bluuebunny/crossref_metadata_2025 huggingface dataset:
Higher order arrangement of the eukaryotic nuclear bodies
Authors: Wang I-Fan , Reddy Narsa M. , Shen C.-K. James
Published: October 2002

The nuclei of eukaryotic cells consist of discrete substructures. These substructures include the nuclear bodies, which have been implicated in a number of biological processes such as transcription and splicing. However, for most nuclear bodies, the details of involvement in these processes in relation to their three-dimensional distributions in the nucleus are still unclear. Through the analysis of TDP, a protein functional in both transcriptional repression and alternative splicing, we have identified a new category of nuclear bodies within which the TDP molecules reside. Remarkably, the TDP bodies (TBs) colocalize or overlap with several different types of nuclear bodies previously suggested to function in transcription or splicing. Of these nuclear bodies, the Gemini of coiled bodies (GEM) seems to associate with TB through the interaction between survival motor neuron (SMN) protein and TDP. Furthermore, TB sometimes appears to be the bridge of two or more of these other nuclear bodies. Our data suggest the existence of a hierarchy and possibly functional arrangement of the nuclear bodies within the eukaryotic nuclei.
Regulation of a xenobiotic sulfonation cascade by nuclear pregnane X receptor (PXR)
Authors: Sonoda Junichiro , Xie Wen , Rosenfeld John M. , Barwick Joyce L. , Guzelian Philip S. , Evans Ronald M.
Published: October 2002

The nuclear receptor PXR (pregnane X receptor) protects the body from hepatotoxicity of secondary bile acids such as lithocholic acid (LCA) by inducing expression of the hydroxylating cytochrome P450 enzyme CYP3A and promoting detoxification. We found that activation of PXR also increases the activity and gene expression of the phase II conjugating enzyme dehydroepiandrosterone sulfotransferase (STD) known to sulfate LCA to facilitate its elimination. This activation is direct and appears to extend to other xenobiotic sulfotransferases as well as to 3′-phosphoadenosine 5′-phosphosulfate synthetase 2 (PAPSS2), an enzyme that generates the donor cofactor for the reaction. Because sulfation plays an important role in the metabolism of many xenobiotics, prescription drugs, and toxins, we propose that PXR serves as a master regulator of the phase I and II responses to facilitate rapid and efficient detoxification and elimination of foreign chemicals.
Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA
+
hexamers
Authors: Hansson A. , Willows R. D. , Roberts T. H. , Hansson M.
Published: October 2002

Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg
2+
into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA
+
) family of proteins. AAA
+
proteins are Mg
2+
-dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.
Class I negative CD8 T cells reveal the confounding role of peptide-transfer onto CD8 T cells stimulated with soluble H2-Kbmolecules
Authors: Schott Eckart , Bertho Nicolas , Ge Qing , Maurice Madelon M. , Ploegh Hidde L.
Published: October 2002

Crosslinking of the T cell receptor has been proposed to be a prerequisite for T cell activation. Although the evidence supports this notion for CD4 T cells, the situation for CD8 T cells is less clear. Soluble class I monomers have been used to determine activation requirementsin vitrowith contradictory results. The possibility of transfer of peptide from soluble class I molecules onto class I molecules present on the surface of CD8 T cells, with ensuing presentation to other CD8 T cells, has been widely ignored. We show that monomeric and tetrameric class I molecules as well as free peptide can stimulate naïve CD8 T cellsin vitro. We generate and characterize CD8 T cells that express the OT-I T cell receptor (for Kb/SIINFEKL) yet lack Kband Dbmolecules, and show that their activation requirements differ from their class I positive counterparts when stimulated with soluble Kbmolecules. By eliminating the confounding effect of peptide transfer, we unmask the true activation requirements for naïve CD8 T cells and show that multivalent engagement of T cell receptors, as well as costimulation, is required for optimal stimulation.
B cells develop in the zebrafish pancreas
Authors: Danilova Nadia , Steiner Lisa A.
Published: October 2002

The zebrafish, with its transparent free-living embryo, is a useful organism for investigating early stages in lymphopoiesis. Previously, we showed that T cells differentiate in the thymus by day 4, but no sites for B cell differentiation were seen until 3 weeks. We report here that on day 4, we detect rearrangements of genes encoding B cell receptors in DNA extracted from whole fish. Also by day 4, rag1 transcripts are seen in the pancreas, an organ not previously associated with lymphopoiesis; by day 10, Igμ transcripts are detected here. Thus, in zebrafish, the pancreas assumes the role of both the liver in fetal mice and the spleen in neonatal mice.
DBC2
, a candidate for a tumor suppressor gene involved in breast cancer
Authors: Hamaguchi Masaaki , Meth Jennifer L. , von Klitzing Christine , Wei Wen , Esposito Diane , Rodgers Linda , Walsh Tom , Welcsh Piri , King Mary-Claire , Wigler Michael H.
Published: October 2002

