bluuebunny/crossref_metadata_2025 huggingface dataset:
CTLA4 polymorphisms are associated with vitiligo, in patients with concomitant autoimmune diseases
Authors: Blomhoff Anne , Helen Kemp E. , Gawkrodger David J. , Weetman Anthony P. , Husebye Eystein S. , Akselsen Hanne E. , Lie Benedicte A. , Undlien Dag E.
Published: January 2005

The cytotoxic T lymphocyte antigen4 (CTLA4) gene plays a critical role in the control of T cell activation. The gene encodes a surface molecule with inhibitory effects on activated T cells. Several studies have disclosed an association between the previously known variants of the CTLA4 gene and autoimmune disorders, but no study has as yet found any definite association between vitiligo and the CTLA4 polymorphisms. A recent study identified new candidate susceptibility polymorphisms in this region, associated with differential gene splicing and thereby the relative abundance of soluble CTLA4. To assess these new polymorphisms in patients with vitiligo, we genotyped 100 vitiligo patients and 140 healthy controls from the UK, for these novel polymorphisms. No association was found in patients with isolated vitiligo, but a significant association was seen in patients with vitiligo and other autoimmune diseases. The results indicate that the polymorphisms in the CTLA4 gene region confer susceptibility to vitiligo when occurring together with other autoimmune diseases, but not in patients with isolated vitiligo. This raises the possibility that there are two distinct forms of vitiligo where only a subgroup of patients may have a disease caused by the autoimmune destruction of melanocytes.
Metal–ion interactions and the structural organization of Sepia eumelanin
Authors: Liu Yan , Simon John D.
Published: January 2005

The structural organization of melanin granules isolated from ink sacs of Sepia officinalis was examined as a function of metal ion content by scanning electron microscopy and atomic force microscopy. Exposing Sepia melanin granules to ethelenediaminetetraacetic acid (EDTA) solution or to metal salt solutions changed the metal content in the melanin, but did not alter granular morphology. Thus ionic forces between the organic components and metal ions in melanin are not required to sustain the natural morphology once the granule is assembled. However, when aqueous suspensions of Sepia melanin granules of varying metal content are ultra‐sonicated, EDTA‐washed and Fe‐saturated melanin samples lose material to the solution more readily than the corresponding Ca(II) and Mg(II)‐loaded samples. The solubilized components are found to be 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA)‐rich constituents. Associated with different metal ions, Na(I), Ca(II) and Mg(II) or Fe(III), these DHICA‐rich entities form distinct two‐dimensional aggregation structures when dried on the flat surface of mica. The data suggest multiply‐charged ions play an important role in assisting or templating the assembly of the metal‐free organic components to form the three‐dimensional substructure distributed along the protein scaffold within the granule.
Role of keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes
Authors: Hirobe Tomohisa
Published: January 2005

Melanocytes characterized by the activities of tyrosinase, tyrosinase‐related protein (TRP)‐1 and TRP‐2 as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by keratinocytes is indispensable in addition to the regulation by genetic factors in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify factors derived from keratinocytes. Alpha‐melanocyte‐stimulating hormone, adrenocorticotrophic hormone, basic fibroblast growth factor, nerve growth factor, endothelins, granulocyte‐macrophage colony‐stimulating factor, steel factor, leukemia inhibitory factor and hepatocyte growth factor have been suggested to be the keratinocyte‐derived factors and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous factors may be produced in and released from keratinocytes and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor‐mediated signaling pathways.
Pigment cell‐related manifestations in neurofibromatosis type 1: an overview
Authors: Schepper Sofie De , Boucneau Joachim , Lambert Jo , Messiaen Ludwine , Naeyaert Jean‐Marie
Published: January 2005

Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder, affecting approximately 1 in 3500 individuals. The most commonly seen tumors in NF1 patients are the (sub)cutaneous neurofibromas. However, individuals with NF1 typically present in childhood with well‐defined pigmentary defects, including café‐au‐lait macules (CALMs), intertriginous freckling and iris Lisch nodules. NF1 is considered a neurocristopathy, primarily affecting tissues derived from the neural crest. Since the pigment producing melanocyte originates in the neural crest, the presence of (hyper)pigmentary lesions in the NF1 phenotype because of changes in melanocyte cell growth and differentiation is to be expected. We want to discuss the pigmentary cutaneous manifestations of NF1 represented by CALMs and intertriginous freckles and the pigmentary non‐cutaneous manifestations represented by iris Lisch nodules. Several hypotheses have been suggested in explaining the poorly understood etiopathogenesis of CALMs. Whether other pigmentary manifestations might share similar etiopathogenic mechanisms remains obscure. Additional attention will be drawn to a readily seen phenomenon in NF1: hyperpigmentation overlying (plexiform) neurofibromas, which could suggest common etiopathogenetic‐environmental cues or mechanisms underlying CALMs and neurofibromas. Finally, we want to address the relationship between malignant melanoma and NF1.
MAGNETIC RESONANCE IMAGING FINDINGS OF PRESUMED CEREBELLAR CEREBROVASCULAR ACCIDENT IN TWELVE DOGS
Authors: McConnell J. F. , Garosi L. , Platt S. R.
Published: January 2005

