bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of salivary agglutinin to IgA
Authors: LIGTENBERG Antoon J. M. , BIKKER Floris J. , DE BLIECK-HOGERVORST Jolanda M. A. , VEERMAN Enno C. I. , NIEUW AMERONGEN Arie V.
Published: September 2004

SAG (salivary agglutinin), which is identical to gp-340 (glycoprotein-340) from the lung, is encoded by DMBT1 (deleted in malignant brain tumours 1). It is a member of the SRCR (scavenger receptor cysteine-rich) superfamily and contains 14 SRCR domains, 13 of which are highly similar. SAG in saliva is partially complexed with IgA, which may be necessary for bacterial binding. The goal of the present study was to characterize the binding of purified SAG to IgA. SAG binds to a variety of proteins, including serum and secretory IgA, alkaline phosphatase-conjugated IgGs originating from rabbit, goat, swine and mouse, and lactoferrin and albumin. Binding of IgA to SAG is calcium dependent and is inhibited by 0.5 M KCl, suggesting that electrostatic interactions are involved. Binding of IgA was destroyed after reduction of SAG, suggesting that the protein moiety is involved in binding. To pinpoint further the binding domain for IgA on SAG, a number of consensus-based peptides of the SRCR domains and SRCR interspersed domains were designed and synthesized. ELISA binding studies with IgA indicated that only one of the peptides tested, comprising amino acids 18–33 (QGRVEVLYRGSWGTVC) of the 109-amino-acid SRCR domain, exhibited binding to IgA. This domain is identical to the domain of SAG that is involved in binding to bacteria. Despite this similar binding site, IgA did not inhibit binding of Streptococcus mutans to SAG or peptide. These results show that the binding of IgA to SAG is specifically mediated by a peptide sequence on the SRCR domains.
Heparan sulphate proteoglycans interact with neurocan and promote neurite outgrowth from cerebellar granule cells
Authors: AKITA Kaoru , TODA Munetoyo , HOSOKI Yuki , INOUE Mizue , FUSHIKI Shinji , OOHIRA Atsuhiko , OKAYAMA Minoru , YAMASHINA Ikuo , NAKADA Hiroshi
Published: September 2004

We found that neurocan, a major brain chondroitin sulphate proteoglycan, interacts with HSPGs (heparan sulphate proteoglycans) such as syndecan-3 and glypican-1. Binding of these HSPGs to neurocan was prevented by treatment of the HSPGs with heparitinases I and II, but not by treatment of neurocan with chondroitinase ABC. Scatchard plot analysis indicated that neurocan has two binding sites for these HSPGs with different affinities. It is known that neurocan in the rodent brain is proteolytically processed with aging into N- and C-terminal fragments. When a mixture of whole neurocan and N- and C-terminal fragments prepared from neonatal mouse brains or recombinant N- and C-terminal fragments was applied to a heparin column, the whole molecule and both the N- and C-terminal fragments bound to heparin. A centrifugation cell adhesion assay indicated that both the N- and C-terminal neurocan fragments could interact with these HSPGs expressed on the cell surface. To examine the biological significance of the HSPG–neurocan interaction, cerebellar granule cells expressing these HSPGs were cultured on the recombinant neurocan substrate. A significant increase in the rate of neurite outgrowth was observed on the wells coated with the C-terminal neurocan fragment, but not with the N-terminal one. Neurite outgrowth-promoting activity was inhibited by pretreatment of neurocan substrate with heparin or the addition of heparitinase I to culture medium. These results suggest that HSPGs such as syndecan-3 and glypican-1 serve as the cell-surface receptor of neurocan, and that the interaction of these HSPGs with neurocan through its C-terminal domain is involved in the promotion of neurite outgrowth.
Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism
Authors: RICE Gillian I. , THOMAS Daniel A. , GRANT Peter J. , TURNER Anthony J. , HOOPER Nigel M.
Published: September 2004

In the RAS (renin–angiotensin system), Ang I (angiotensin I) is cleaved by ACE (angiotensin-converting enzyme) to form Ang II (angiotensin II), which has effects on blood pressure, fluid and electrolyte homoeostasis. We have examined the kinetics of angiotensin peptide cleavage by full-length human ACE, the separate N- and C-domains of ACE, the homologue of ACE, ACE2, and NEP (neprilysin). The activity of the enzyme preparations was determined by active-site titrations using competitive tight-binding inhibitors and fluorogenic substrates. Ang I was effectively cleaved by NEP to Ang (1–7) (kcat/Km of 6.2×105 M−1·s−1), but was a poor substrate for ACE2 (kcat/Km of 3.3×104 M−1·s−1). Ang (1–9) was a better substrate for NEP than ACE (kcat/Km of 3.7×105 M−1·s−1 compared with kcat/Km of 6.8×104 M−1·s−1). Ang II was cleaved efficiently by ACE2 to Ang (1–7) (kcat/Km of 2.2×106 M−1·s−1) and was cleaved by NEP (kcat/Km of 2.2×105 M−1·s−1) to several degradation products. In contrast with a previous report, Ang (1–7), like Ang I and Ang (1–9), was cleaved with a similar efficiency by both the N- and C-domains of ACE (kcat/Km of 3.6×105 M−1·s−1 compared with kcat/Km of 3.3×105 M−1·s−1). The two active sites of ACE exhibited negative co-operativity when either Ang I or Ang (1–7) was the substrate. In addition, a range of ACE inhibitors failed to inhibit ACE2. These kinetic data highlight that the flux of peptides through the RAS is complex, with the levels of ACE, ACE2 and NEP dictating whether vasoconstriction or vasodilation will predominate.
A novel retinoic acid-response element requires an enhancer element mediator for transcriptional activation
Authors: HARRIS Laura R. , KAMARAINEN Olli-Pekka , SEVAKIVI Minna , MILLER Gwen C. , CLARKE James W. , POTTER Jennifer L. , BRIDGEWATER Laura C.
Published: September 2004