A previously uncharacterized gene,
DBC2
(
deleted in breast cancer
), was cloned from a homozygously deleted region at human chromosome 8p21.
DBC2
contains a highly conserved RAS domain and two putative protein interacting domains. Our analyses indicate that
DBC2
is the best candidate tumor suppressor gene from this region. It lies within the epicenter of the deletions and is homozygously deleted in 3.5% (7/200) of breast tumors. Mutation analysis of
DBC2
led to discovery of two instances of somatic missense mutations in breast tumor specimens, whereas no missense mutations were found in other candidates from the region. Unlike other genes in the region, expression of
DBC2
is often extinguished in breast cancer cells or tissues. Moreover, our functional analysis revealed that
DBC2
expression in breast cancer cells lacking
DBC2
transcripts causes growth inhibition. By contrast, expression of a somatic mutant discovered in a breast cancer specimen does not suppress the growth of breast cancer cells.
A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity
Authors: Bufler Philip , Azam Tania , Gamboni-Robertson Fabia , Reznikov Leonid L. , Kumar Sanjay , Dinarello Charles A. , Kim Soo-Hyun
Published: October 2002

IL-1F7 was discovered in expressed sequence tag databases as a member of the increasing family of proteins sharing sequence homology to IL-1α/β, IL-1Ra, and IL-18. In the present study using immunohistochemical staining, IL-1F7 was localized in human peripheral monocytic cells, suggesting its role in immune regulation. Recombinant human IL-1F7b was shown to bind to the IL-18Rα but without IL-18 agonistic or antagonistic function. Using chemical cross-linking, we observed that, unlike IL-18, IL-1F7b fails to recruit the IL-18Rβ chain to form a functionally active, ternary complex with the IL-18Rα chain. IL-1F7b shares two conserved amino acids with IL-18 (Glu-35 and Lys-124), which participate in the interaction of IL-18 with the IL-18Rα chain as well as the IL-18-binding protein (IL-18BP), a secreted protein that neutralizes IL-18 activity. In testing whether IL-1F7b interacts with IL-18BP, we unexpectedly observed that IL-1F7b enhanced the ability of IL-18BP to inhibit IL-18-induced IFNγ by 25–30% in a human natural killer cell line. This effect was observed primarily at limiting concentrations of IL-18BP (3.12–12.5 ng/ml) and at a 50- to 100-fold molar excess of IL-1F7b. Similar results were obtained by using isolated human peripheral blood mononuclear cells. To study the molecular basis of this effect we performed binding studies of IL-1F7b and IL-18BP. After cross-linking, a high molecular weight complex consisting of IL-1F7b and IL-18BP was observed on SDS/PAGE. We propose that after binding to IL-18BP, IL-1F7b forms a complex with IL-18Rβ, depriving the β-chain of forming a functional receptor complex with IL-18Rα and thus inhibiting IL-18 activity.
Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible
N
-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line
Authors: Reeves Philip J. , Callewaert Nico , Contreras Roland , Khorana H. Gobind
Published: October 2002

An HEK293S cell line resistant to ricin was prepared by mutagenesis by using ethyl methanesulfonate. It was shown to lack
N
-acetylglucosaminyltransferase I (GnTI) activity, and consequently unable to synthesize complex N-glycans. The tetracycline-inducible opsin expression system was assembled into this GnTI
−
HEK293S cell line. Stable cell lines were isolated that gave tetracycline/sodium butyrate-inducible expression of the WT opsin gene at levels comparable with those observed in the parent tetracycline-inducible HEK293S cell line. Analysis of the N-glycan in rhodopsin expressed by the HEK293S GnTI
−
stable cell line showed it to be Man
5
GlcNAc
2
. In a larger-scale expression experiment (1.1 liter) a WT opsin production level of 6 mg/liter was obtained. Further, the toxic constitutively active rhodopsin mutant, E113Q/E134Q/M257Y, previously shown to require inducible expression, has now been expressed in an HEK293S GNTI
−
-inducible cell line at levels comparable with those obtained with WT rhodopsin.
Mice with chronic norepinephrine deficiency resemble amphetamine-sensitized animals
Authors: Weinshenker David , Miller Nicole S. , Blizinsky Katherine , Laughlin Marc L. , Palmiter Richard D.
Published: October 2002

