bluuebunny/crossref_metadata_2025 huggingface dataset:
Camalexin is synthesized from indole-3-acetaldoxime, a key branching point between primary and secondary metabolism in
Arabidopsis
Authors: Glawischnig Erich , Hansen Bjarne Gram , Olsen Carl Erik , Halkier Barbara Ann
Published: May 2004

Characteristic for cruciferous plants is their production of N- and S-containing indole phytoalexins with disease resistance and cancer-preventive properties, previously proposed to be synthesized from indole independently of tryptophan. We show that camalexin, the indole phytoalexin of
Arabidopsis thaliana
, is synthesized from tryptophan via indole-3-acetaldoxime (IAOx) in a reaction catalyzed by CYP79B2 and CYP79B3.
Cyp79B2
/
cyp79B3
double knockout mutant is devoid of camalexin, as it is also devoid of indole glucosinolates [Zhao, Y., Hull, A. K., Gupta, N. R., Goss, K. A., Alonso, J., Ecker, J. R., Normanly, J., Chory, J. & Celenza, J. L. (2002)
Genes Dev.
16, 3100–3112], and isotope-labeled IAOx is incorporated into camalexin. These results demonstrate that only CYP79B2 and CYP79B3 contribute significantly to the IAOx pool from which camalexin and indole glucosinolates are synthesized. Furthermore, production of camalexin in the
sur1
mutant devoid of glucosinolates excludes the possibility that camalexin is derived from indole glucosinolates. CYP79B2 plays an important role in camalexin biosynthesis in that the transcript level of
CYP79B2
, but not
CYP79B3
, is increased upon induction of camalexin by silver nitrate as evidenced by microarray analysis and promoter–β-glucuronidase data. The structural similarity between cruciferous indole phytoalexins suggests that these compounds are biogenetically related and synthesized from tryptophan via IAOx by CYP79B homologues. The data show that IAOx is a key branching point between several secondary metabolic pathways as well as primary metabolism, where IAOx has been shown to play a critical role in IAA homeostasis.
Membrane growth can generate a transmembrane pH gradient in fatty acid vesicles
Authors: Chen Irene A. , Szostak Jack W.
Published: May 2004

Electrochemical proton gradients are the basis of energy transduction in modern cells, and may have played important roles in even the earliest cell-like structures. We have investigated the conditions under which pH gradients are maintained across the membranes of fatty acid vesicles, a model of early cell membranes. We show that pH gradients across such membranes decay rapidly in the presence of alkali-metal cations, but can be maintained in the absence of permeable cations. Under such conditions, when fatty acid vesicles grow through the incorporation of additional fatty acid, a transmembrane pH gradient is spontaneously generated. The formation of this pH gradient captures some of the energy released during membrane growth, but also opposes and limits further membrane area increase. The coupling of membrane growth to energy storage could have provided a growth advantage to early cells, once the membrane composition had evolved to allow the maintenance of stable pH gradients.
The twilight of Heliozoa and rise of Rhizaria, an emerging supergroup of amoeboid eukaryotes
Authors: Nikolaev Sergey I. , Berney Cédric , Fahrni José F. , Bolivar Ignacio , Polet Stephane , Mylnikov Alexander P. , Aleshin Vladimir V. , Petrov Nikolai B. , Pawlowski Jan
Published: May 2004

Recent molecular phylogenetic studies revealed the extraordinary diversity of single-celled eukaryotes. However, the proper assessment of this diversity and accurate reconstruction of the eukaryote phylogeny are still impeded by the lack of molecular data for some major groups of easily identifiable and cultivable protists. Among them, amoeboid eukaryotes have been notably absent from molecular phylogenies, despite their diversity, complexity, and abundance. To partly fill this phylogenetic gap, we present here combined small-subunit ribosomal RNA and actin sequence data for the three main groups of “Heliozoa” (Actinophryida, Centrohelida, and Desmothoracida), the heliozoan-like
Sticholonche
, and the radiolarian group Polycystinea. Phylogenetic analyses of our sequences demonstrate the polyphyly of heliozoans, which branch either as an independent eukaryotic lineage (Centrohelida), within stramenopiles (Actinophryida), or among cercozoans (Desmothoracida), in broad agreement with previous ultrastructure-based studies. Our data also provide solid evidence for the existence of the Rhizaria, an emerging supergroup of mainly amoeboid eukaryotes that includes desmothoracid heliozoans, all radiolarians,
Sticholonche
, and foraminiferans, as well as various filose and reticulose amoebae and some flagellates.
A new activity for an old enzyme:
Escherichia coli
bacterial alkaline phosphatase is a phosphite-dependent hydrogenase
Authors: Yang Kechao , Metcalf William W.
Published: May 2004