The Col11a2 gene codes for α2(XI), a subunit of type XI collagen that is a critical component of the cartilage extracellular matrix. The 5′ regulatory region of Col11a2 was subjected to deletional analysis to detect any regulatory element in addition to the two known chondrocyte-specific enhancer elements B/C and D/E. Deletion of the region from −342 to −242 bp reduced transcriptional activity to less than 50% of wild-type, but the sequence showed no independent ability to increase transcription from a minimal promoter. When cloned downstream of the D/E enhancer, however, a subsection of the sequence nearly doubled transcriptional activity and produced an additional 3-fold activation in response to RA (retinoic acid). A 6-bp direct repeat, separated by 4 bp (a DR-4 element) near the 5′-end of this region, was found to be essential for its activity, and was further shown to bind the RA X receptor β in electrophoretic mobility-shift assays. The present study has revealed a novel RA-response element in Col11a2 that does not interact directly with the promoter, but instead requires the D/E enhancer to mediate transcriptional activation. Proteins bound at the enhancer, therefore, would be expected to affect the transcriptional response to RA. Such a system of regulation, particularly if found to be operating in other cartilage genes, could explain the conflicting responses RA produces in chondrocytes under different experimental conditions.
Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells
Authors: KOREEN Irina V. , ELSAYED Wafaa A. , LIU Yu J. , HARRIS Andrew L.
Published: September 2004

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10–20 μg of pure connexin protein from 2.5×108 HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)–agarose make this affinity tagging and purification procedure easily applicable to other proteins.
Absolute transcript levels of thioredoxin- and glutathione-dependent redox systems in Saccharomyces cerevisiae: response to stress and modulation with growth
Authors: MONJE-CASAS Fernando , MICHÁN Carmen , PUEYO Carmen
Published: September 2004

We report the co-ordinated fine-tune of mRNA molecules that takes place in yeast (Saccharomyces cerevisiae) in response to diverse environmental stimuli. We performed a systematic and refined quantification of the absolute expression patterns of 16 genes coding for thioredoxin- and glutathione-dependent redox system components. Quantifications were performed to examine the response to oxidants, to sudden temperature upshifts and in association with metabolic changes accompanying culture growth and to explore the contribution of mRNA decay rates to the differences observed in basal expression levels. Collectively, these quantifications show (i) vast differences in the steady-state amounts of the investigated transcripts, cTPxI being largely overexpressed compared with GPX1 during the exponential phase and GPX2 beyond this growth stage; (ii) drastic changes in the relative abundance of the transcripts in response to oxidants and heat shock; and (iii) a unique temporal expression profile for each transcript as cells proceed from exponential to stationary growth phase, yet with some general trends such as maximal or near-maximal basal amounts of most mRNA species at early growth stages when glucose concentration is high and cells are actively growing. Moreover, the results indicate that (i) the half-lives of the investigated transcripts are longer and distributed within a narrower range than previously reported global mRNA half-lives and (ii) transcriptional initiation may play an important role in modulating the significant alterations that most mRNAs exhibit in their steady-state levels along with culture growth.
Gts1p stabilizes oscillations in energy metabolism by activating the transcription of TPS1 encoding trehalose-6-phosphate synthase 1 in the yeast Saccharomyces cerevisiae
Authors: XU Zhaojun , YAGUCHI So-ichi , TSURUGI Kunio
Published: September 2004

We reported previously that Gts1p regulates oscillations of heat resistance in concert with those of energy metabolism in continuous cultures of the yeast Saccharomyces cerevisiae by inducing fluctuations in the levels of trehalose, but not in those of Hsp104 (heat shock protein 104). Further, the expression of TPS1, encoding trehalose-6-phosphate synthase 1, and HSP104 was activated by Gts1p in combination with Snf1 kinase, a transcriptional activator of glucose-repressible genes, in batch cultures under derepressed conditions. Here we show that, in continuous cultures, the mRNA level of TPS1 increased 6-fold in the early respiro–fermentative phase, while that of HSP104 did not change. The expression of SUC2, a representative glucose-repressible gene encoding invertase, also fluctuated, suggesting the involvement of the Snf1 kinase in the periodic activation of these genes. However, this possibility was proven to be unlikely, since the oscillations in both TPS1 and SUC2 mRNA expression were reduced by approx. 3-fold during the transient oscillation in gts1Δ (GTS1-deleted) cells, in which the energy state determined by extracellular glucose and intracellular adenine nucleotide levels was comparable with that in wild-type cells. Furthermore, neither the mRNA level nor the phosphorylation status of Snf1p changed significantly during the oscillation. Thus we suggest that Gts1p plays a major role in the oscillatory expression of TPS1 and SUC2 in continuous cultures of Saccharomyces cerevisiae, and hypothesized that Gts1p stabilizes oscillations in energy metabolism by activating trehalose synthesis to facilitate glycolysis at the shift from the respiratory to the respiro–fermentative phase.
Plasma from women with severe pre-eclampsia increases microvascular permeability in an animal model in vivo
Authors: NEAL Christopher R. , HUNTER Alyson J. , HARPER Steven J. , SOOTHILL Peter W. , BATES David O.
Published: September 2004