Acute pharmacological blockade of α1 adrenoreceptors (ARs) attenuates the locomotor response to amphetamine (LRA). We took a genetic approach to study how norepinephrine (NE) signaling modulates psychostimulant responses by testing LRA in dopamine β-hydroxylase knockout (
Dbh
−/−) mice that lack NE. Surprisingly,
Dbh
−/− animals were hypersensitive to the behavioral effects of amphetamine. Amphetamine (2 mg/kg) elicited greater locomotor activity in
Dbh
−/− mice compared to controls, whereas 5 mg/kg caused stereotypy in
Dbh
−/− mice, which is only observed in control mice at higher doses. Prazosin, an α1AR antagonist, attenuated LRA in
Dbh
+/− mice but had no effect in
Dbh
−/− mice. Changes in the sensitivity of dopamine (DA)-signaling pathways may contribute to the altered amphetamine responses of
Dbh
−/− mice because they were relatively insensitive to a D1 agonist and hypersensitive to a D2 agonist. Daily amphetamine administration resulted in behavioral sensitization in both
Dbh
+/− and
Dbh
−/− mice, demonstrating that NE is not required for the development or expression of behavioral sensitization. Daily prazosin administration blunted but did not completely block locomotor sensitization in
Dbh
+/− mice, suggesting that α1AR signaling contributes to, but is not required for sensitization in
Dbh
+/− control animals. We conclude that in contrast to acute α1AR blockade, chronic NE deficiency induces changes similar to sensitization, perhaps by altering DA-signaling pathways.
Long-term systemic therapy of Fabry disease in a knockout mouse by adeno-associated virus-mediated muscle-directed gene transfer
Authors: Takahashi Hiroshi , Hirai Yukihiko , Migita Makoto , Seino Yoshihiko , Fukuda Yuh , Sakuraba Hitoshi , Kase Ryoichi , Kobayashi Toshihide , Hashimoto Yasuhiro , Shimada Takashi
Published: October 2002

Fabry disease is a systemic disease caused by genetic deficiency of a lysosomal enzyme, α-galactosidase A (α-gal A), and is thought to be an important target for enzyme replacement therapy. We studied the feasibility of gene-mediated enzyme replacement for Fabry disease. The adeno-associated virus (AAV) vector containing the α-gal A gene was injected into the right quadriceps muscles of Fabry knockout mice. A time course study showed that α-gal A activity in plasma was increased to ≈25% of normal mice and that this elevated activity persisted for up to at least 30 weeks without development of anti-α-gal A antibodies. The α-gal A activity in various organs of treated Fabry mice remained 5–20% of those observed in normal mice. Accumulated globotriaosylceramide in these organs was completely cleared by 25 weeks after vector injection. Reduction of globotriaosylceramide levels was also confirmed by immunohistochemical and electronmicroscopic analyses. Echocardiographic examination of treated mice demonstrated structural improvement of cardiac hypertrophy 25 weeks after the treatment. AAV vector-mediated muscle-directed gene transfer provides an efficient and practical therapeutic approach for Fabry disease.
Suppressors of the egg-laying defective phenotype of sel-12 presenilin mutants implicate the CoREST corepressor complex in LIN-12/Notch signaling in C. elegans
Authors: Jarriault Sophie , Greenwald Iva
Published: October 2002

Presenilin is an essential component of the LIN-12/Notch signaling pathway and also plays a critical role in the genesis of Alzheimer's disease. Previously, a screen for suppressors of the egg-laying defective phenotype caused by partial loss of presenilin activity inCaenorhabditis elegans identified a number of new sprgenes that are potentially involved in the regulation of LIN-12/Notch signaling or presenilin activity. Here we report the molecular identity of two spr genes, spr-1 and spr-5. Our genetic analysis indicates that loss of spr-1 elevateslin-12/Notch gene activity in many different cell fate decisions, suggesting that spr-1 is a negative regulator of LIN-12/Notch signaling. Sequence analysis revealed that spr-1is an ortholog of human CoREST, a known corepressor. SPR-1 is localized to the nucleus and acts in a cell-autonomous manner; furthermore, human CoREST can substitute for SPR-1 in C. elegans. We also show that spr-5 encodes a homolog of p110b, another known member of the CoREST corepressor complex. Our results suggest that the CoREST corepressor complex might be functionally conserved in worms, and we discuss the potential role of SPR-1 and SPR-5 in the repression of transcription of genes involved in, or downstream of, LIN-12/Notch signal transduction.
Lethality of Drosophila lacking TSC tumor suppressor function rescued by reducing dS6K signaling
Authors: Radimerski Thomas , Montagne Jacques , Hemmings-Mieszczak Maja , Thomas George
Published: October 2002

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in one of two tumor suppressor genes, TSC1 andTSC2. Here, we show that absence of Drosophila Tsc1/2 leads to constitutive dS6K activation and inhibition of dPKB, the latter effect being relieved by loss of dS6K. In contrast, the dPTEN tumor suppressor, a negative effector of PI3K, has little effect on dS6K, but negatively regulates dPKB. More importantly, we demonstrate that reducing dS6K signaling rescues early larval lethality associated with loss of dTsc1/2 function, arguing that the S6K pathway is a promising target for the treatment of TSC.
A cellular memory module conveys epigenetic inheritance of hedgehog expression during Drosophila wing imaginal disc development
Authors: Maurange Cédric , Paro Renato
Published: October 2002