Genetic analysis indicates that
Escherichia coli
possesses two independent pathways for oxidation of phosphite (Pt) to phosphate. One pathway depends on the 14-gene
phn
operon, which encodes the enzyme C-P lyase. The other pathway depends on the
phoA
locus, which encodes bacterial alkaline phosphatase (BAP). Transposon mutagenesis studies strongly suggest that BAP is the only enzyme involved in the
phoA
-dependent pathway. This conclusion is supported by purification and biochemical characterization of the Pt-oxidizing enzyme, which was proven to be BAP by N terminus protein sequencing. Highly purified BAP catalyzed Pt oxidation with specific activities of 62–242 milliunits/mg and phosphate ester hydrolysis with specific activities of 41–61 units/mg. Surprisingly, BAP catalyzes the oxidation of Pt to phosphate and molecular H
2
. Thus, BAP is a unique Pt-dependent, H
2
-evolving hydrogenase. This reaction is unprecedented in both P and H biochemistry, and it is likely to involve direct transfer of hydride from the substrate to water-derived protons.
The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition
Authors: Okamoto Ken , Matsumoto Koji , Hille Russ , Eger Bryan T. , Pai Emil F. , Nishino Takeshi
Published: May 2004

Molybdenum is widely distributed in biology and is usually found as a mononuclear metal center in the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems catalyzing hydroxylation reactions in that the oxygen atom incorporated into the product is derived from water rather than molecular oxygen. Here, we present the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon–oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable broad charge–transfer band at ≈640 nm. The crystal structure of the complex indicates that the catalytically labile Mo—OH oxygen has formed a bond with a carbon atom of the substrate. In addition, the Mo⋕S group of the oxidized enzyme has become protonated to afford Mo—SH on reduction of the molybdenum center. In contrast to previous assignments, we find this last ligand at an equatorial position in the square-pyramidal metal coordination sphere, not the apical position. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.
A gene cluster for secondary metabolism in oat: Implications for the evolution of metabolic diversity in plants
Authors: Qi X. , Bakht S. , Leggett M. , Maxwell C. , Melton R. , Osbourn A.
Published: May 2004

The evolution of the ability to synthesize specialized metabolites is likely to have been key for survival and diversification of different plant species. Oats (
Avena
spp.) produce antimicrobial triterpenoids (avenacins) that protect against disease. The oat β-amyrin synthase gene
AsbAS1
, which encodes the first committed enzyme in the avenacin biosynthetic pathway, is clearly distinct from other plant β-amyrin synthases. Here we show that
AsbAS1
has arisen by duplication and divergence of a cycloartenol synthase-like gene, and that its properties have been refined since the divergence of oats and wheat. Strikingly, we have also found that
AsbAS1
is clustered with other genes required for distinct steps in avenacin biosynthesis in a region of the genome that is not conserved in other cereals. Because the components of this gene cluster are required for at least four clearly distinct enzymatic processes (2,3-oxidosqualene cyclization, β-amyrin oxidation, glycosylation, and acylation), it is unlikely that the cluster has arisen as a consequence of duplication of a common ancestor. Although clusters of paralogous genes are common in plants (e.g., gene clusters for rRNA and specific disease resistance), reports of clusters of genes that do not share sequence relatedness and whose products contribute to a single selectable function are rare [Gierl, A. & Frey, M. (2001)
Planta
213, 493–498]. Taken together, our evidence has important implications for the generation of metabolic diversity in plants.
Computational design of receptors for an organophosphate surrogate of the nerve agent soman
Authors: Allert Malin , Rizk Shahir S. , Looger Loren L. , Hellinga Homme W.
Published: May 2004

We report the computational design of soluble protein receptors for pinacolyl methyl phosphonic acid (PMPA), the predominant hydrolytic product of the nerve agent soman. Using recently developed computational protein design techniques, the ligand-binding pockets of two periplasmic binding proteins, glucose-binding protein and ribose-binding protein, were converted to bind PMPA instead of their cognate sugars. The designs introduce 9–12 mutations in the parent proteins. Twelve of 20 designs tested exhibited PMPA-dependent changes in emission intensity of a fluorescent reporter with affinities between 45 nM and 10 μM. The contributions to ligand binding by individual residues were determined in two designs by alanine-scanning mutagenesis, and are consistent with the molecular models. These results demonstrate that designed receptors with radically altered binding specificities and affinities that rival or exceed those of the parent proteins can be successfully predicted. The designs vary in parent scaffold, sequence diversity, and orientation of docked ligand, suggesting that the number of possible solutions to the design problem is large and degenerate. This observation has implications for the genesis of biological function by random processes. The designed receptors reported here may have utility in the development of fluorescent biosensors for monitoring nerve agents.
Inhibitor of κB kinase is required to activate a subset of interferon γ-stimulated genes
Authors: Sizemore Nywana , Agarwal Anju , Das Kingshuk , Lerner Natalia , Sulak Michael , Rani Sandhya , Ransohoff Richard , Shultz David , Stark George R.
Published: May 2004