Pre-eclampsia results in oedema, hypertension and proteinuria, and is associated with increased vascular permeability. A number of studies have pointed to the existence of a circulating macromolecule that induces this endothelial dysfunction. To test whether this circulating factor could increase vascular permeability, we have measured the effect of dialysed human plasma from pregnant women with mild or severe pre-eclampsia (pre-eclamptic toxaemia). Plasma was collected from patients with mild or severe pre-eclampsia and from normotensive women. Plasma was dialysed against frog Ringer's solution using a 12–14 kDa molecular-mass cut-off dialysis tubing. πc (colloid osmotic pressure) was measured with a modified Hansen oncometer. Lp (hydraulic conductivity) and σ (oncotic reflection coefficient) were measured in individually perfused frog mesenteric microvessels using the Landis–Michel technique during perfusion with dialysed plasma. Perfusion of vessels with normal plasma or plasma from patients with mild pre-eclampsia did not alter either Lp or σ. However, plasma from patients with severe pre-eclampsia resulted in a 3.8±0.3-fold increase in Lp and a reduction in σ from 0.96±0.03 to 0.80±0.11. There was a significant correlation between the change in σ and the change in Lp, suggesting that the increase in permeability was due to an increase in pore size in these vessels. A circulating macromolecule in human plasma in severe pre-eclampsia is therefore able to increase vascular permeability in an animal model. The nature of the circulating macromolecule is not known, except that it is, or is bound to, a molecule greater than 12 kDa.
Mitochondrial DNA and aging
Authors: ALEXEYEV Mikhail F. , LeDOUX Susan P. , WILSON Glenn L.
Published: September 2004

Among the numerous theories that explain the process of aging, the mitochondrial theory of aging has received the most attention. This theory states that electrons leaking from the ETC (electron transfer chain) reduce molecular oxygen to form O2•− (superoxide anion radicals). O2•−, through both enzymic and non-enzymic reactions, can cause the generation of other ROS (reactive oxygen species). The ensuing state of oxidative stress results in damage to ETC components and mtDNA (mitochondrial DNA), thus increasing further the production of ROS. Ultimately, this ‘vicious cycle’ leads to a physiological decline in function, or aging. This review focuses on recent developments in aging research related to the role played by mtDNA. Both supportive and contradictory evidence is discussed.
Arabidopsis
C-terminal domain phosphatase-like 1 and 2 are essential Ser-5-specific C-terminal domain phosphatases
Authors: Koiwa Hisashi , Hausmann Stéphane , Bang Woo Young , Ueda Akihiro , Kondo Naoko , Hiraguri Akihiro , Fukuhara Toshiyuki , Bahk Jeong Dong , Yun Dae-Jin , Bressan Ray A. , Hasegawa Paul M. , Shuman Stewart
Published: September 2004

Transcription and mRNA processing are regulated by phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II, which consists of tandem repeats of a Y
1
S
2
P
3
T
4
S
5
P
6
S
7
heptapeptide. Previous studies showed that members of the plant CTD phosphatase-like (CPL) protein family differentially regulate osmotic stress-responsive and abscisic acid-responsive transcription in
Arabidopsis thaliana
. Here we report that AtCPL1 and AtCPL2 specifically dephosphorylate Ser-5 of the CTD heptad in
Arabidopsis
RNA polymerase II, but not Ser-2. An N-terminal catalytic domain of CPL1, which suffices for CTD Ser-5 phosphatase activity
in vitro
, includes a signature DXDXT acylphosphatase motif, but lacks a breast cancer 1 CTD, which is an essential component of the fungal and metazoan Fcp1 CTD phosphatase enzymes. The CTD of CPL1, which contains two putative double-stranded RNA binding motifs, is essential for the
in vivo
function of CPL1 and includes a C-terminal 23-aa signal responsible for its nuclear targeting. CPL2 has a similar domain structure but contains only one double-stranded RNA binding motif. Combining mutant alleles of
CPL1
and
CPL2
causes synthetic lethality of the male but not the female gametes. These results indicate that CPL1 and CPL2 exemplify a unique family of CTD Ser-5-specific phosphatases with an essential role in plant growth and development.
Visual expertise with nonface objects leads to competition with the early perceptual processing of faces in the human occipitotemporal cortex
Authors: Rossion Bruno , Kung Chun-Chia , Tarr Michael J.
Published: September 2004