In Drosophila, the Trithorax-group (trxG) and Polycomb-group (PcG) proteins interact with chromosomal elements, termed Cellular Memory Modules (CMMs). By modifying chromatin, this ensures a stable heritable maintenance of the transcriptional state of developmental regulators, like the homeotic genes, that is defined embryonically. We asked whether such CMMs could also control expression of genes involved in patterning imaginal discs during larval development. Our results demonstrate that expression of the hedgehog gene, once activated, is maintained by a CMM. In addition, our experiments indicate that the switching of such CMMs to an active state during larval stages, in contrast to embryonic stages, may require specifictrans-activators. Our results suggest that the patterning of cells in particular developmental fields in the imaginal discs does not only rely on external cues from morphogens, but also depends on the previous history of the cells, as the control by CMMs ensures a preformatted gene expression pattern.
An empirical investigation of context‐dependent communications between auditors and bankers
Authors: Dreike Almer Elizabeth , Brody Richard G.
Published: October 2002

Despite attempts to close the expectation gap, researchers in the USA and other countries continue to demonstrate the existence of differences between auditors’ beliefs about their responsibilities and the public’s understanding of the audit function. This study considers how ambiguous language in auditor communications, and the context in which it occurs, contribute to the expectation gap. An experiment examined auditors’ and bankers’ understandings of the phrase “substantial doubt” used in a going concern audit report. It was predicted and found that auditors and bankers had different general understandings of the meaning of “substantial doubt” and their understandings were conditioned on ownership structure, a contextual variable. The results demonstrate the potentially crucial role of context in ambiguous communications with users. Implications of these results are discussed, as are suggested areas for future research.
Auditors’, bankers’ and insolvency practitioners’ “going‐concern” opinion logit model
Authors: Constantinides Sylvia
Published: October 2002

“Going‐concern” opinions are judgemental and therefore fraught with numerous problems. The inability to establish the “correctness” or otherwise of the “going‐concern” opinion decision has also been puzzling practitioners throughout the years. Exploratory interviews revealed that a “going‐concern” opinion decision involves not only auditors but also bankers and insolvency practitioners particularly under conditions of financial distress. The purpose of this paper is to determine the factors influencing “going‐concern” opinion decisions of auditors, bankers and insolvency practitioners (IPs) for financially distressed client‐firms. Using data collected via a postal questionnaire, a logistic regression model is developed with an overall correct classification of 81.11 per cent. The model uses indicators of “going‐concern” uncertainties and events/action triggers that invalidate the “going‐concern” status of a financially distressed firm. Further research examining the impact of other (behavioural) factors is required but also highlighting any differences between these three professional user groups of company accounts.
Market‐oriented behavior
Authors: Cadogan John W. , Sundqvist Sanna , Salminen Risto T. , Puumalainen Kaisu
Published: October 2002

The activities of service and product firms are compared in terms of their market‐oriented behavior in their export operations (i.e. their export market‐oriented (EMO) behavior). Empirical analysis conducted on a sample of 783 Finnish exporters containing both service and product firms uncovered several interesting differences: service and product firms differed in their level of EMO behavior; the direct effects from EMO behavior to various dimensions of export success were invariant across the samples; however, the export environment moderated the link between EMO behavior and export profit performance in different ways across the samples. The results indicate that EMO behavior may be more appropriate under certain environmental conditions, and less appropriate under others. However, the nature of the relationship between EMO behavior and export success may also depend on whether the firm’s core export market offerings are services or physical products.
Redefining market orientation from a relationship perspective
Authors: Helfert Gabriele , Ritter Thomas , Walter Achim
Published: October 2002

Many studies have shown that market orientation is important for firms because it has a positive impact on performance. However, several studies have indicated that the relation between a firm’s market orientation and its success is sometimes weak and that moderating variables need to be considered at least under certain circumstances. As such the overall message from the market orientation studies is not clear. The usefulness of the market orientation concept must also be questioned when looking at the realities of business markets. In most if not all cases the firms’ “surroundings” should be seen as a network of inter‐organizational relationships rather than an anonymous market. Therefore, in this paper the notion of market orientation is explored with particular focus on inter‐organizational relationships. Hereby, it is argued that the relationships are important and that the overall market orientation of firms needs to be translated to a relationship level in order to be effective. It is further argued that market orientation on a relationship level can be interpreted in terms of a firm’s employed resources and executed activities dedicated to relational exchange processes.
Functional aspects of co‐variant surface charges in an antibody fragment
Authors: Hugo Nicolas , Lafont Virginie , Beukes Mervyn , Altschuh Danièle
Published: October 2002