The magnetic resonance imaging (MRI) findings of presumed cerebrovascular accident in 12 dogs are described. Fourteen lesions were seen, commonly (11 of 14) within the gray matter of the cerebellar hemispheres or vermis. Thirteen lesions were hyperintense on T2‐weighted images (in 11 dogs) and one was hypointense. Eleven of 14 lesions were within the region supplied by the rostral cerebellar artery or one of its main branches and there was no, or minimal, mass effect. Contrast enhancement was only seen in six lesions and was mild in all. Gradient‐echo images provided additional information in two dogs. The appearance of infarction in dogs with diffusion‐weighted images (DWI) is similar to that in humans, and provided supportive evidence for the diagnosis of infarction in five dogs. The use of gradient‐echo and DWI is recommended for the evaluation of suspected cerebrovascular accidents in dogs. Six of the 12 affected animals were spaniels or spaniel crosses, suggesting a possible breed predisposition.
MAGNETIC RESONANCE IMAGING FINDINGS IN 25 DOGS WITH INFLAMMATORY CEREBROSPINAL FLUID
Authors: Lamb Christopher R. , Croson Penny J. , Cappello Rodolfo , Cherubini Giunio Bruto
Published: January 2005

To describe the signs that may be associated with intracranial inflammatory conditions, magnetic resonance (MR) images of 25 dogs that had inflammatory cerebrospinal fluid (CSF) were mixed with those of a control group of 40 dogs that had CSF negative for inflammatory disease and reviewed without knowledge of the clinical signs or diagnosis. CSF was considered inflammatory if the protein level was >0.25 g/l and the white cell count was >5 mm−3. Abnormalities were found by MR imaging in 19 (76%) dogs with inflammatory CSF. Two dogs had focal lesions, 10 had multifocal lesions, and seven had diffuse lesions. Lesions affected all divisions of the brain. Mass effect was identified in seven (28%) dogs, including one that had a choroid plexus carcinoma. Lesions were hyperintense in T2‐weighted images in 18 dogs and hypointense in T1‐weighted images in six dogs. Multifocal or diffuse intraaxial lesions that were hyperintense in T2‐weighted images were observed in 17 (68%) dogs with inflammatory CSF. Administration of gadolinium resulted in enhancement of intraaxial lesions in nine (36%) dogs and enhancement of meninges in seven (28%) dogs. Six (24%) dogs with inflammatory CSF had images interpreted as normal.
COMMUNICATIONS OF NORMAL NASAL AND PARANASAL CAVITIES IN COMPUTED TOMOGRAPHY OF HORSES
Authors: Probst Alexander , Henninger Wolfgang , Willmann Michael
Published: January 2005

Heads from 15 male and female horses of various breeds and ages were examined with computed tomography under general anesthesia in dorsal recumbency. The main interest was the evaluation of the entire paranasal sinus system. Special attention was paid to the location, size, and shape of the conchomaxillary, nasomaxillary, frontomaxillary, and sphenopalatinal openings. The frontomaxillary opening was the largest aperture and was found to be age‐dependent in size mostly. Orientation and shape of the openings were sagittal or horizontal; the narrowest of them was the slit‐like nasomaxillary aperture. The thickness of the vascularized mucous membranes in the nasal cavity was obtained up to 6 mm; the surface was smooth and well defined. The paranasal lining was invisible in computed tomography (CT) images because of its thin layer. CT proved to be a very useful method for documentation of the paranasal openings and for evaluation of the state of the mucous membranes in the nasal and paranasal cavities.
FAT IS RESPONSIBLE FOR AN INTRAMURAL RADIOLUCENT BAND IN THE FELINE STOMACH WALL
Authors: Heng Hock Gan , Wrigley Robert H. , Kraft Susan L. , Powers Barbara E.
Published: January 2005

An intramural radiolucent band in the stomach wall can be seen on abdominal radiographs of cats without concurrent clinical gastrointestinal signs. A retrospective study of cats with normal abdominal radiographs and computed tomography (CT) was performed to determine the prevalence of this finding. This gastric radiolucent band was seen in 13 of 37 (35%) radiographs. There was no relationship between age, weight and relative obesity and the presence of this radiolucent band. Using CT, an intramural hypoattenuating layer was detected in the stomach wall of four of 15 (27%) cats. The intramural radiolucent band was visible on the radiographs in all four of those cats. This radiographic and CT finding was histologically determined to be normal fat in the gastric submucosa in one cat cadaver.
BIPARTITE DISTAL PHALANX AND NAVICULAR BONE IN AN ANDALUSIAN STALLION
Authors: Benninger Monika I. , Deiss Erwin , Ueltschi Gottlieb
Published: January 2005