Human electrophysiological studies have found that the processing of faces and other objects differs reliably at ≈150 ms after stimulus onset, faces giving rise to a larger occipitotemporal field potential on the scalp, termed the N170. We hypothesize that visual expertise with nonface objects leads to the recruitment of early face-related categorization processes in the occipitotemporal cortex, as reflected by the N170. To test this hypothesis, the N170 in response to laterally presented faces was measured while subjects concurrently viewed centrally presented, novel, nonface objects (asymmetric “Greebles”). The task was simply to report the side of the screen on which each face was presented. Five subjects were tested during three event-related potential sessions interspersed throughout a training protocol during which they became experts with Greebles. After expertise training, the N170 in response to faces was substantially decreased (≈20% decrease in signal relative to that when subjects were novices) when concurrently processing a nonface object in the domain of expertise, but not when processing untrained objects of similar complexity. Thus, faces and nonface objects in a domain of expertise compete for early visual categorization processes in the occipitotemporal cortex.
Molecular profiling reveals synaptic release machinery in Merkel cells
Authors: Haeberle Henry , Fujiwara Mika , Chuang Jody , Medina Michael M. , Panditrao Mayuri V. , Bechstedt Susanne , Howard Jonathon , Lumpkin Ellen A.
Published: September 2004

Merkel cell-neurite complexes are somatosensory receptors that initiate the perception of gentle touch. The role of epidermal Merkel cells within these complexes is disputed. To ask whether Merkel cells are genetically programmed to be excitable cells that may participate in touch reception, we purified Merkel cells from touch domes and used DNA microarrays to compare gene expression in Merkel cells and other epidermal cells. We identified 362 Merkel-cell-enriched transcripts, including neuronal transcription factors, presynaptic molecules, and ion-channel subunits. Antibody staining of skin sections showed that Merkel cells are immunoreactive for presynaptic proteins, including piccolo, Rab3C, vesicular glutamate transporter 2, and cholecystokinin 26-33. These data indicate that Merkel cells are poised to release glutamate and neuropeptides. Finally, by using Ca
2+
imaging, we discovered that Merkel cells have L- and P/Q-type voltage-gated Ca
2+
channels, which have been shown to trigger vesicle release at synapses. These results demonstrate that Merkel cells are excitable cells and suggest that they release neurotransmitters to shape touch sensitivity.
wEMBOSS: a web interface for EMBOSS
Authors: Sarachu Martín , Colet Marc
Published: September 2004

Summary: wEMBOSS provides a web environment from which the user can access EMBOSS in a user-friendly way. wEMBOSS supplies each user with space and tools to organize and review his or her work.

Availability: wEMBOSS can be downloaded at http://www.wemboss.org

Contact:  msarachu@biol.unlp.edu.ar
GLAD: a system for developing and deploying large-scale bioinformatics grid
Authors: Teo Yong-Meng , Wang Xianbing , Ng Yew-Kwong
Published: September 2004

Motivation: Grid computing is used to solve large-scale bioinformatics problems with gigabytes database by distributing the computation across multiple platforms. Untill now in developing bioinformatics grid applications, it is extremely tedious to design and implement the component algorithms and parallelization techniques for different classes of problems, and to access remotely located sequence database files of varying formats across the grid. In this study, we propose a grid programming toolkit, GLAD (Grid Life sciences Applications Developer), which facilitates the development and deployment of bioinformatics applications on a grid.

Results: GLAD has been developed using ALiCE (Adaptive scaLable Internet-based Computing Engine), a Java-based grid middleware, which exploits the task-based parallelism. Two bioinformatics benchmark applications, such as distributed sequence comparison and distributed progressive multiple sequence alignment, have been developed using GLAD.

Availability: GLAD and ALiCE are available at http://www.comp.nus.edu.sg/~teoym/alice.htm

Contact:  teoym@comp.nus.edu.sg
Feline foamy virus Tas protein is a DNA-binding transactivator
Authors: Omoto Shinya , Brisibe Ebiamadon Andi , Okuyama Harumi , Fujii Yoichi R.
Published: September 2004

Foamy viruses (FVs) harbour a transcriptional transactivator (Tas) and two Tas-responsive promoter regions, one in the 5′ long terminal repeat (LTR) and the other an internal promoter (IP) in the envelope gene. To analyse the mechanism of transactivation of the FVs, the specificity of feline FV (FFV) Tas protein, which is more distantly related to the respective proteins of non-human primate origin, were investigated. FFV Tas has been shown specifically to activate gene expression from the cognate promoters. No cross-transactivation was noted of the prototype foamy virus and human immunodeficiency virus type 1 LTR. The putative transactivation response element of FFV Tas was mapped to the 5′ LTR U3 region (approximately nt −228 to −195). FFV Tas binds to this element in addition to a previously described sequence (position −66 to −51). It is therefore concluded that FFV Tas is a DNA-binding transactivator that interacts with at least two regions in the virus LTR.
Anticarsia gemmatalis multicapsid nucleopolyhedrovirus v-trex gene encodes a functional 3′ to 5′ exonuclease
Authors: Slack Jeffrey M. , Shapiro Martin
Published: September 2004