A mutational analysis of three co‐variant pairs of residues, located at the surface of a single‐chain fragment, variable (scFv), remote from the antigen‐binding site, was performed to investigate the tolerance of these positions to amino acid changes. The replacements consisted of the elimination or addition of charges, or in their replacement by a charge of opposite sign. As measured by Biacore, antigen‐binding kinetics and specificity were essentially unaffected by the mutations. The purified scFvs remained mostly 100% active for 14 h, and their sensitivity to guanidinium‐chloride denaturation was similar. These observations indicate that the mutations did not affect antigen‐binding properties and that protein folding was conserved. However, the various scFvs differed greatly in half‐life in periplasmic extracts (<4 h to >16 h at 25°C). The deleterious effect on half‐life produced by single mutations could be reversed by introducing a second mutation that restores the natural combination of amino acids in the co‐variant pair, indicating that the consequence of charge modifications at these locations depends on the sequence context. We propose that the differences in half‐life result from differences in aggregation propensities with other periplasmic proteins, related to the presence of charged patches at the surface of the scFvs. The practical implication is that changes in surface charge may drastically affect the level of active molecules in complex protein mixtures, a potentially important consideration in engineering scFvs for biotechnological or medical purposes.
MAMMOTH (Matching molecular models obtained from theory): An automated method for model comparison
Authors: Ortiz Angel R. , Strauss Charlie E.M. , Olmea Osvaldo
Published: October 2002

Advances in structural genomics and protein structure prediction require the design of automatic, fast, objective, and well benchmarked methods capable of comparing and assessing the similarity of low‐resolution three‐dimensional structures, via experimental or theoretical approaches. Here, a new method for sequence‐independent structural alignment is presented that allows comparison of an experimental protein structure with an arbitrary low‐resolution protein tertiary model. The heuristic algorithm is given and then used to show that it can describe random structural alignments of proteins with different folds with good accuracy by an extreme value distribution. From this observation, a structural similarity score between two proteins or two different conformations of the same protein is derived from the likelihood of obtaining a given structural alignment by chance. The performance of the derived score is then compared with well established, consensus manual‐based scores and data sets. We found that the new approach correlates better than other tools with the gold standard provided by a human evaluator. Timings indicate that the algorithm is fast enough for routine use with large databases of protein models. Overall, our results indicate that the new program (MAMMOTH) will be a good tool for protein structure comparisons in structural genomics applications. MAMMOTH is available from our web site at http://physbio.mssm.edu/∼ortizg/.
A statistically derived parameterization for the collagen triple‐helix
Authors: Rainey Jan K. , Goh M. Cynthia
Published: October 2002

The triple‐helix is a unique secondary structural motif found primarily within the collagens. In collagen, it is a homo‐ or hetero‐tripeptide with a repeating primary sequence of (Gly‐X‐Y)n, displaying characteristic peptide backbone dihedral angles. Studies of bulk collagen fibrils indicate that the triple‐helix must be a highly repetitive secondary structure, with very specific constraints. Primary sequence analysis shows that most collagen molecules are primarily triple‐helical; however, no high‐resolution structure of any entire protein is yet available. Given the drastic morphological differences in self‐assembled collagen structures with subtle changes in assembly conditions, a detailed knowledge of the relative locations of charged and sterically bulky residues in collagen is desirable. Its repetitive primary sequence and highly conserved secondary structure make collagen, and the triple‐helix in general, an ideal candidate for a general parameterization for prediction of residue locations and for the use of a helical wheel in the prediction of residue orientation. Herein, a statistical analysis of the currently available high‐resolution X‐ray crystal structures of model triple‐helical peptides is performed to produce an experimentally based parameter set for predicting peptide backbone and Cβ atom locations for the triple‐helix. Unlike existing homology models, this allows easy prediction of an entire triple‐helix structure based on all existing high‐resolution triple‐helix structures, rather than only on a single structure or on idealized parameters. Furthermore, regional differences based on the helical propensity of residues may be readily incorporated. The parameter set is validated in terms of the predicted bond lengths, backbone dihedral angles, and interchain hydrogen bonding.
Prediction of the disulfide‐bonding state of cysteines in proteins at 88% accuracy
Authors: Martelli Pier Luigi , Fariselli Piero , Malaguti Luca , Casadio Rita
Published: October 2002

The task of predicting the cysteine‐bonding state in proteins starting from the residue chain is addressed by implementing a new hybrid system that combines a neural network and a hidden Markov model (hidden neural network). Training is performed using 4136 cysteine‐containing segments extracted from 969 nonhomologous proteins of well‐resolved three‐dimensional structure. After a 20‐fold cross‐validation procedure, the efficiency of the prediction scores as high as 88% and 84%, when measured on cysteine and protein basis, respectively. These results outperform previously described methods for the same task.
Structural‐based mutational analysis of d‐aminoacylase from Alcaligenes faecalis DA1
Authors: Hsu Cheng‐Sheng , Lai Wen‐Lin , Chang Wei‐Wei , Liaw Shwu‐Huey , Tsai Ying‐Chieh
Published: October 2002

