bluuebunny/crossref_metadata_2025 huggingface dataset:
Starch-Branching Enzyme I-Deficient Mutation Specifically Affects the Structure and Properties of Starch in Rice Endosperm
Authors: Satoh Hikaru , Nishi Aiko , Yamashita Kazuhiro , Takemoto Yoko , Tanaka Yasumasa , Hosaka Yuko , Sakurai Aya , Fujita Naoko , Nakamura Yasunori
Published: November 2003

We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) ≥ 37 and short chains with DP 12 to 21, marked increase in short chains with DP ≤ 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2–3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.
Overexpression of the FRO2 Ferric Chelate Reductase Confers Tolerance to Growth on Low Iron and Uncovers Posttranscriptional Control
Authors: Connolly Erin L. , Campbell Nathan H. , Grotz Natasha , Prichard Charis L. , Guerinot Mary Lou
Published: November 2003

The Arabidopsis FRO2 gene encodes the low-iron-inducible ferric chelate reductase responsible for reduction of iron at the root surface. Here, we report that FRO2 and IRT1, the major transporter responsible for high-affinity iron uptake from the soil, are coordinately regulated at both the transcriptional and posttranscriptional levels. FRO2 and IRT1 are induced together following the imposition of iron starvation and are coordinately repressed following iron resupply. Steady-state mRNA levels of FRO2 and IRT1 are also coordinately regulated by zinc and cadmium. Like IRT1, FRO2 mRNA is detected in the epidermal cells of roots, consistent with its proposed role in iron uptake from the soil. FRO2 mRNA is detected at high levels in the roots and shoots of 35S-FRO2 transgenic plants. However, ferric chelate reductase activity is only elevated in the 35S-FRO2 plants under conditions of iron deficiency, indicating that FRO2 is subject to posttranscriptional regulation, as shown previously for IRT1. Finally, the 35S-FRO2 plants grow better on low iron as compared with wild-type plants, supporting the idea that reduction of ferric iron to ferrous iron is the rate-limiting step in iron uptake.
Cleavage of thePseudomonas syringaeType III Effector AvrRpt2 Requires a Host Factor(s) Common among Eukaryotes and Is Important for AvrRpt2 Localization in the Host Cell
Authors: Jin Ping , Wood Michelle D. , Wu Yan , Xie Zhiyi , Katagiri Fumiaki
Published: November 2003

Many phytopathogenic bacteria use a type III secretion system to deliver type III effector proteins into the host plant cell. The Pseudomonas syringae type III effector AvrRpt2 is cleaved at a specific site when translocated into the host cell. In this study, we first demonstrate that the factor(s) required for AvrRpt2 cleavage is present in extracts from animal and yeast cells, as well as plant cells. The cleavage factor in animal and plant cell extracts was heat labile but relatively insensitive to protease inhibitors. Second, mutational analysis of AvrRpt2 was applied to identify features important for its cleavage. In addition to two of the amino acid residues in the immediate vicinity of the cleavage site, a large part of the region C-terminal to the cleavage site was required when AvrRpt2 was cleaved in animal cell extract. Most of these features were also important when AvrRpt2 was cleaved in plant cells. Third, we investigated the effect of cleavage in interactions of AvrRpt2 with plant cells. Cleavage of AvrRpt2 appeared to be important for proper interactions with Arabidopsis cells that lack the resistance gene product corresponding to AvrRpt2, RPS2. In addition, removal of the region N-terminal to the cleavage site was important for the correct localization of the C-terminal effector region of the protein in the host cell. We speculate that the virulence function of AvrRpt2 requires removal of the N-terminal region to redirect the effector protein to a specific subcellular location in the host cell after translocation of the protein.
Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration
Authors: Chilton Mary-Dell M. , Que Qiudeng
Published: November 2003

Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome.
A Semidwarf Phenotype of Barley uzu Results from a Nucleotide Substitution in the Gene Encoding a Putative Brassinosteroid Receptor
Authors: Chono Makiko , Honda Ichiro , Zeniya Haruko , Yoneyama Koichi , Saisho Daisuke , Takeda Kazuyoshi , Takatsuto Suguru , Hoshino Tsuguhiro , Watanabe Yoshiaki
Published: November 2003

Brassinosteroids (BRs) play important roles throughout plant growth and development. Despite the importance of clarifying the mechanism of BR-related growth regulation in cereal crops, BR-related cereal mutants have been identified only in rice (Oryza sativa). We previously found that semidwarf barley (Hordeum vulgare) accessions carrying the “uzu” gene, called “uzu” barley in Japan, are non-responding for brassinolide (BL). We then performed chemical and molecular analyses to clarify the mechanisms of uzu dwarfism using isogenic line pairs of uzu gene. The response of the uzu line to BL was significantly lower than that of its corresponding normal line. Measurement of BRs showed that the uzu line accumulates BRs, similar to known BR-insensitive mutants. The marker synteny of rice and barley chromosomes suggests that the uzu gene may be homologous to rice D61, a rice homolog of Arabidopsis BR-insensitive 1 (BRI1), encoding a BR-receptor protein. A barley homolog of BRI1, HvBRI1, was isolated by using degenerate primers. A comparison of HvBRI1 sequences in uzu and normal barley varieties showed that the uzu phenotype is correlated with a single nucleotide substitution. This substitution results in an amino acid change at a highly conserved residue in the kinase domain of the BR-receptor protein. These results may indicate that uzu dwarfism is caused by the missense mutation in HvBRI1. The uzu gene is being introduced into all hull-less barley cultivars in Japan as an effective dwarf gene for practical use, and this is the first report about an agronomically important mutation related to BRs.
Genome-Wide Characterization of the Lignification Toolbox in Arabidopsis
Authors: Raes Jeroen , Rohde Antje , Christensen Jørgen Holst , Van de Peer Yves , Boerjan Wout
Published: November 2003

Lignin, one of the most abundant terrestrial biopolymers, is indispensable for plant structure and defense. With the availability of the full genome sequence, large collections of insertion mutants, and functional genomics tools, Arabidopsis constitutes an excellent model system to profoundly unravel the monolignol biosynthetic pathway. In a genome-wide bioinformatics survey of the Arabidopsis genome, 34 candidate genes were annotated that encode genes homologous to the 10 presently known enzymes of the monolignol biosynthesis pathway, nine of which have not been described before. By combining evolutionary analysis of these 10 gene families with in silico promoter analysis and expression data (from a reverse transcription-polymerase chain reaction analysis on an extensive tissue panel, mining of expressed sequence tags from publicly available resources, and assembling expression data from literature), 12 genes could be pinpointed as the most likely candidates for a role in vascular lignification. Furthermore, a possible novel link was detected between the presence of the AC regulatory promoter element and the biosynthesis of G lignin during vascular development. Together, these data describe the full complement of monolignol biosynthesis genes in Arabidopsis, provide a unified nomenclature, and serve as a basis for further functional studies.
The Maize Single myb histone 1 Gene, Smh1, Belongs to a Novel Gene Family and Encodes a Protein That Binds Telomere DNA Repeats in Vitro
Authors: Marian Calin O. , Bordoli Stefano J. , Goltz Marion , Santarella Rachel A. , Jackson Leisa P. , Danilevskaya Olga , Beckstette Michael , Meeley Robert , Bass Hank W.
Published: November 2003