We describe the clinical findings and radiographic abnormalities of a horse with a bipartite distal phalanx and a bipartite navicular bone of the right forelimb. Associated findings including suspected keratoma and degenerative joint disease of the distal interphalangeal joint are described and possible etiologies are discussed.
Defining the QP‐site of Escherichia coli fumarate reductase by site‐directed mutagenesis, fluorescence quench titrations and EPR spectroscopy
Authors: Rothery Richard A. , Seime Andrea M. , Spiers A.‐M. Caroline , Maklashina Elena , Schröder Imke , Gunsalus Robert P. , Cecchini Gary , Weiner Joel H.
Published: January 2005

We have used fluorescence quench titrations, EPR spectroscopy and steady‐state kinetics to study the effects of site‐directed mutants of FrdB, FrdC and FrdD on the proximal menaquinol (MQH2) binding site (QP) of Escherichia coli fumarate reductase (FrdABCD) in cytoplasmic membrane preparations. Fluorescence quench (FQ) titrations with the fluorophore and MQH2 analog 2‐n‐heptyl‐4‐hydroxyquinoline‐N‐oxide (HOQNO) indicate that the QP site is defined by residues from FrdB, FrdC and FrdD. In FQ titrations, wild‐type FrdABCD binds HOQNO with an apparent Kd of 2.5 nm, and the following mutations significantly increase this value: FrdB‐T205H (Kd = 39 nm); FrdB‐V207C (Kd = 20 nm); FrdC‐E29L (Kd = 25 nm); FrdC‐W86R (no detectable binding); and FrdD‐H80K (Kd = 20 nm). In all titrations performed, data were fitted to a monophasic binding equation, indicating that no additional high‐affinity HOQNO binding sites exist in FrdABCD. In all cases where HOQNO binding is detectable by FQ titration, it can also be observed by EPR spectroscopy. Steady‐state kinetic studies of fumarate‐dependent quinol oxidation indicate that there is a correlation between effects on HOQNO binding and effects on the observed Km and kcat values, except in the FrdC‐E29L mutant, in which HOQNO binding is observed, but no enzyme turnover is detected. In this case, EPR studies indicate that the lack of activity arises because the enzyme can only remove one electron from reduced MQH2, resulting in it being trapped in a form with a bound menasemiquinone radical anion. Overall, the data support a model for FrdABCD in which there is a single redox‐active and dissociable Q‐site.
Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity
Authors: Seydel Ulrich , Schromm Andra B. , Brade Lore , Gronow Sabine , Andrä Jörg , Müller Mareike , Koch Michel H. J. , Fukase Koichi , Kataoka Mikayo , Hashimoto Masaya , Kusumoto Shoichi , Brandenburg Klaus
Published: January 2005

Lipopolysaccharide (LPS) from the outer membrane of Gram‐negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the ‘endotoxic principle’ of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition.

The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM‐506 and Bis‐CM‐506) and of tetraacyl lipid A (Bis‐CM‐406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular (‘endotoxic’) conformation and with that, a particular aggregate structure is a prerequisite for high cytokine‐inducing capacity and antagonistic activity, respectively.

In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.
Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis
Authors: Sasso Severin , Ramakrishnan Chandra , Gamper Marianne , Hilvert Donald , Kast Peter
Published: January 2005

The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases (CMs) was cloned from the genome of Mycobacterium tuberculosis and expressed in Escherichia coli. The gene product (*MtCM) complements a CM‐deficient E. coli strain, but only if produced without the predicted N‐terminal signal sequence typical of M. tuberculosis. The mature *MtCM, which was purified by exploiting its resistance to irreversible thermal denaturation, possesses high CM activity in vitro. The enzyme follows simple Michaelis–Menten kinetics, having a kcat of 50 s−1 and a Km of 180 µm (at 30 °C and pH 7.5). *MtCM was shown to be a dimer by analytical ultracentrifugation and size‐exclusion chromatography. Secondary‐structure prediction and CD spectroscopy confirmed that *MtCM is a member of the all‐α‐helical AroQ class of CMs, but it seems to have a topologically rearranged AroQ fold. Because CMs are normally intracellular metabolic enzymes required for the biosynthesis of phenylalanine and tyrosine, the existence of an exported CM in Gram‐positive M. tuberculosis is puzzling. The observation that homologs of *MtCM with a predicted export sequence are generally only present in parasitic or pathogenic organisms suggests that secreted CMs may have evolved to participate in some aspect of parasitism or pathogenesis yet to be unraveled.
Annexin A2 binds to the localization signal in the 3′ untranslated region of c‐myc mRNA
Authors: Mickleburgh Ian , Burtle Brian , Hollås Hanne , Campbell Gill , Chrzanowska‐Lightowlers Zofia , Vedeler Anni , Hesketh John
Published: January 2005