The viral three-prime repair exonuclease (v-trex) gene of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) is the first baculovirus gene to be described with significant homology to a 3′ exonuclease. v-trex is an early gene that is expressed by AgMNPV from 3 h post-infection. In the present study, the AgMNPV v-trex ORF was cloned into the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) under the control of a polyhedrin promoter. The resulting virus produced an abundant, soluble protein that migrated with an apparent size of 23·7 kDa. The 3′ to 5′ exonuclease activity associated with this v-trex-expressing recombinant AcMNPV was 2000-fold above that of wild-type AcMNPV. This exonuclease activity was inhibited by EDTA and was activated in the presence of Mg2+ and, to a lesser extent, Mn2+. From these results, the AgMNPV v-trex gene is concluded to encode an independently active 3′ to 5′ exonuclease.
Single point mutation in tick-borne encephalitis virus prM protein induces a reduction of virus particle secretion
Authors: Yoshii Kentarou , Konno Akihiro , Goto Akiko , Nio Junko , Obara Mayumi , Ueki Tomotaka , Hayasaka Daisuke , Mizutani Tetsuya , Kariwa Hiroaki , Takashima Ikuo
Published: September 2004

Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway. Virus envelope proteins play important roles in this process. In this study, the effect of mutations in the envelope proteins of tick-borne encephalitis (TBE) virus on secretion of virus-like particles (VLPs), using a recombinant plasmid expression system was analysed. It was found that a single point mutation at position 63 in prM induces a reduction in secretion of VLPs. The mutation in prM did not affect the folding of the envelope proteins, and chaperone-like activity of prM was maintained. As observed by immunofluorescence microscopy, viral envelope proteins with the mutation in prM were scarce in the Golgi complex, and accumulated in the ER. Electron microscopic analysis of cells expressing the mutated prM revealed that many tubular structures were present in the lumen. The insertion of the prM mutation at aa 63 into the viral genome reduced the production of infectious virus particles. This data suggest that prM plays a crucial role in the virus budding process.
Cell-cycle dependence of foamy virus vectors
Authors: Patton Gillian S. , Erlwein Otto , McClure Myra O.
Published: September 2004

Retroviruses differ in the extent to which they are dependent on host-cell proliferation for their replication, an aspect of their replication that impacts on their vector potential. Foamy viruses offer distinct advantages over other retroviruses for development as vectors for gene therapy. A vector derived from the prototypic foamy virus (PFV), formerly known as human foamy virus (HFV), transduced aphidicolin-arrested cells five- to tenfold more efficiently than one derived from murine leukemia virus (MLV), but several-fold less efficiently than a human immunodeficiency virus type 1 (HIV-1) vector. The same relative efficiency was found following transduction of cells that had been arrested by γ-irradiation or with mitomycin C. Cells that were exposed to vector during aphidicolin arrest and were subsequently allowed to cycle were transduced significantly better by PFV than by MLV. Quiescent human CD34+ progenitor cells were transduced as efficiently by PFV as by HIV vectors (40–50 %) when transduction was assayed after the cells were allowed to cycle.
Sequential modification of translation initiation factor eIF4GI by two different foot-and-mouth disease virus proteases within infected baby hamster kidney cells: identification of the 3Cpro cleavage site
Authors: Strong Rebecca , Belsham Graham J.
Published: September 2004

Infection of cells by foot-and-mouth disease virus (FMDV) causes the rapid inhibition of cellular cap-dependent protein synthesis that results from cleavage of the translation initiation factor eIF4G, a component of the cap-binding complex eIF4F. Two FMDV proteins, the leader (L) and 3C proteases, have been shown individually to induce cleavage of eIF4GI at distinct sites within baby hamster kidney (BHK) cells. Here, sequential cleavage of eIF4GI by the L and 3C proteases was demonstrated in FMDV-infected BHK cells. The FMDV 3C cleavage site within hamster eIF4GI was localized to a small region (about 40 aa) of the protein, between the sites cleaved by the poliovirus 2A protease and the human immunodeficiency virus type 2 protease. Human eIF4GI was found to be resistant to the action of the FMDV 3C protease. On the basis of amino acid sequence alignments, it was predicted and then verified that substitution of a single amino acid residue within this region of human eIF4GI conferred sensitivity to cleavage by the FMDV 3C protease within cells. Full-length eIF4GI and both forms of the C-terminal cleavage product must be capable of supporting the activity of the FMDV internal ribosome entry site in directing translation initiation.
Genomic classification of new betanodavirus isolates by phylogenetic analysis of the coat protein gene suggests a low host-fish species specificity
Authors: Thiéry Richard , Cozien Joëlle , de Boisséson Claire , Kerbart-Boscher Soasig , Névarez Laurent
Published: September 2004

Viral encephalopathy and retinopathy is a devastating disease that causes neurological disorders and high mortality in a large number of cultivated marine fish species around the world. It is now established that several viral strains classified in the genus Betanodavirus of the family Nodaviridae are the aetiological agents of this disease. Betanodaviruses can be classified into four genotypes based on the coat protein gene sequence. Here, the coat protein genes of the three major strains isolated from sea bass (Dicentrarchus labrax) in France were found to be different. In addition, 21 novel strains of betanodavirus from several fish species from France, Spain, Tunisia and Tahiti were classified by using phylogenetic analysis of a partial sequence (383 nt) of the coat protein gene. Most of the isolates were grouped in the red-spotted grouper nervous necrosis virus type, which was subdivided into two subtypes, one of them containing only French isolates. Furthermore, an isolate obtained from sea bass during an outbreak at low temperature (15 °C) was classified as the barfin flounder nervous necrosis virus type. This is the first reported isolation from sea bass of such a strain, which is known to infect several cold-water marine fish species. In addition, a betanodavirus belonging to the striped jack nervous necrosis virus type was detected in Senagalese sole (Solea senegalensis) farmed in Spain, which is the first indication of the presence of this genotype outside Japan. These findings suggest that the different genotypes can infect a variety of fish species and thus have a low host-fish species specificity.
Glycosyltransferases encoded by viruses
Authors: Markine-Goriaynoff Nicolas , Gillet Laurent , Van Etten James L. , Korres Haralambos , Verma Naresh , Vanderplasschen Alain
Published: September 2004