d‐Aminoacylase is an attractive candidate for commercial production of d‐amino acids through its catalysis in the zinc‐assistant hydrolysis of N‐acyl‐d‐amino acids. We report here the cloning, expression, and structural‐based mutation of the d‐aminoacylase from Alcaligenes faecalis DA1. A 1,007‐bp PCR product amplified with degenerate primers, was used to isolate a 4‐kb genomic fragment, encoding a 484‐residue d‐aminoacylase. The enzyme amino‐terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D‐PSSM to be similar to urease and dihydroorotase, which have grouped into a novel α/β‐barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His‐67, His‐69, and His‐250, putative metal ligands in d‐aminoacylase, have been mutated previously, the remaining histidine (His‐220) and aspartate (Asp‐366) Asp‐65, and four cysteines were then characterized. Substitution of Asp‐65, Cys‐96, His‐220, and Asp‐366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc‐binding ability, revealing that Cys‐96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino‐terminal segment including Asp‐65 may be responsible for structural stabilization.
13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching
Authors: Asakura Tetsuo , Yao Juming
Published: October 2002

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk‐like fiber. The silk I structure has been recently resolved by 13C solid‐state NMR as a “repeated β‐turn type II structure.” Here, we used 13C solid‐state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the 13C CP/MAS NMR revealed a broad and asymmetric peak for the Ala Cβ carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala‐Gly‐Ser‐Gly‐Ala‐Gly sequences), 18% distorted β‐turn, 13% β‐sheet (parallel Ala residues), and 25% β‐sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr‐rich region, 22% in both distorted β‐turn and distorted β‐sheet. Such a heterogeneous structure including distorted β‐turn can be observed for the peptides (AG)n (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala‐Gly‐Ser‐Gly‐Ala‐Gly)n in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala Cβ peak of 13C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios.
Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP‐independent control of ATP‐sensitive K+ channels
Authors: Ainscow Edward K. , Mirshamsi Shirin , Tang Teresa , Ashford Michael L. J. , Rutter Guy A.
Published: October 2002

Glucose‐responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP‐sensitive K+ (KATP) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP]c) was monitored in vivo using adenoviral‐driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of KATP channels in GR neurons, glucose (3 or 15 mm) caused no detectable increase in [ATP]c, monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25‐luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mm) with a significant increase in [ATP]c. Both neurons and glia from the cerebellum, a glucose‐unresponsive region of the brain, responded robustly to 3 or 15 mm glucose with increases in [ATP]c. Further implicating an ATP‐independent mechanism of KATP channel closure in hypothalamic neurons, removal of extracellular glucose (10 mm) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mm) of intracellular ATP. Neurons from both brain regions responded to 5 mm lactate (but not pyruvate) with an oligomycin‐sensitive increase in [ATP]c. High levels of the plasma membrane lactate‐monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed KATP channels in GR neurons. These data suggest that (1) ATP‐independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells.
Individual mouse taste cells respond to multiple chemical stimuli
Authors: Caicedo Alejandro , Kim Kyung‐Nyun , Roper Stephen D.
Published: October 2002

Sensory organs are specialized to detect and decode stimuli in terms of intensity and quality. In the gustatory system, the process of identifying and distinguishing taste qualities (e.g. bitter versus sweet) begins in taste buds. A central question in gustatory research is how information about taste quality is extracted by taste receptor cells. For instance, whether and how individual taste cells respond to multiple chemical stimuli is still a matter for debate. A recent study showed that taste cells expressing bitter‐responsive taste receptors do not also express sweet‐responsive taste receptors and vice versa. These results suggest that the gustatory system may use separate cellular pathways to process bitter and sweet signals independently. Results from electrophysiological studies, however, reveal that individual taste receptor cells respond to stimuli representing multiple taste qualities. Here we used non‐invasive Ca2+ imaging in slices of lingual tissue containing taste buds to address the issue of quality detection in murine taste receptor cells. We recorded calcium transients elicited by chemical stimuli representing different taste qualities (sweet, salty, sour and bitter). Many receptor cells (38 %) responded to multiple taste qualities, with some taste cells responding to both appetitive (‘sweet’) and aversive (‘bitter’) stimuli. Thus, there appears to be no strict and separate detection of taste qualities by distinct subpopulations of taste cells in peripheral gustatory sensory organs in mice.
Intestinal Nematode Infection Ameliorates Experimental Colitis in Mice
Authors: Khan W. I. , Blennerhasset P. A. , Varghese A. K. , Chowdhury S. K. , Omsted P. , Deng Y. , Collins S. M.
Published: October 2002