IκB kinase (IKK), discovered as the major activator of NF-κB, plays additional roles in signaling. By using mouse embryo fibroblasts (MEFs) lacking both the α and β subunits of IKK, we find that these proteins are required for induction of a major subset of IFNγ-stimulated genes and that this requirement is independent of NF-κB activation. Furthermore, there is no defect in IFNγ-stimulated signal transducer and activator of transcription 1 (Stat1) activation or function in the IKKα/β-null MEFs. Therefore, although activated Stat1 dimers are necessary for the activation of these genes in response to IFNγ, they are not sufficient. These results reveal an important additional pathway for IFNγ-stimulated gene expression in which an NF-κB-independent function of IKK is required.
Deletion of the Parkin coregulated gene causes male sterility in the

quaking
viable

mouse mutant
Authors: Lorenzetti Diego , Bishop Colin E. , Justice Monica J.
Published: May 2004

Quaking
viable

(

qk
v

) is a recessive neurological mouse mutation with severe dysmyelination of the CNS and spermiogenesis failure. The molecular lesion in the

qk
v

mutant is a deletion of ≈1 Mb on mouse chromosome 17 that alters the expression of the
qk
gene in oligodendrocytes. Complementation analysis between the

qk
v

mutation and
qk
mutant alleles generated through chemical mutagenesis showed that the male sterility is a distinctive feature of the

qk
v

allele. This observation suggested that the sperm differentiation defect in

qk
v

is due to the deletion of a gene(s) distinct from
qk
. Here, we demonstrate that the deletion of
Pacrg
is the cause of male sterility in the

qk
v

mutant.
Pacrg
is the mouse homologue of the human
PARKIN
-coregulated gene (
PACRG
), which encodes for a protein whose biochemical function remains unclear. We show that
Pacrg
is highly expressed in the testes in both mice and humans. In addition, the expression pattern of
Pacrg
during spermiogenesis suggests that it plays a role in sperm differentiation. In support of this hypothesis, we show that transgenic expression of
Pacrg
in testes restores spermiogenesis and fertility in

qk
v

males. This finding provides the first
in vivo
evidence, to our knowledge, for the function of Pacrg in a model organism. Immunolocalization experiments on isolated spermatozoa show that the Pacrg protein is present in mature sperm. Remarkably, the mammalian Pacrg protein shares significant sequence similarities with gene products from flagellated protozoans, suggesting that Pacrg may be necessary for proper flagellar formation in many organisms.
Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria
Authors: Arnoult Damien , Bartle Laura M. , Skaletskaya Anna , Poncet Delphine , Zamzami Naoufal , Park Peter U. , Sharpe Juanita , Youle Richard J. , Goldmacher Victor S.
Published: May 2004

We report that the cytomegalovirus-encoded cell death suppressor vMIA binds Bax and prevents Bax-mediated mitochondrial membrane permeabilization by sequestering Bax at mitochondria in the form of a vMIA–Bax complex. vMIA mutants with a defective mitochondria-targeting domain retain their Bax-binding function but not their ability to suppress mitochondrial membrane permeabilization or cell death. vMIA does not seem to either specifically associate with Bak or suppress Bak-mediated mitochondrial membrane permeabilization. Recent evidence suggests that the contribution of Bax and Bak in the mitochondrial apoptotic signaling pathway depends on the distinct phenotypes of cells, and it appears from our data that vMIA is capable of suppressing apoptosis in cells in which this pathway is dominated by Bax, but not in cells where Bak also plays a role.
Flint mining in prehistory recorded by
in situ
-produced cosmogenic
10
Be
Authors: Verri G. , Barkai R. , Bordeanu C. , Gopher A. , Hass M. , Kaufman A. , Kubik P. , Montanari E. , Paul M. , Ronen A. , Weiner S. , Boaretto E.
Published: May 2004

The development of mining to acquire the best raw materials for producing stone tools represents a breakthrough in human technological and intellectual development. We present a new approach to studying the history of flint mining, using
in situ
-produced cosmogenic
10
Be concentrations. We show that the raw material used to manufacture flint artifacts ≈300,000 years old from Qesem Cave (Israel) was most likely surface-collected or obtained from shallow quarries, whereas artifacts of the same period from Tabun Cave (Israel) were made of flint originating from layers 2 or more meters deep, possibly mined or quarried by humans.
Predicting indirect readout effects in protein–DNA interactions
Authors: Zhang Yongli , Xi Zhiqun , Hegde Rashmi S. , Shakked Zippora , Crothers Donald M.
Published: May 2004