Studies of cellular biology in recent decades have highlighted the crucial roles of glycans in numerous important biological processes, raising the concept of glycomics that is now considered as important as genomics, transcriptomics and proteomics. For millions of years, viruses have been co-evolving with their hosts. Consequently, during this co-evolution process, viruses have acquired mechanisms to mimic, hijack or sabotage host processes that favour their replication, including mechanisms to modify the glycome. The importance of the glycome in the regulation of host–virus interactions has recently led to a new concept called ‘glycovirology’. One fascinating aspect of glycovirology is the study of how viruses affect the glycome. Viruses reach that goal either by regulating expression of host glycosyltransferases or by expressing their own glycosyltransferases. This review describes all virally encoded glycosyltransferases and discusses their established or putative functions. The description of these enzymes illustrates several intriguing aspects of virology and provides further support for the importance of glycomics in biological processes.
Correlation of psoriasis activity with abundance of CD25+CD8+ T cells: conditions for cloning T cells from psoriatic plaques
Authors: Kohlmann Wayan M. , Urban Wiebke , Sterry Wolfram , Foerster John
Published: September 2004

Abstract:  The role of T cells in the pathogenesis of psoriasis is widely acknowledged. However, key aspects of their precise function in the disease as well as the relative pathogenic contribution of T‐cell subsets are still unknown. T‐cell clones have been isolated from psoriatic plaques but a study of conditions affecting the isolation and expansion of T‐cell clones from psoriatic skin has not been reported to date. Here, we observe a correlation of disease activity with the frequency of the CD3+CD8+CD25+ subset. We show that prolonged in vitro culture changes the phenotypic subset distribution of T‐cell lines derived from psoriatic skin and that T‐cell clones can be isolated by sorting of CD25+ cells emigrated from skin fragments after 7 days. We evaluate various conditions affecting expansion of psoriatic T‐cell clones in vitro and show that blocking apoptosis can facilitate proliferation of activated T‐cell clones in vitro. Our results indicate a prominent role of the CD8+CD25+ T‐cell subset in disease pathogenesis and should be useful in the design of experiments aiming at a systematic analysis of the specificity of clones present in psoriatic plaques.
Therapeutic effects of gemcitabine on cutaneous manifestations in an Adamantiades–Behçet's disease‐like mouse model
Authors: Sohn Seonghyang , Lutz Manfred , Kwon Hyuk Jae , Konwalinka Guenther , Lee Sungnack , Schirmer Michael
Published: September 2004

Abstract:  The aim of this work was to study the effects and side effects of gemcitabine (2′,2′‐difluorodeoxycytidine, dFdC), a pyrimidine synthesis inhibitor, on skin lesions of a herpes simplex virus (HSV)‐induced Adamantiades–Behçet's disease (ABD)‐like mouse model. For the dose‐escalation study, ICR mice were treated intraperitoneally with dFdC over 5 days. For the efficacy study, ICR mice were inoculated with HSV and classified as having ABD according to a revised Japanese classification, and then 18 ABD mice were randomly assigned to placebo, 0.06 or 0.12 µg of dFdC/day over 5 days. Serum levels of interleukin‐4 (IL‐4), IL‐6, IL‐10, interferon‐γ (IFN‐γ), and tumor necrosis factor‐α (TNF‐α) were determined using enzyme‐linked immunosorbent assay. After application of 3 µg of dFdC over 5 days, alanine aminotransferase increased (P = 0.032), but all other kidney and liver parameters were unchanged. In ABD mice, 5 days of dFdC treatment with 0.06 or 0.12 µg of dFdC/day resulted in a dose‐dependent improvement of cutaneous manifestations by more than 60% (P = 0.017). There was no significant change in cytokine levels, and none of the cytokine levels correlated with response to treatment. Moreover, dFdC shows promising effects to improve cutaneous lesions in the HSV‐induced ABD‐like mouse model. In this animal model, effects of dFdC on the cytokine profile remained inconclusive.
Rejection Sensitivity and the Defensive Motivational System: Insights From the Startle Response to Rejection Cues
Authors: Downey Geraldine , Mougios Vivian , Ayduk Ozlem , London Bonita E. , Shoda Yuichi
Published: September 2004

Rejection sensitivity (RS) is the disposition to anxiously expect, readily perceive, and intensely react to rejection. This study used the startle probe paradigm to test whether the affect-based defensive motivational system is automatically activated by rejection cues in people who are high in RS. Stimuli were representational paintings depicting rejection (by Hopper) and acceptance (by Renoir), as well as nonrepresentational paintings of either negative or positive valence (by Rothko and Miro, respectively). Eyeblink startle magnitude was potentiated in people high in RS when they viewed rejection themes, compared with when they viewed nonrepresentational negative themes. Startle magnitude was not attenuated during viewing of acceptance themes in comparison with nonrepresentational positive themes. Overall, the results provide evidence that for people high in RS, rejection cues automatically activate the defensive motivational system, but acceptance cues do not automatically activate the appetitive motivational system.
Formation of an SCFZTL complex is required for proper regulation of circadian timing
Authors: Han Linqu , Mason Mary , Risseeuw Eddy P. , Crosby William L. , Somers David E.
Published: September 2004