Epidemiological studies suggest that inflammatory bowel disease (IBD) is common in developed countries and rare in countries where intestinal nematode infections are common. T cells are critical in many immune responses, including those associated with IBD and nematode infection. Among the distinct T helper (Th) cell subsets, Th1-type immune response is predominantly associated with Crohn's disease, while many nematode infections generate a strong Th2 response. The reciprocal cross regulation between Th1 and Th2 cells suggests that generation of a Th2 response by nematodes could prevent or reduce the effects of Th1-mediated diseases. In the present study, we investigated the effect of polarizing the immune response toward the Th2 type, using intestinal nematode infection, on subsequent experimental colitis. Mice were infected with the intestinal nematodeTrichinella spiralisand allowed to recover before colitis was induced with dinitrobenzene sulfonic acid. The mice were sacrificed postcolitis to assess colonic damage macroscopically, histologically, and by myeloperoxidase (MPO) activity and Th cytokines. Prior nematode infection reduced the severity of colitis both macroscopically and histologically together with a decreased mortality and was correlated with a down-regulation of MPO activity, Th1-type cytokine expression in colonic tissue, and emergence of a Th2-type immune response. These results indicate a protective role of nematode infection in Th1 cell-driven inflammation and prompt consideration of a novel therapeutic strategy in IBD based on immunological distraction.
Characterization of the
Pasteurella multocida hgbA
Gene Encoding a Hemoglobin-Binding Protein
Authors: Bosch Montserrat , Garrido M. Elena , Llagostera Montserrat , Pérez de Rozas Ana M. , Badiola Ignacio , Barbé Jordi
Published: October 2002

Reverse transcriptase PCR analyses have demonstrated that open reading frames (ORFs) PM0298, PM0299, and PM0300 of the animal pathogen
Pasteurella multocida
constitute a single transcriptional unit. By cloning and overexpression studies in
Escherichia coli
cells, the product of ORF PM0300 was shown to bind hemoglobin in vitro; this ORF was therefore designated
hgbA.
In vitro and in vivo quantitative assays demonstrated that the
P. multocida hgbA
mutant bound hemoglobin to the same extent as the wild-type strain, although the adsorption kinetics was slightly slower for the
hgbA
cells. In agreement with this, the virulence of
P. multocida hgbA
cells was not affected, suggesting that other functional hemoglobin receptor proteins must be present in this organism. On the other hand,
P. multocida
mutants defective in PM0298 and PM0299 could be isolated only when a plasmid containing an intact copy of the gene was present in the cells, suggesting that these genes are essential for the viability of this bacterial pathogen. By adapting the recombinase-based expression technology in vivo to
P. multocida
, we also demonstrated that the transcriptional PM0298-PM0299-
hgbA
unit is iron regulated and that its expression is triggered in the first 2 h following infection in a mouse model. Furthermore, hybridization experiments showed that the
hgbA
gene is widespread in
P. multocida
strains regardless of their serotype or the animal from which they were isolated.
Nitric Oxide Limits the Expansion of Antigen-Specific T Cells in Mice Infected with the Microfilariae ofBrugia pahangi
Authors: O'Connor Richard A. , Devaney Eileen
Published: October 2002

Infection of BALB/c mice with the microfilariae (Mf) of the filarial nematodeBrugia pahangiresults in an antigen-specific proliferative defect that is induced by high levels of NO. Using carboxyfluorescein diacetate succinimydl ester and cell surface labeling, it was possible to identify a population of antigen-specific T cells from Mf-infected BALB/c mice that expressed particularly high levels of CD4 (CD4hi). These cells proliferated in culture only when inducible NO synthase was inhibited and accounted for almost all of the antigen-specific proliferative response under those conditions. CD4hicells also expressed high levels of CD44, consistent with their status as activated T cells. A similar population of CD4hicells was observed in cultures from Mf-infected gamma interferon receptor knockout (IFN-γR−/−) mice. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining revealed that the CD4+T cells from Mf-infected wild-type mice were preferentially susceptible to apoptosis compared to CD4+T cells from IFN-γR−/−mice. These studies suggest that the expansion of antigen-specific T cells in Mf-infected mice is limited by NO.
Acquisition of Mn(II) in Addition to Fe(II) Is Required for Full Virulence of
Salmonella enterica
Serovar Typhimurium
Authors: Boyer E. , Bergevin I. , Malo D. , Gros P. , Cellier M. F. M.
Published: October 2002