Recognition of DNA by proteins relies on direct interactions with specific DNA-functional groups, along with indirect effects that reflect variable energetics in the response of DNA sequences to twisting and bending distortions induced by proteins. Predicting indirect readout requires knowledge of the variations in DNA curvature and flexibility in the affected region, which we have determined for a series of DNA-binding sites for the E2 regulatory protein by using the cyclization kinetics method. We examined 16 sites containing different noncontacted spacer sequences, which vary by more than three orders of magnitude in binding affinity. For 15 of these sites, the variation in affinity was predicted within a factor of 3, by using experimental curvature and flexibility values and a statistical mechanical theory. The sole exception was traced to differential magnesium ion binding.
Role of Polo-like kinase in the degradation of early mitotic inhibitor 1, a regulator of the anaphase promoting complex/cyclosome
Authors: Moshe Yakir , Boulaire Jérôme , Pagano Michele , Hershko Avram
Published: May 2004

Early mitotic inhibitor 1 (Emi1) inhibits the activity of the anaphase promoting complex/cyclosome (APC/C), which is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. Levels of Emi1 oscillate in the cell cycle: it accumulates in the S phase and is rapidly degraded in prometaphase. The degradation of Emi1 in early mitosis is necessary for the activation of APC/C in late mitosis. Previous studies have shown that Emi1 is targeted for degradation in mitosis by a Skp1–Cullin1 F-box protein (SCF) ubiquitin ligase complex that contains the F-box protein β-TrCP. As with other substrates of SCF
β-TrCP
, the phosphorylation of Emi1 on a DSGxxS sequence is required for this process. However, the protein kinase(s) involved has not been identified. We find that Polo-like kinase 1 (Plk1), a protein kinase that accumulates in mitosis, markedly stimulates the ligation of Emi1 to ubiquitin by purified SCF
β-TrCP
. Cdk1-cyclin B, another major mitotic protein kinase, has no influence on this process by itself but stimulates the action of Plk1 at low, physiological concentrations. Plk1 phosphorylates serine residues in the DSGxxS sequence of Emi1, as suggested by the reduced phosphorylation of a derivative in which the two serines were mutated to nonphosphorylatable amino acids. Transfection with an small interfering RNA duplex directed against Plk1 caused the accumulation of Emi1 in mitotically arrested HeLa cells. It is suggested that phosphorylation of Emi1 by Plk1 is involved in its degradation in mitosis.
H5N1 influenza: A protean pandemic threat
Authors: Guan Y. , Poon L. L. M. , Cheung C. Y. , Ellis T. M. , Lim W. , Lipatov A. S. , Chan K. H. , Sturm-Ramirez K. M. , Cheung C. L. , Leung Y. H. C. , Yuen K. Y. , Webster R. G. , Peiris J. S. M.
Published: May 2004

Infection with avian influenza A virus of the H5N1 subtype (isolates A/HK/212/03 and A/HK/213/03) was fatal to one of two members of a family in southern China in 2003. This incident was preceded by lethal outbreaks of H5N1 influenza in waterfowl, which are the natural hosts of these viruses and, therefore, normally have asymptomatic infection. The hemagglutinin genes of the A/HK/212/03-like viruses isolated from humans and waterfowl share the lineage of the H5N1 viruses that caused the first known cases of human disease in Hong Kong in 1997, but their internal protein genes originated elsewhere. The hemagglutinin of the recent human isolates has undergone significant antigenic drift. Like the 1997 human H5N1 isolates, the 2003 human H5N1 isolates induced the overproduction of proinflammatory cytokines by primary human macrophages
in vitro
, whereas the precursor H5N1 viruses and other H5N1 reassortants isolated in 2001 did not. The acquisition by the viruses of characteristics that enhance virulence in humans and waterfowl and their potential for wider distribution by infected migrating birds are causes for renewed pandemic concern.
Cretaceous flowers of Nymphaeaceae and implications for complex insect entrapment pollination mechanisms in early Angiosperms
Authors: Gandolfo M. A. , Nixon K. C. , Crepet W. L.
Published: May 2004

Based on recent molecular systematics studies, the water lily lineage (Nymphaeales) provides an important key to understanding ancestral angiosperm morphology and is of considerable interest in the context of angiosperm origins. Therefore, the fossil record of Nymphaeales potentially provides evidence on both the timing and nature of diversification of one of the earliest clades of flowering plants. Recent fossil evidence of Turonian age (≈90 million years B.P.) includes fossil flowers with characters that, upon rigorous analysis, firmly place them within Nymphaeaceae. Unequivocally the oldest floral record of the Nymphaeales, these fossils are closely related to the modern Nymphaealean genera
Victoria
(the giant Amazon water lily) and
Euryale
. Although the fossils are much smaller than their modern relatives, the precise and dramatic correspondence between the fossil floral morphology and that of modern
Victoria
flowers suggests that beetle entrapment pollination was present in the earliest part of the Late Cretaceous.
E-selectin is required for the antiangiogenic activity of endostatin
Authors: Yu Ying , Moulton Karen S. , Khan Mohamed K. , Vineberg Sabrina , Boye Eileen , Davis Vannessa M. , O'Donnell Peter E. , Bischoff Joyce , Milstone David S.
Published: May 2004