Messenger RNA trafficking, which provides a mechanism for local protein synthesis, is dependent on cis‐acting sequences in the 3′ untranslated regions (3′UTRs) of the mRNAs concerned acting together with trans‐acting proteins. The C‐MYC transcription factor is a proto‐oncogene product involved in cell proliferation, differentiation and apoptosis. Localization of c‐myc mRNA to the perinuclear cytoplasm and its association with the cytoskeleton is determined by a signal in the 3′UTR. Here we show the specific binding of a trans‐acting factor to the perinuclear localization element in the 3′UTR of c‐myc mRNA and identify this protein as annexin A2. Gel retardation and UV cross‐linking experiments showed that proteins in fibroblast extracts formed complexes with the region of c‐myc 3′UTR implicated in localization; a protein of ≈ 36 kDa exhibited specific, Ca2+‐dependent binding. Binding was reduced by introduction of a mutation that abrogates localization. Using RNA‐affinity columns followed by gel electrophoresis and mass spectrometry this protein was identified as annexin A2. The RNA–protein complex formed by cell extracts was further retarded by anti‐(annexin A2). Purified annexin A2 bound to the same region of the c‐myc 3′UTR but binding was reduced by introduction of a mutation, as with cell extracts. It is proposed that binding of annexin A2 to the localization signal in the c‐myc mRNA leads to association with the cytoskeleton and perinuclear localization. The data indicate a novel functional role for the RNA‐binding properties of annexin A2 in perinuclear localization of mRNA and the association with the cytoskeleton.
Effect of deletion of the DNase I hypersensitive sites on the transcription of chicken Ig‐β gene and on the maintenance of active chromatin state in the Ig‐β locus
Authors: Matsudo Hiroki , Osano Kyoichi , Arakawa Hiroshi , Ono Masao
Published: January 2005

The role of DNase I hypersensitive sites (DHSs) in transcription of the B cell‐specific Ig‐β gene and in maintenance of active chromatin state in the Ig‐β locus were examined. A total of 10 DHSs were divided into four regions, and each region was deleted separately in chicken B lymphocyte‐derived DT40 cells. Deletion of three DHSs located between the Ig‐β promoter and its upstream Na channelgene, resulted in the absence of Ig‐β mRNA. Three regions except the region in the Na channel gene were involved in the transcription of Ig‐β gene. The enhancing activity of DHSs as determined by transient transfection assays did not always correlate with the effect of DHS deletion on the expression level of Ig‐β mRNA. In each deletion, cells contained the same DHSs as observed in the predeletion cells, indicating that deleted DHSs did not participate in the maintenance of DT40‐specific DHSs. Enhanced acetylation of H3 and H4 histones at the Ig‐β promoter and at DT40‐specific DHSs was observed in cells in which DHSs between the Na channel gene and Ig‐β promoter were deleted; therefore, these DHSs are prerequisite for transcription of the Ig‐β gene but not required for the maintenance of active chromatin state in the Ig‐β locus. Thus, epigenetic factors required for the maintenance of the active chromatin state are suggested to reside in other regions than those deleted in this study.
Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells
Authors: Rumbo Martin , Courjault‐Gautier Françoise , Sierro Frédéric , Sirard Jean‐Claude , Felley‐Bosco Emanuela
Published: January 2005

Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exists as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated with the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were seen in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization‐dependent processes.
Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails
Authors: Folichon Marc , Allemand Frédéric , Régnier Philippe , Hajnsdorf Eliane
Published: January 2005

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3′ end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq–RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).
The N‐terminus of B96Bom, a Bombyx mori G‐protein‐coupled receptor, is N‐myristoylated and translocated across the membrane
Authors: Utsumi Toshihiko , Ohta Hiroto , Kayano Yoshiyuki , Sakurai Nagisa , Ozoe Yoshihisa
Published: January 2005