The roles of the genes
feoB
(ABC ferrous iron transporter),
mntH
(proton-dependent manganese transporter), and
sitABCD
(putative ABC iron and/or manganese transporter) in
Salmonella
pathogenicity were investigated by using mutant strains deficient in one, two, or three transporters. Our results indicated that
sitABCD
encodes an important transporter of Mn(II) and Fe(II) which is required for full virulence in susceptible animals (
Nramp1
−/−
) and for replication inside
Nramp1
−/−
macrophages in vitro. The
mntH sitABCD
double mutant (mutant MS) showed minimal Mn(II) uptake and increased sensitivity to H
2
O
2
and to the divalent metal chelator 2,2′-dipyridyl (DP) and was defective for replication in macrophages. In vivo MS appeared to be as virulent as the
sitABCD
mutant in
Nramp1
−/−
animals. The ferrous iron transporter Feo was required for full virulence in 129/Sv
Nramp1
−/−
mice, and infection with multiple mutants lacking FeoB was not fatal. The
sitABCD feoB
mutant (mutant SF) and the
mntH sitABCD feoB
mutant (mutant MSF) showed minimal Fe(II) uptake and were slightly impaired for replication in susceptible macrophages. MSF showed reduced growth in minimal medium deficient in divalent cations. The role of the
mntH
gene, which is homologous to mammalian
Nramp
genes, was also investigated after overexpression in the double mutant MS. MntH preferred Mn(II) over Fe(II) and could suppress MS sensitivity to H
2
O
2
and to DP, and it also improved the intracellular survival in
Nramp1
−/−
macrophages. This study indicates that acquisition of Mn(II), in addition to Fe(II), is required for intracellular survival and replication of
Salmonella enterica
serovar Typhimurium in macrophages in vitro and for virulence in vivo.
The Transcriptional Regulator AlgR Is Essential for
Pseudomonas aeruginosa
Pathogenesis
Authors: Lizewski Stephen E. , Lundberg Derek S. , Schurr Michael J.
Published: October 2002

Chronic
Pseudomonas aeruginosa
lung infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. One
P. aeruginosa
virulence factor unique to CF isolates is overproduction of alginate, phenotypically termed mucoidy. Mucoidy is the result of increased transcription from the
algD
gene and is activated by the transcriptional regulator AlgR. Mutations in
algR
result in a nonmucoid phenotype and loss of twitching motility. Additionally, AlgR controls transcription of
algC
, encoding a dual-function enzyme necessary for both lipopolysaccharide (LPS) and alginate production. Therefore, to determine the effect of
algR
on
P. aeruginosa
virulence, an
algR
mutant was examined for sensitivity to reactive oxygen intermediates, killing by phagocytes, systemic virulence, and the ability to maintain a murine lung infection. We found that
P. aeruginosa
PAO700 (
algR
::Gm
r
) was less lethal than PAO1, as tested in an acute septicemia infection mouse model, and was cleared more efficiently in a mouse pneumonia model. Additionally, the
algR
mutant (PAO700) was more sensitive to hypochlorite. However, PAO700 was more resistant to hydrogen peroxide and killed less readily in an acellular myeloperoxidase assay than PAO1. There was little difference in killing between PAO1 and PAO700 with macrophage-like J774 cells and human polymorhonuclear leukocytes. Two-dimensional gel analysis of
P. aeruginosa algR
mutant and wild-type protein extracts revealed 47 differentially regulated proteins, suggesting that AlgR plays both a positive role and a negative role in gene expression. Together, these results imply that AlgR is necessary for virulence and regulates genes in addition to the genes associated with alginate and LPS production and pilus function.
Characterization of the Gene Encoding the Major Secreted Lysophospholipase A of
Legionella pneumophila
and Its Role in Detoxification of Lysophosphatidylcholine
Authors: Flieger Antje , Neumeister Birgid , Cianciotto Nicholas P.
Published: October 2002

We previously showed that
Legionella pneumophila
secretes, via its type II secretion system, phospholipase A activities that are distinguished by their specificity for certain phospholipids. In this study, we identified and characterized
plaA
, a gene encoding a phospholipase A that cleaves fatty acids from lysophospholipids. The
plaA
gene encoded a 309-amino-acid protein (PlaA) which had homology to a group of lipolytic enzymes containing the catalytic signature GDSL. In
Escherichia coli
, the cloned gene conferred trypsin-resistant hydrolysis of lysophosphatidylcholine and lysophosphatidylglycerol. An
L. pneumophila plaA
mutant was generated by allelic exchange. Although the mutant grew normally in standard buffered yeast extract broth, its culture supernatants lost greater than 80% of their ability to release fatty acids from lysophosphatidylcholine and lysophosphatidylglycerol, implying that PlaA is the major secreted lysophospholipase A of
L. pneumophila
. The mutant's reduced lipolytic activity was confirmed by growth on egg yolk agar and thin layer chromatography and was complemented by reintroduction of an intact copy of
plaA
. Overexpression of
plaA
completely protected
L. pneumophila
from the toxic effects of lysophosphatidylcholine, suggesting a role for PlaA in bacterial detoxification of lysophospholipids. The
plaA
mutant grew like the wild type in U937 cell macrophages and
Hartmannella vermiformis
amoebae, indicating that PlaA is not essential for intracellular infection of
L. pneumophila
. In the course of characterizing
plaA
, we discovered that wild-type legionellae secrete a phospholipid cholesterol acyltransferase activity, highlighting the spectrum of lipolytic enzymes produced by
L. pneumophila
.