Endostatin, a 20-kDa fragment of collagen XVIII, is a potent angiogenesis inhibitor. E-selectin, an inducible leukocyte adhesion molecule specifically expressed by endothelial cells, has also been implicated in angiogenesis. By usingin vivo,ex vivo, andin vitroangiogenic assays, we investigated the functional relationship between endostatin and E-selectin. In corneal micropocket assays, recombinant endostatin administered i.p. by osmotic pump inhibited basic fibroblast growth factor-induced angiogenesis in WT, but not E-selectin-deficient, mice. Similarly, endostatin inhibited vascular endothelial growth factor-stimulated endothelial sprout formation from aortic rings dissected from WT but not from E-selectin-deficient mice. To further explore this apparent requirement for E-selectin in endostatin action, we manipulated E-selectin expression in cultured human endothelial cells. When E-selectin was induced by IL-1β, or lipopolysaccharide, human umbilical vein endothelial cells and human dermal microvascular endothelial cells each became markedly more sensitive to inhibition by endostatin in a vascular endothelial growth factor-induced cell migration assay. To dissociate E-selectin expression from other consequences of endothelial activation, human umbilical vein endothelial cells were transduced with an adenoviral human E-selectin expression construct; these cells also showed increased sensitivity to endostatin, and this effect required the E-selectin cytoplasmic domain. Taken together, these results indicate that E-selectin is required for the antiangiogenic activity of endostatinin vivoandex vivoand confers endostatin sensitivity to nonresponsive human endothelial cellsin vitro. E-selectin may be a useful predictor and modulator of endostatin efficacy in antiangiogenic therapy.
The dynamic processivity of the T4 DNA polymerase during replication
Authors: Yang Jingsong , Zhuang Zhihao , Roccasecca Rosa Maria , Trakselis Michael A. , Benkovic Stephen J.
Published: May 2004

The polymerase (gp43) processivity during T4 replisome mediated DNA replication has been investigated. The size of the Okazaki fragments remains constant over a wide range of polymerase concentrations. A dissociation rate constant of ≈0.0013 sec
-1
was measured for the polymerases from both strands, consistent with highly processive replication on both the leading and lagging strands. This processive replication, however, can be disrupted by a catalytically inactive mutant D408N gp43 that retains normal affinity for DNA and the clamp. The inhibition kinetics fit well to an active exchange model in which the mutant polymerase (the polymerase trap) displaces the replicating polymerase. This kinetic model was further strengthened by the observation that the sizes of both the Okazaki fragments and the extension products on a primed M13mp18 template were reduced in the presence of the mutant polymerase. The effects of the trap polymerase therefore suggest a dynamic processivity of the polymerase during replication, namely, a solution/replisome polymerase exchange takes place without affecting continued DNA synthesis. This process mimics the polymerase switching recently suggested during the translesion DNA synthesis, implies the multiple functions of the clamp in replication, and may play a potential role in overcoming the replication barriers by the T4 replisome.
Unique CD40-mediated biological program in B cell activation requires both type 1 and type 2 NF-κB activation pathways
Authors: Zarnegar Brian , He Jeannie Q. , Oganesyan Gagik , Hoffmann Alexander , Baltimore David , Cheng Genhong
Published: May 2004

B lymphocytes can be activated by many different stimuli. However, the mechanisms responsible for the signaling and functional specificities of individual stimuli remain to be elucidated. Here, we have compared the contribution of the type 1 (p50-dependent) and type 2 (p52-dependent) NF-κB activation pathways to cell survival, proliferation, homotypic aggregation, and specific gene regulation of murine primary B lymphocytes. Whereas lipopolysaccharide (LPS) and B cell activation factor (BAFF) mainly activate the type 1 or type 2 pathways, respectively, CD40 ligand (CD40L) strongly activates both. Rescue of spontaneous apoptosis is diminished in p52–/–B cells after BAFF stimulation and in p50–/–c-Rel–/–B cells after LPS stimulation. Interestingly, significant CD40-induced B cell survival is still observed even in p50–/–c-Rel–/–p65–/+B cells, which is correlated with the ability of CD40L to up-regulate Bcl-xLexpression in these cells. CD40L- and LPS-induced B cell proliferation, as well as up-regulation of proliferation-related genes, however, are greatly reduced in c-Rel–/–and p50–/–c-Rel–/–B cells but are normal in p52–/–B cells. We have further demonstrated that both c-Rel and p52 are required for CD40-mediated B cell homotypic aggregation, which explains well why neither LPS nor BAFF has this function. Overall, our studies suggest that both type 1 and type 2 NF-κB pathways contribute to the gene expression and biological program unique for CD40 in B cell activation.
Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone
Authors: Magnusson Olafur Th. , Toyama Hirohide , Saeki Megumi , Rojas Ana , Reed John C. , Liddington Robert C. , Klinman Judith P. , Schwarzenbacher Robert
Published: May 2004