In eukaryotic cellular proteins, protein N‐myristoylation has been recognized as a protein modification that occurs mainly on cytoplasmic or nucleoplasmic proteins. In this study, to search for a eukaryotic N‐myristoylated transmembrane protein, the susceptibility of the N‐terminus of several G‐protein‐coupled receptors (GPCRs) to protein N‐myristoylation was evaluated by in vitro and in vivo metabolic labeling. It was found that the N‐terminal 10 residues of B96Bom, a Bombyx mori GPCR, efficiently directed the protein N‐myristoylation. Analysis of a tumor necrosis factor (TNF) fusion protein with the N‐terminal 90 residues of B96Bom at its N‐terminus revealed that (a) transmembrane domain 1 of B96Bom functioned as a type I signal anchor sequence, (b) the N‐myristoylated N‐terminal domain (58 residues) was translocated across the membrane, and (c) two N‐glycosylation motifs located in this domain were efficiently N‐glycosylated. In addition, when Ala4 in the N‐myristoylation motif of B96Bom90‐TNF, Met‐Gly‐Gln‐Ala‐Ala‐Thr(1–6), was replaced with Asn to generate a new N‐glycosylation motif, Asn‐Ala‐Thr(4–6), efficient N‐glycosylation was observed on this newly introduced N‐glycosylation site in the expressed protein. These results indicate that the N‐myristoylated N‐terminus of B96Bom is translocated across the membrane and exposed to the extracellular surface. To our knowledge, this is the first report showing that a eukaryotic transmembrane protein can be N‐myristoylated and that the N‐myristoylated N‐terminus of the protein can be translocated across the membrane.
Molecular characterization, phylogenetic relationships, and developmental expression patterns of prion genes in zebrafish (Danio rerio)
Authors: Cotto Emmanuelle , André Michèle , Forgue Jean , Fleury Hervé J , Babin Patrick J
Published: January 2005

Prion diseases are characterized by the accumulation of a pathogenic misfolded form of a prion protein (PrP) encoded by the Prnp gene in humans. In the present study in zebrafish, two transcripts and the corresponding genes encoding prion proteins, PrP1 and PrP2, related to human PrP have been characterized with a relatively divergent deduced amino acid sequence, but a well preserved overall organization of structural prion protein motifs. Whole‐mount in situ hybridization analysis performed during embryonic and larval development showed a high level of PrP1 mRNA spatially restricted to the anterior floor‐plate of the central nervous system and in ganglia. Transcripts of prp2 were detected in embryonic cells from the mid‐blastula transition to the end of the segmentation period. From 24 h postfertilization up to larval stages, prp2 transcripts were localized in distinct anatomical structures, including a major expression in the brain, eye, kidney, lateral line neuromasts, liver, heart, pectoral fins and posterior intestine. The observed differential developmental expression patterns of the two long PrP forms, prp1 and prp2, and the short PrP form prp3, a more divergent prion‐related gene previously identified in zebrafish, should contribute to understanding of the phylogenetic and functional relationships of duplicated prion gene forms in the fish genome. Together, the complex history of prion‐related genes, reflected in the deduced structural features, conserved amino acid sequence and repeat motifs of the corresponding proteins, and the presence of differential developmental expression patterns suggest possible acquisition or loss of prion protein functions during vertebrate evolution.
Comparison of the substrate specificity of two potyvirus proteases
Authors: Tözsér József , Tropea Joseph E. , Cherry Scott , Bagossi Peter , Copeland Terry D. , Wlodawer Alexander , Waugh David S.
Published: January 2005

The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate‐binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.
2‐Hydroxyisocaproyl‐CoA dehydratase and its activator from Clostridium difficile
Authors: Kim Jihoe , Darley Daniel , Buckel Wolfgang
Published: January 2005

The hadBC and hadI genes from Clostridium difficile were functionally expressed in Escherichia coli and shown to encode the novel 2‐hydroxyisocaproyl‐CoA dehydratase HadBC and its activator HadI. The activated enzyme catalyses the dehydration of (R)‐2‐hydroxyisocaproyl‐CoA to isocaprenoyl‐CoA in the pathway of leucine fermentation. The extremely oxygen‐sensitive homodimeric activator as well as the heterodimeric dehydratase, contain iron and inorganic sulfur; besides varying amounts of zinc, other metal ions, particularly molybdenum, were not detected in the dehydratase. The reduced activator transfers one electron to the dehydratase concomitant with hydrolysis of ATP, a process similar to that observed with the unrelated nitrogenase. The thus activated dehydratase was separated from the activator and ATP; it catalyzed about 104 dehydration turnovers until the enzyme became inactive. Adding activator, ATP, MgCl2, dithionite and dithioerythritol reactivated the enzyme. This is the first demonstration with a 2‐hydroxyacyl‐CoA dehydratase that the catalytic electron is recycled after each turnover. In agreement with this observation, only substoichiometric amounts of activator (dehydratase/activator = 10 mol/mol) were required to generate full activity.
Detection of native peptides as potent inhibitors of enzymes
Authors: Singh Nagendra , Jabeen Talat , Sharma Sujata , Roy Ipsita , Gupta Munishwar N. , Bilgrami Sameeta , Somvanshi Rishi K. , Dey Sharmistha , Perbandt Marcus , Betzel Christian , Srinivasan A. , Singh Tej P.
Published: January 2005