The circadian timing system involves an autoregulatory transcription/translation feedback loop that incorporates a diverse array of factors to maintain a 24‐h periodicity. In Arabidopsis a novel F‐box protein, ZEITLUPE (ZTL), plays an important role in the control of the free‐running period of the circadian clock. As a class, F‐box proteins are well‐established components of the Skp/Cullin/F‐box (SCF) class of E3 ubiquitin ligases that link the target substrates to the core ubiquitinating activity of the ligase complex via direct association with the Skp protein. Here we identify and characterize the SCFZTL complex in detail. Yeast two‐hybrid tests demonstrate the sufficiency and necessity of the F‐box domain for Arabidopsis Skp‐like protein (ASK) interactions and the dispensability of the unique N‐terminal LOV domain in this association. Co‐immunoprecipitation of full‐length (FL) ZTL with the three known core components of SCF complexes (ASK1, AtCUL1 and AtRBX1) demonstrates that ZTL can assemble into an SCF complex in vivo. F‐box‐containing truncated versions of ZTL (LOV‐F and F‐kelch) can complex with SCF components in vivo, whereas stably expressed LOV or kelch domains alone cannot. Stable expression of F‐box‐mutated FL ZTL eliminates the shortened period caused by mild ZTL overexpression and also abolishes ASK1 interaction in vivo. Reduced levels of the core SCF component AtRBX1 phenocopy the long period phenotype of ztl loss‐of‐function mutations, demonstrating the functional significance of the SCFZTL complex. Taken together, our data establish SCFZTL as an essential SCF class E3 ligase controlling circadian period in plants.
Agrodrench: a novel and effective agroinoculation method for virus‐induced gene silencing in roots and diverse Solanaceous species
Authors: Ryu Choong‐Min , Anand Ajith , Kang Li , Mysore Kirankumar S.
Published: September 2004

Virus‐induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics. We usedTobacco rattle virus(TRV)‐derived VIGS vectors expressed from binary vectors withinAgrobacteriumto induce RNA silencing in plants. Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large‐scale screening and some plants are difficult to infiltrate. Here we have developed a novel and simple method of agroinoculation, called ‘agrodrench’, where soil adjacent to the plant root is drenched with anAgrobacteriumsuspension carrying the TRV‐derived VIGS vectors. By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg‐protoporphyrin chelatase (Chl H) encoding genes inNicotiana benthamianaand in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family. An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration. We also demonstrated that VIGS functioned to silence target genes in plant roots. The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots. Agrodrench will facilitate rapid large‐scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.
The role of sugars and hexose phosphorylation in regulating the re‐establishment of desiccation tolerance in germinated radicles of Cucumis sativa and Medicago truncatula
Authors: Leprince Olivier , Satour Pascale , Vu Benoît Ly , Buitink Julia
Published: September 2004

This study examined whether sugars and hexose phosphorylation participate in the regulatory mechanisms that induce desiccation tolerance (DT) in seeds. In germinated desiccation‐sensitive radicles of Cucumis sativa and Medicago truncatula, DT was re‐established by an osmotic treatment using polyethylene glycol (PEG) for several days. In cucumber, Glc kinase activity (EC 2.7.1.1) transiently peaked early during PEG incubation before the induction of DT in protruded radicles, whereas Fru kinase activity (EC 2.7.1.4) increased progressively during the re‐establishment of DT. Glucosamine (GAM, a competitive inhibitor of HXK) was able to repress the PEG‐induced DT in both species, whereas hexose and poorly metabolizable hexose analogues had no effect. GAM was effective in repressing DT only early during PEG incubation, indicating that this effect is transient. Both Glc and Man fully rescued GAM‐inhibited DT. PEG‐induced accumulation of Suc was not affected by GAM. Isocitrate lyase (ICL) gene expression, which is known to be regulated by hexoses, responded to the re‐establishment of DT and GAM feeding. In cucumber, expression of ICL was repressed after 6 h of PEG incubation whereas GAM feeding led to ICL de‐repression. When GAM could no longer inhibit the re‐establishment of DT, neither were steady‐state levels of ICL influenced. The implication of HXK as a catalytic regulator and sugar‐sensor in DT is discussed.
Molecular characterization of plasma membrane H+‐ATPase in a carrot mutant cell line with enhanced citrate excretion
Authors: Ohno Takashi , Nakahira Shinobu , Suzuki Yuji , Kani Tomomi , Hara Tetsuo , Koyama Hiroyuki
Published: September 2004