The biosynthesis of pyrroloquinoline quinone (PQQ), a vitamin and redox cofactor of quinoprotein dehydrogenases, is facilitated by an unknown pathway that requires the expression of six genes,
pqqA
to -
F
. PqqC, the protein encoded by
pqqC
, catalyzes the final step in the pathway in a reaction that involves ring cyclization and eight-electron oxidation of 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic-acid to PQQ. Herein, we describe the crystal structures of PqqC and its complex with PQQ and determine the stoichiometry of H
2
O
2
formation and O
2
uptake during the reaction. The PqqC structure(s) reveals a compact seven-helix bundle that provides the scaffold for a positively charged active site cavity. Product binding induces a large conformational change, which results in the active site recruitment of amino acid side chains proposed to play key roles in the catalytic mechanism. PqqC is unusual in that it transfers redox equivalents to molecular oxygen without the assistance of a redox active metal or cofactor. The structure of the enzyme-product complex shows additional electron density next to R179 and C5 of PQQ, which can be modeled as O
2
or H
2
O
2
, indicating a site for oxygen binding. We propose a reaction sequence that involves base-catalyzed cyclization and a series of quinone-quinol tautomerizations that are followed by cycles of O
2
/H
2
O
2
-mediated oxidations.
Discovery and characterization of an epithelial-specific galectin in the endometrium that forms crystals in the trophectoderm
Authors: Gray C. Allison , Adelson David L. , Bazer Fuller W. , Burghardt Robert C. , Meeusen Els N. T. , Spencer Thomas E.
Published: May 2004

Secretions of the uterus support survival and growth of the conceptus (embryo/fetus and associated membranes) during pregnancy. Galectin-15, also known as OVGAL11 and a previously uncharacterized member of the galectin family of secreted β-galactoside lectins containing a conserved carbohydrate recognition domain and a separate putative integrin binding domain, was discovered in the uterus of sheep. In endometria of cyclic and pregnant sheep, galectin-15 mRNA was expressed specifically in the endometrial luminal epithelium but not in the conceptus. In pregnant sheep, galectin-15 mRNA expression appeared in the epithelia between days 10 and 12 and increased between days 12 and 16. Progesterone induced and IFN-τ stimulated galectin-15 mRNA in the endometrial epithelium. Galectin-15 protein was concentrated near and on the apical surface of the endometrial luminal epithelia and localized within discrete cytoplasmic crystalline structures of conceptus trophectoderm (Tr). In the uterine lumen, secreted galectin-15 protein increased between days 14 and 16 of pregnancy. Galectin-15 protein was functional in binding lactose and mannose sugars and immunologically identical to the unnamedMr14,000 (14K) protein from the ovine uterus that forms crystalline inclusion bodies in endometrial epithelia and conceptus Tr. Based on the functional studies of other galectins, galectin-15 is hypothesized to function extracellularly to regulate Tr migration and adhesion to the endometrial epithelium and intracellularly to regulate Tr cell survival, growth, and differentiation. Galectins may be useful as cellular and molecular markers for endometrial function and receptivity, to enhance conceptus survival and development, and to evaluate and enhance fertility.
Previous-year reproduction reduces photosynthetic capacity and slows lifetime growth in females of a neotropical tree
Authors: Wheelwright Nathaniel T. , Logan Barry A.
Published: May 2004

Females of dioecious plant species typically invest more in reproduction than males because they produce seeds, fruits, and associated structures in addition to flowers. If females are unable to compensate by up-regulating rates of photosynthesis or by reproducing less frequently than males, their greater reproductive investment may result in reduced growth or higher mortality. Here we provide evidence of the cost of reproduction in
Ocotea tenera
(Lauraceae), a dioecious neotropical tree common in lower montane forests of Monteverde, Costa Rica. Over periods of 12–21 years, females grew more slowly than males in a natural population and in two experimental plots where we were able to control for genotype, age, habitat, and reproductive history. Simultaneous measurements of 10 matched pairs of sibling trees of the opposite sex but same age demonstrated that the photosynthetic capacities of females were 13% lower than those of males. Among females, photosynthetic capacity was negatively correlated with fruit production during the most recent reproductive season but not with lifetime fruit production. Sexual size dimorphism in adult
O. tenera
trees appears to be a nonadaptive consequence of trading off recent reproduction against maintenance of the photosynthetic apparatus, with long-term negative effects on growth.
c-Abl phosphorylates Dok1 to promote filopodia during cell spreading
Authors: Woodring Pamela J. , Meisenhelder Jill , Johnson Sam A. , Zhou Guo-Lei , Field Jeffrey , Shah Kavita , Bladt Friedhelm , Pawson Tony , Niki Masaru , Pandolfi Pier Paolo , Wang Jean Y.J. , Hunter Tony
Published: May 2004