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in α‐chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 ± 0.3 × 10−11 m and thus works as a highly potent competitive inhibitor. The commercially available α‐chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP‐treated α‐chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mm sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 m ammonium sulfate. The rectangular crystals of small dimensions of 0.24 × 0.15 × 0.10 mm3 were obtained. The X‐ray intensity data were collected at 2.2 Å resolution and the structure was refined to an R‐factor of 0.192. An extra electron density was observed at the binding site of α‐chymotrypsin, which was readily interpreted as a 14 residue fragment of α‐chymotrypsin corresponding to Ile‐Val‐Asn‐Gly‐Glu‐Glu‐Ala‐Val‐Pro‐Gly‐Ser‐Trp‐Pro‐Trp(16–29). The electron density for the eight residues of the C‐terminus, i.e. Ala22–Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N‐terminus, Ile16–Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 σ between the active site Ser195 Oγ and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 Å between Ser195 Oγ and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16–Trp29. In addition to the O–C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of α‐chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of α‐chymotrypsin.
Binding of the volatile general anesthetics halothane and isoflurane to a mammalian β‐barrel protein
Authors: Johansson Jonas S. , Manderson Gavin A. , Ramoni Roberto , Grolli Stefano , Eckenhoff Roderic G.
Published: January 2005

A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic–protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large β‐barrel internal cavity (515 ± 30 Å3) lined predominantly by aromatic and aliphatic residues. Halothane binding to the β‐barrel cavity was determined using fluorescence quenching of Trp16, and a competitive binding assay with 1‐aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using isothermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the β‐barrel of porcine odorant binding protein with dissociation constants of 0.46 ± 0.10 mm and 0.43 ± 0.12 mm determined using fluorescence quenching and competitive binding with 1‐aminoanthracene, respectively. Isothermal titration calorimetry revealed that halothane and isoflurane bound with Kd values of 80 ± 10 µm and 100 ± 10 µm, respectively. Halothane and isoflurane binding resulted in an overall stabilization of the folded conformation of the protein by −0.9 ± 0.1 kcal·mol−1. In addition to indicating specific binding to the native protein conformation, such stabilization may represent a fundamental mechanism whereby anesthetics reversibly alter protein function. Because porcine odorant binding protein has been successfully analyzed by X‐ray diffraction to 2.25 Å resolution [1], this represents an attractive system for atomic‐level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules.
Functional implications of pigments bound to a cyanobacterial cytochromeb6fcomplex
Authors: Wenk Stephan‐Olav , Schneider Dirk , Boronowsky Ute , Jäger Cornelia , Klughammer Christof , de Weerd Frank L. , van Roon Henny , Vermaas Wim F. J. , Dekker Jan P. , Rögner Matthias
Published: January 2005

A highly purified cytochromeb6fcomplex from the cyanobacteriumSynechocystissp. PCC 6803 selectively binds one chlorophyll a and one carotenoid in analogy to the recent published structure from two otherb6fcomplexes. The unknown function of these pigments was elucidated by spectroscopy and site‐directed mutagenesis. Low‐temperature redox difference spectroscopy showed red shifts in the chlorophyll and carotenoid spectra upon reduction of cytochromeb6, which indicates coupling of these pigments with the heme groups and thereby with the electron transport. This is supported by the correlated kinetics of these redox reactions and also by the distinct orientation of the chlorophyll molecule with respect to the heme cofactors as shown by linear dichroism spectroscopy. The specific role of the carotenoid echinenone for the cytochromeb6fcomplex ofSynechocystis6803 was elucidated by a mutant lacking the last step of echinenone biosynthesis. The isolated mutant complex preferentially contained a carotenoid with 0, 1 or 2 hydroxyl groups (most likely 9‐cisisomers of β‐carotene, a monohydroxy carotenoid and zeaxanthin, respectively) instead. This indicates a substantial role of the carotenoid – possibly for strucure and assembly – and a specificity of its binding site which is different from those in most other oxygenic photosynthetic organisms. In summary, both pigments are probably involved in the structure, but may also contribute to the dynamics of the cytochromeb6fcomplex.
Colonization by arbuscular mycorrhizal and fine endophytic fungi in herbaceous vegetation in the Canadian High Arctic
Authors: Olsson Pål Axel , Eriksen Bente , Dahlberg Anders
Published: January 2005

