchrist J. F. , McCarthy J. J. , Ottino J. M.
Published: July 2002

An important industrial problem that provides fascinating puzzles in pattern formation is the tendency for granular mixtures to de-mix or segregate. Small differences in either size or density lead to flow-induced segregation. Similar to fluids, noncohesive granular materials can display chaotic advection; when this happens chaos and segregation compete with each other, giving rise to a wealth of experimental outcomes. Segregated structures, obtained experimentally, display organization in the presence of disorder and are captured by a continuum flow model incorporating collisional diffusion and density-driven segregation. Under certain conditions, structures never settle into a steady shape. This may be the simplest experimental example of a system displaying competition between chaos and order.
Activity-dependent regulation of synaptic clustering in a hippocampal culture system
Authors: Kavalali Ege T. , Klingauf Jürgen , Tsien Richard W.
Published: July 2002

Currently, there is a limited understanding of the factors that influence the localization and density of individual synapses in the central nervous system. Here we have studied the effects of activity on synapse formation between hippocampal dentate granule cells and CA3 pyramidal neurons in culture, taking advantage of FM1–43 as a fluorescent marker of synaptic boutons. We observed an early tendency for synapses to group together, quickly followed by the appearance of synaptic clusters on dendritic processes. These events were strongly influenced by
N
-methyl-
d
-aspartic acid receptor- and cyclic AMP-dependent signaling. The microstructure and localization of the synaptic clusters resembled that found in hippocampus, at mossy fiber synapses of stratum lucidum. Activity-dependent clustering of synapses represents a means for synaptic targeting that might contribute to synaptic organization in the brain.
Perceptual learning in motion discrimination that generalizes across motion directions
Authors: Liu Zili
Published: July 2002

When human subjects discriminate motion directions of two visual stimuli, their discrimination improves with practice. This improved performance has been found to be specific to the practiced directions and does not transfer to new motion directions. Indeed, such stimulus-specific learning has become a trademark finding in almost all perceptual learning studies and has been used to infer the loci of learning in the brain. For example, learning in motion discrimination has been inferred to occur in the visual area MT (medial temporal cortex) of primates, where neurons are selectively tuned to motion directions. However, such motion discrimination task is extremely difficult, as is typical of most perceptual learning tasks. When the difficulty is moderately reduced, learning transfers to new motion directions. This result challenges the idea of using simple visual stimuli to infer the locus of learning in low-level visual processes and suggests that higher-level processing is essential even in “simple” perceptual learning tasks.
Coevolutionary analysis of resistance-evading peptidomimetic inhibitors of HIV-1 protease
Authors: Rosin Christopher D. , Belew Richard K. , Morris Garrett M. , Olson Arthur J. , Goodsell David S.
Published: July 2002

We have developed a coevolutionary method for the computational
design of HIV-1 protease inhibitors selected for their ability to
retain efficacy in the face of protease mutation. For HIV-1 protease,
typical drug design techniques are shown to be ineffective for the
design of resistance-evading inhibitors: An inhibitor that is a direct
analogue of one of the natural substrates will be susceptible to
resistance mutation, as will inhibitors designed to fill the active
site of the wild-type or a mutant enzyme. Two design principles are
demonstrated: (
i
) For enzymes with broad substrate
specificity, such as HIV-1 protease, resistance-evading inhibitors are
best designed against the immutable properties of the active site—the
properties that must be conserved in any mutant protease to retain the
ability to bind and cleave all of the native substrates.
(
ii
) Robust resistance-evading inhibitors can be designed
by optimizing activity simultaneously against a large set of mutant
enzymes, incorporating as much of the mutational space as possible.
Inhibitory sites in enzymes: Zinc removal and reactivation by thionein
Authors: Maret Wolfgang , Jacob Claus , Vallee Bert L. , Fischer Edmond H.
Published: July 2002

Thionein (T) has not been isolated previously from biological material. However, it is generated transiently
in situ
by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.