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.
Mammalian Fat1 cadherin regulates actin dynamics and cell–cell contact
Authors: Tanoue Takuji , Takeichi Masatoshi
Published: May 2004

Fat cadherins form a distinct subfamily of the cadherin gene superfamily, and are featured by their unusually large extracellular domain. In this work, we investigated the function of a mammalian Fat cadherin. Fat1 was localized at filopodial tips, lamellipodial edges, and cell–cell boundaries, overlapping with dynamic actin structures. RNA interference–mediated knockdown of Fat1 resulted in disorganization of cell junction–associated F-actin and other actin fibers/cables, disturbance of cell–cell contacts, and also inhibition of cell polarity formation at wound margins. Furthermore, we identified Ena/vasodilator-stimulated phosphoproteins as a potential downstream effector of Fat1. These results suggest that Fat1 regulates actin cytoskeletal organization at cell peripheries, thereby modulating cell contacts and polarity.
ENaC–Membrane Interactions
Authors: Awayda Mouhamed S. , Shao Weijian , Guo Fengli , Zeidel Mark , Hill Warren G.
Published: May 2004

Recently, it was reported that the epithelial Na+ channel (ENaC) is regulated by temperature (Askwith, C.C., C.J. Benson, M.J. Welsh, and P.M. Snyder. 2001. Proc. Natl. Acad. Sci. USA. 98:6459–6463). As these changes of temperature affect membrane lipid order and lipid–protein interactions, we tested the hypothesis that ENaC activity can be modulated by membrane lipid interactions. Two approaches were used to modulate membrane anisotropy, a lipid order–dependent parameter. The nonpharmacological approach used temperature changes, while the pharmacological one used chlorpromazine (CPZ), an agent known to decrease membrane order, and Gd+3. Experiments used Xenopus oocytes expressing human ENaC. Methods of impedance analysis were used to determine whether the effects of changing lipid order indirectly altered ENaC conductance via changes of membrane area. These data were further corroborated with quantitative morphology on micrographs from oocytes membranes studied via electron microscopy. We report biphasic effects of cooling (stimulation followed by inhibition) on hENaC conductance. These effects were relatively slow (minutes) and were delayed from the actual bath temperature changes. Peak stimulation occurred at a calculated Tmax of 15.2. At temperatures below Tmax, ENaC conductance was inhibited with cooling. The effects of temperature on gNa were distinct from those observed on ion channels endogenous to Xenopus oocytes, where the membrane conductance decreased monoexponentially with temperature (t = 6.2°C). Similar effects were also observed in oocytes with reduced intra- and extracellular [Na+], thereby ruling out effects of self or feedback inhibition. Addition of CPZ or the mechanosensitive channel blocker, Gd+3, caused inhibition of ENaC. The effects of Gd+3 were also attributed to its ability to partition into the outer membrane leaflet and to decrease anisotropy. None of the effects of temperature, CPZ, or Gd+3 were accompanied by changes of membrane area, indicating the likely absence of effects on channel trafficking. However, CPZ and Gd+3 altered membrane capacitance in an opposite manner to temperature, consistent with effects on the membrane-dielectric properties. The reversible effects of both Gd+3 and CPZ could also be blocked by cooling and trapping these agents in the rigidified membrane, providing further evidence for their mechanism of action. Our findings demonstrate a novel regulatory mechanism of ENaC.
A Gastropod Toxin Selectively Slows Early Transitions in the Shaker K Channel's Activation Pathway
Authors: Sack Jon T. , Aldrich Richard W. , Gilly William F.
Published: May 2004

A toxin from a marine gastropod's defensive mucus, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT), was found to inhibit voltage-gated potassium channels by a novel mechanism. Voltage-clamp experiments with Shaker K channels reveal that externally applied BrMT slows channel opening but not closing. BrMT slows K channel activation in a graded fashion: channels activate progressively slower as the concentration of BrMT is increased. Analysis of single-channel activity indicates that once a channel opens, the unitary conductance and bursting behavior are essentially normal in BrMT. Paralleling its effects against channel opening, BrMT greatly slows the kinetics of ON, but not OFF, gating currents. BrMT was found to slow early activation transitions but not the final opening transition of the Shaker ILT mutant, and can be used to pharmacologically distinguish early from late gating steps. This novel toxin thus inhibits activation of Shaker K channels by specifically slowing early movement of their voltage sensors, thereby hindering channel opening. A model of BrMT action is developed that suggests BrMT rapidly binds to and stabilizes resting channel conformations.
The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis
Authors: Loïodice Isabelle , Alves Annabelle , Rabut Gwénaël , van Overbeek Megan , Ellenberg Jan , Sibarita Jean-Baptiste , Doye Valérie
Published: May 2004