The occurrence of arbuscular mycorrhizal (AM) fungi was surveyed along a latitudinal gradient in Arctic Canada including Banks Island (73°N), Devon Island (74°N), Ellesmere Island (76°N), and the Magnetic North Pole at Ellef Ringnes Island (78°N). At Banks Island, AM fungi were present and colonized at a high intensity in all specimens of Potentilla hookeriana Lehm. – Potentilla pulchella R.Br., Arnica angustifolia Vahl, and Erigeron uniflorus L. ssp. eriocephalus (Vahl ex Hornen.) Cronq. sampled. The soil collected under these plants showed a high inoculum potential when tested at greenhouse conditions using Plantago lanceolata L. as a bait plant. Occasional occurrence of AM fungi was recorded in Festuca hyperborea Holmen ex Frederiksen, Trisetum spicatum (L.) Richt., and Potentilla hookeriana – Potentilla pulchella at Devon Island. Despite the fact that potential AM plants are present, no AM was found at the two most northern sites, Ellesmere Island and Ellef Ringnes Island. There seems to be climatic or dispersal limitations to AM colonization at these northern sites. Fine endophytic fungi, formerly named Glomus tenue (Grenall) I.R. Hall, were recorded at all four sites, but most frequently at Banks Island. We thereby provide further evidence that fine endophytes are more frequent in harsh climatic conditions than AM fungi. There was a relatively high proportion of nonmycorrhizal plant species at all sites, and this proportion increased towards the north.Key words: arctic, arbuscular mycorrhiza, fine endophytes, dark septate fungi.
Carbohydrate and amino acid composition in phloem sap of Lolium perenne L. before and after defoliation
Authors: Amiard Véronique , Morvan-Bertrand Annette , Cliquet Jean-Bernard , Billard Jean-Pierre , Huault Claude , Sandström Jonas P , Prud'homme Marie-Pascale
Published: January 2005

Carbohydrate and amino acid composition of phloem sap was studied in the grass Lolium perenne L., before and after defoliation. Leaf exudate was collected in a 5 mmol·L1 EDTA solution from cut leaf blades or stubble, and phloem sap was obtained through excised aphid (Rhopalosiphum padi L.) stylets. Results indicate that leaf exudates obtained from leaves devoid of petiole might not be relevant predictors of carbohydrate content of pure phloem sap. Sucrose was the dominating carbohydrate, accounting for 93% of the total soluble sugars in the phloem sap. Myo-inositol, glucose, and fructose were present in trace amounts, while fructans, raffinose, and loliose have never been detected. Predominant amino acid in the phloem sap was glutamine followed by glutamate, aspartate, and serine. Phloem sap component concentration declined during the first hours following defoliation. Sucrose was the main sugar transported in the phloem sap of Lolium perenne, despite the fact that the product of fructan degradation was fructose and not sucrose. The results are discussed in relation with the physiological mechanisms that contribute to plant recovery after defoliation.Key words: fructan, sucrose, loliose, defoliation, phloem sap, amino acids.
Secondary vegetation and floristic succession within a boreal aspen (Populus tremuloides Michx.) clearcut
Authors: Strong W L
Published: January 2005

The composition and abundance of vascular plants within a boreal Populus tremuloides Michx. stand in west-central Alberta were assessed three, five, nine, and 20 years after clearcutting. Rapid re-establishment of Populus tremuloides and other species such as Amelanchier alnifolia (Nutt.) Nutt. ex M. Roemer and Rosa acicularis Lindl. occurred primarily in response to vegetative propagation. Some minor compositional variability occurred among the year 3 plots (n = 88), but they were considered members of the same plant community, with 88% of the plots undergoing similar developmental processes during the course of the study. Some dominant species substantially increased in canopy cover, although most species maintained a relatively consistent level of abundance over time. Floristic richness was similar in year 3 (64 species) and year 20 (62 species) with the survival of 52 initiating species in year 20. The variability in floristic richness included the invasion of 32 species and the extirpation of 34 species. Stand development over a 20-year period best fit a tolerance model of succession because of the rapid development of dominant species, the lack of a major understory composition change, and a similar degree of dominance concentration among species. Understory vegetation growth was constrained by overstory shading.Key words: plant community, tolerance model, secondary succession.
Spatial variability ofAspergillusflavussoil populations under different crops and corn grain colonization and aflatoxins
Authors: Abbas H K , Zablotowicz R M , Locke M A
Published: January 2005

Aflatoxin contamination in corn caused by Aspergillus flavus Link is a serious constraint on economical corn (Zea mays L.) production in the Mississippi Delta. The ecology of A. flavus was evaluated in a 3-year study assessing the spatial variability of soil populations of A. flavus in a Mississippi Delta field under different crops. A 1.07-ha section of the field was laid out in 126 9.2-m2plots, and soil was sampled in May 2000, March 2001, and April 2002. Aspergillus flavus populations were determined by plating on selective media, and A. flavus colonization was assessed in corn during 2000. Aspergillus flavus populations in soil were significantly (P < 0.01 level) influenced by previous crop. The highest propagule density (794 cfu·g1) was found following the corn crop in 2001 versus 251 cfu·g1soil in 2000 following cotton and 457 cfu·g1following wheat in 2002. Aspergillus flavus populations in 2001 and 2002 exhibited a moderate degree of spatial structure, described by spherical and exponential models, respectively, but populations in 2000 exhibited little spatial structure. Colonization of corn kernels by A. flavus in 2000 ranged from 0% to 100% (mean = 15% colonized kernels), and aflatoxin levels ranged from 0 to 1590 ppb (mean = 57 ppb). Aflatoxin levels were randomly distributed in the field and not correlated with A. flavus colonization. Aflatoxin production was found in 43% to 59% of A. flavus soil isolates with the highest incidence in soil populations following corn in 2001. However, 84% of A. flavus isolated from corn kernels produced aflatoxin. Results indicate that within a single field there was a wide range of A. flavus soil propagule densities varying in potential to produce aflatoxin.Key words: Aspergillus flavus, aflatoxins, soil, corn (Zea mays), cotton, wheat, spatial variability.
Uptake of heavy metal salt solutions by pollen grains ofMagnolia×soulangeana(Magnoliaceae)
Authors: Dinis Augusto M , Mesquita José F
Published: January 2005