The citrate efflux of a carrot mutant cell line (IPG; insoluble phosphate grower) with enhanced citrate excretion is concomitant with an electrically equivalent H+ release. Furthermore, plasma membrane H+‐ATPase activity (EC 3.6.1.35) in the IPG cell line is known to be greater than in the wild‐type cell line. In the present study, molecular characterization of plasma membrane H+‐ATPase in the IPG cell line was conducted to examine the involvement of this enzyme in enhanced citrate excretion. Four type‐II (DcPA 1, 2, 4 and 5) and two type‐I (DcPA 3 and 6) full‐sized cDNAs encoding Daucus carota plasma membrane H+‐ATPase were cloned. The transcript level of the most abundant isoform, DcPA 1, was three times higher in the IPG cell line than in the wild‐type cells. The transcript levels of the other isoforms showed no differences between cell types. The plasma membrane H+‐ATPase protein level in the IPG cell line was 1.6‐fold that of the wild‐type cell line when analysed by western blot analysis. These results indicate that the higher activity of plasma membrane H+‐ATPase in the IPG cell line is due to up‐regulation of DcPA 1. Antisense inhibition of the DcPA 1 gene also led to decreased citrate excretion from the transgenic IPG cell line, suggesting that the up‐regulation of plasma membrane H+‐ATPase is involved in enhanced citrate excretion.
Functional recovery and axonal growth following spinal cord transection is accelerated by sustained l‐DOPA administration
Authors: Doyle L. M. F. , Roberts B. L.
Published: September 2004

The eel, Anguilla anguilla, as with other fish species, recovers well from spinal cord injury. We assessed the quality of locomotion of spinally transected eels from measurements made from video recordings of individuals swimming at different speeds in a water tunnel. Following transection of the spinal cord just caudal to the anus, the animals displayed higher tail beat frequencies and lower tail beat amplitudes than before surgery, owing to the loss of power in this region. Swimming performance then progressively recovered, appearing normal within 1 month of surgery. Eels with similar transections, but given regular, repeated intraperitoneal injections (50 mg/kg) of l‐3,4‐dihydroxyphenylalanine (l‐DOPA) showed an equivalent pattern of decline and recovery that was 10–20 days shorter than that seen in non‐treated fish. Axonal growth into the denervated cord, as determined from anterograde labelling experiments, was also more rapid in the drug‐treated fish. l‐DOPA treatment increased the activity of all fish for up to 18 h, and accelerated the spontaneous movements (‘spinal swimming’) made by the denervated, caudal portion of the animal that appeared following transection. We suggest that this enhancement of locomotion underlies the accelerated axonal growth and, hence, functional recovery.
Thyroid hormone participates in the regulation of neural stem cells and oligodendrocyte precursor cells in the central nervous system of adult rat
Authors: M. Fernandez , S. Pirondi , M. Manservigi , L. Giardino , L. Calzà
Published: September 2004

Oligodendrocyte development and myelination are under thyroid hormone control. In this study we analysed the effects of chronic manipulation of thyroid status on the expression of a wide spectrum of oligodendrocyte precursor cells (OPCs) markers and myelin basic protein (MBP) in the subventricular zone (SVZ), olfactory bulb and optic nerve, and on neural stem cell (NSC) lineage in adult rats. Hypo‐ and hyperthyroidism were induced in male rats, by propyl‐thio‐uracil (PTU) and l‐thyroxin (T4) treatment, respectively. Hypothyroidism increased and hyperthyroidism downregulated proliferation in the SVZ and olfactory bulb (Ki67 immunohistochemistry and Western blotting, bromodeoxyuridine uptake). Platelet‐derived growth factor receptor alpha (PDGFα‐R) and MBP mRNA levels decreased in the optic nerve of hypothyroid rats; the same also occurred at the level of MBP protein. Hyperthyroidism slightly upregulates selected markers such as NG2 in the olfactory bulb. The lineage of cells derived from primary cultures of NSC prepared from the forebrain of adult hypo‐ and hyperthyroid also differs from those derived from control animals. Although no difference of in vitro proliferation of NSCs was observed in the presence of epidermal growth factor, maturation of oligodendrocytes (defined by process number and length) was enhanced in hyperthyroidism, suggesting a more mature state than in control animals. This difference was even greater when compared with the hypothyroid group, the morphology of which suggested a delay in differentiation. These results indicate that thyroid hormone affects NSC and OPC proliferation and maturation also in adulthood.
Transcranial magnetic stimulation of the human motor cortex influences the neuronal activity of subthalamic nucleus
Authors: Strafella Antonio P. , Vanderwerf Ysbrand , Sadikot Abbas F.
Published: September 2004

The critical role of the subthalamic nucleus (STN) in the control of movement and parkinsonian symptoms is well established. Research in animals suggests that the cerebral cortex plays an important role in regulating the activity of the STN but this control is not known in humans. The most extensive cortical innervation of the STN originates from motor areas. Here, we used transcranial magnetic stimulation (TMS) during intraoperative single‐unit recordings from STN, in six patients with Parkinson's disease (PD) undergoing implantation of deep brain stimulators, to determine whether TMS of the motor cortex (MC) modulates the activity of STN and to investigate in vivo the functional organization of the corticosubthalamic circuit in the human brain. Single‐pulse TMS of the MC induced an excitation in 74.9% of neurons investigated. This activation was followed by a long‐lasting inhibition of the STN neuronal activity that did not correlate with PD severity. Responsive neurons to TMS of the hand area of motor cortex were located mainly in the lateral and dorsal region of the subthalamus while unresponsive cells had a prevalently medial distribution. This is the first report of TMS‐induced modulation of STN neuronal activity in humans. These findings open up new avenues for in vivo studies of corticosubthalamic interactions in human brain and PD.