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of ∼30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.
Functional Characterization of Dma1 and Dma2, the Budding Yeast Homologues ofSchizosaccharomyces pombeDma1 and Human Chfr
Authors: Fraschini Roberta , Bilotta Denis , Lucchini Giovanna , Piatti Simonetta
Published: May 2004

Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis. In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected. We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints. We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis. The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation. In addition, dma1Δ dma2Δ cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs. Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase.
Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements
Authors: Naslavsky Naava , Weigert Roberto , Donaldson Julie G.
Published: May 2004

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.
Protein Targeting of an Unusual, Evolutionarily Conserved Adenylate Kinase to a Eukaryotic Flagellum
Authors: Pullen Timothy J. , Ginger Michael L. , Gaskell Simon J. , Gull Keith
Published: May 2004

The eukaryotic flagellum is a large structure into which specific constituent proteins must be targeted, transported and assembled after their synthesis in the cytoplasm. Using Trypanosoma brucei and a proteomic approach, we have identified and characterized a novel set of adenylate kinase proteins that are localized to the flagellum. These proteins represent unique isoforms that are targeted to the flagellum by an N-terminal extension to the protein and are incorporated into an extraaxonemal structure (the paraflagellar rod). We show that the N-terminal extension is both necessary for isoform location in the flagellum and sufficient for targeting of a green fluorescent protein reporter protein to the flagellum. Moreover, these N-terminal extension sequences are conserved in evolution and we find that they allow the identification of novel adenylate kinases in the genomes of humans and worms. Given the existence of specific isoforms of certain central metabolic enzymes, and targeting sequences for these isoforms, we suggest that these isoforms form part of a complex, “solid-phase” metabolic capability that is built into the eukaryotic flagellum.
Cell Cycle-dependent Regulation of a Human DNA Helicase That Localizes in DNA Damage Foci
Authors: Gu Jinming , Xia Xiaobo , Yan Peijun , Liu Hanjian , Podust Vladimir N. , Reynolds Albert B. , Fanning Ellen
Published: May 2004

Mutational studies of human DNA helicase B (HDHB) have suggested that its activity is critical for the G1/S transition of the cell cycle, but the nature of its role remains unknown. In this study, we show that during G1, ectopically expressed HDHB localizes in nuclear foci induced by DNA damaging agents and that this focal pattern requires active HDHB. During S and G2/M, HDHB localizes primarily in the cytoplasm. A carboxy-terminal domain from HDHB confers cell cycle-dependent localization, but not the focal pattern, to a reporter protein. A cluster of potential cyclin-dependent kinase phosphorylation sites in this domain was modified at the G1/S transition and maintained through G2/M of the cell cycle in vivo, coincident with nuclear export of HDHB. Serine 967 of HDHB was the major site phosphorylated in vivo and in vitro by cyclin-dependent kinases. Mutational analysis demonstrated that phosphorylation of serine 967 is crucial in regulating the subcellular localization of ectopically expressed HDHB. We propose that the helicase of HDHB operates primarily during G1 to process endogenous DNA damage before the G1/S transition, and it is largely sequestered in the cytoplasm during S/G2.
Pathway Miner: extracting gene association networks from molecular pathways for predicting the biological significance of gene expression microarray data
Authors: Pandey Ritu , Guru Raghavendra K. , Mount David W.
Published: May 2004

Summary: We have developed a web-based system (Pathway Miner) for visualizing gene expression profiles in the context of biological pathways. Pathway Miner catalogs genes based on their role in metabolic, cellular and regulatory pathways. A Fisher exact test is provided as an option to rank pathways. The genes are mapped onto pathways and gene product association networks are extracted for genes that co-occur in pathways. The networks can be filtered for analysis based on user-selected options.

Availability: Pathway Miner is a freely available web accessible tool at http://www.biorag.org/pathway.html
ClutrFree: cluster tree visualization and interpretation
Authors: Bidaut Ghislain , Ochs Michael F.
Published: May 2004

Summary: ClutrFree facilitates the visualization and interpretation of clusters or patterns computed from microarray data through a graphical user interface that displays patterns, membership information of the genes and annotation statistics simultaneously. ClutrFree creates a tree linking the patterns based on similarity, permitting the navigation among patterns identified by different algorithms or by the same algorithm with different parameters, and aids the inferring of conclusions from a microarray experiment.

Availability: The ClutrFree Java source code and compiled bytecode are available as a package under the GNU General Public License at http://bioinformatics.fccc.edu

Supplementary information: Extended documentation on the software usage, as well as supplemental figures and an appropriate test dataset are provided on the website.