To investigate the role of the plasmalemmic cord in the pollen grains of members of the Magnoliaceae, anthers of Magnolia × soulangeana Soul.-Bod. were incubated for 1, 2.5, and 5 h in lead and lanthanum salt solutions. Ultrastructural analysis revealed differences in the labelling of the pollen grains with the time of exposure to the heavy metal salt, which is likely related to metal tolerance mechanisms operating in the pollen grains. Within 1 h of treatment, both tracers produced a fine precipitate lining the entire vegetative cell (VC) plasmalemma (including the plasmalemmic cord) as well as the generative cell (GC) plasmalemma. Heavy deposits were not found inside both the VC and the GC. At later times, the amount of heavy deposits increased on the pollen wall surface and, particularly, in the two outer intine layers. Also, heavy deposits were found in membrane-bound cell components of the VC and the GC, but never in the cytosol. In the VC, the cell components more frequently labelled were the protein storage vacuoles. In both pollen cells, multivesicular bodies, dictyosome cisternae, and small vesicles were also labelled. We hypothesize that the latter organelles participate in the metal ions accumulation (end of the trip), together with the protein vacuoles, or in the transport of metal ions to the vacuoles following the endocytotic uptake of these ions. The presence of both coated and uncoated pits and vesicles in the treated and the untreated pollen reinforces the latter hypothesis. Another significant result was that the tracers, after diffusing through the microchannels in the exine and crossing the intine layers, moved through the plasmalemmic cord to reach the GC periplasm. We suggest that the plasmalemmic cord may provide a system of communication for the flux of solutes from the anther loculus to both pollen cells and (or) from the VC to the GC.Key words: pollen grain, ultrastructure, metal uptake, metal tolerance, lead nitrate, lanthanum nitrate.
Diversity of ectomycorrhizae on experimentally planted Douglas-fir seedlings in variable retention forestry sites on southern Vancouver Island
Authors: Outerbridge R A , Trofymow J A
Published: January 2005

Studies were done on Vancouver Island of ectomycorrhizal (EM) communities at four distances (545 m) from isolated forest patches in three second-growth (SG) and three old-growth (OG) Douglas-fir sites subject to variable retention harvesting. We tested the hypothesis that retention of mature trees enhances colonization and diversity of EM fungi on seedlings planted in adjacent areas. In total 41 EM morphotypes were described, with mean diversity of 3.47 morphotypes and root colonization of 62% per seedling. Overall, root colonization declined with distance (72% at 5 m vs. 52% at 45 m), as did EM diversity (4.7 at 5 m vs. 2.9 at 45 m). For individual sites, the distance effect was significant for root colonization at four sites and for EM diversity at three to four sites. This suggests that variable retention is important for the recovery of ectomycorrhizal biota in harvested sites. Seedling root colonization was significantly lower in SG sites than in OG sites. Though EM diversity did not differ with stand age, OG sites had potentially more total (34) and unique (14) EM morphotypes than did SG sites (total 27, unique 7). Differences with stand age might be related to the relative abilities of EM fungi to disperse to regenerating second-growth forests.Key words: variable retention silviculture, ectomycorrhizae ecology, Douglas-fir seedlings, old growth, second-growth forests.
What do we know about hybridization among bryophytes in nature?
Authors: Natcheva Rayna , Cronberg Nils
Published: January 2005

Despite being recognized as a widespread and evolutionary important phenomenon among vascular plants, interspecific hybridization among bryophytes has been strongly underestimated. In the present review, we summarize knowledge about bryophyte hybrids that are found in nature. Mechanisms of reproductive isolation in bryophytes are compared with those in vascular plants. The morphological and genetic features of sporophytic hybrids and their gametophytic progeny are discussed, as well as some inferences about hybrid fitness. The data available indicate that spontaneous hybridization among bryophytes is not uncommon and has an important, though still not completely understood, evolutionary significance. The existence of many allopolyploid taxa supports this conclusion. Finally we suggest some methods for further investigation of hybridization among bryophytes in nature.Key words: hybridization, bryophytes, isolating mechanisms, evolution, hybrid fitness, hybrid features.