We screened maize (Zea mays) cDNAs for sequences similar to the single myb-like DNA-binding domain of known telomeric complex proteins. We identified, cloned, and sequenced five full-length cDNAs representing a novel gene family, and we describe the analysis of one of them, the gene Single myb histone 1 (Smh1). The Smh1 gene encodes a small, basic protein with a unique triple motif structure of (a) an N-terminal SANT/myb-like domain of the homeodomain-like superfamily of 3-helical-bundle-fold proteins, (b) a central region with homology to the conserved H1 globular domain found in the linker histones H1/H5, and (c) a coiled-coil domain near the C terminus. The Smh-type genes are plant specific and include a gene family in Arabidopsis and the PcMYB1 gene of parsley (Petroselinum crispum) but are distinct from those (AtTRP1, AtTBP1, and OsRTBP1) recently shown to encode in vitro telomere-repeat DNA-binding activity. The Smh1 gene is expressed in leaf tissue and maps to chromosome 8 (bin 8.05), with a duplicate locus on chromosome 3 (bin 3.09). A recombinant full-length SMH1, rSMH1, was found by band-shift assays to bind double-stranded oligonucleotide probes with at least two internal tandem copies of the maize telomere repeat, TTTAGGG. Point mutations in the telomere repeat residues reduced or abolished the binding, whereas rSMH1 bound nonspecifically to single-stranded DNA probes. The two DNA-binding motifs in SMH proteins may provide a link between sequence recognition and chromatin dynamics and may function at telomeres or other sites in the nucleus.
Urea Transport by Nitrogen-Regulated Tonoplast Intrinsic Proteins in Arabidopsis
Authors: Liu Lai-Hua , Ludewig Uwe , Gassert Brigitte , Frommer Wolf B. , von Wirén Nicolaus
Published: November 2003

Urea is the major nitrogen (N) form supplied as fertilizer in agricultural plant production and also an important N metabolite in plants. Because urea transport in plants is not well understood, the aim of the present study was to isolate urea transporter genes from the model plant Arabidopsis. Using heterologous complementation of a urea uptake-defective yeast (Saccharomyces cerevisiae) mutant allowed to isolate AtTIP1;1, AtTIP1;2, AtTIP2;1, and AtTIP4;1 from a cDNA library of Arabidopsis. These cDNAs encode channel-like tonoplast intrinsic proteins (TIPs) that belong to the superfamily of major intrinsic proteins (or aquaporins). All four genes conferred growth of a urea uptake-defective yeast mutant on 2 mm urea in a phloretin-sensitive and pH-independent manner. Uptake studies using 14C-labeled urea into AtTIP2;1-expressing Xenopus laevis oocytes demonstrated that AtTIP2;1 facilitated urea transport also in a pH-independent manner and with linear concentration dependency. Expression studies showed that AtTIP1;2, AtTIP2;1, and AtTIP4;1 genes were up-regulated during early germination and under N deficiency in roots but constitutively expressed in shoots. Subcellular localization of green fluorescent protein-fused AtTIPs indicated that AtTIP1;2, AtTIP2;1, and AtTIP4;1 were targeted mainly to the tonoplast and other endomembranes. Thus, in addition to their role as water channels, TIP transporters may play a role in equilibrating urea concentrations between different cellular compartments.
Auxin Transport Synchronizes the Pattern of Cell Division in a Tobacco Cell Line
Authors: Campanoni Prisca , Blasius Bernd , Nick Peter
Published: November 2003

The open morphogenesis of plants requires coordination of patterning by intercellular signals. The tobacco (Nicotiana tabacum cv Virginia Bright Italia) cell line VBI-0 provides a simple model system to study the role of intercellular communication in patterning. In this cell line, singular cells divide axially to produce linear cell files of distinct polarity. The trigger for this axial division is exogenous auxin. When frequency distributions of files are constructed over the number of cells per file during the exponential phase of the culture, even numbers are found to be frequent, whereas files consisting of uneven numbers of cells are rare. We can simulate these distributions with a mathematical model derived from nonlinear dynamics, which describes a chain of cell-division oscillators where elementary oscillators are coupled unidirectionally and where the number of oscillators is not conserved. The model predicts several nonintuitive properties of our experimental system. For instance, files consisting of six cells are more frequent than expected from a strictly binary division system. More centrally, the model predicts a polar transport of the coordinating signal. We therefore tested the patterns obtained after treatment with 1-N-naphthylphthalamic acid, an inhibitor of auxin efflux carriers. Using low concentrations of 1-N-naphthylphthalamic acid that leave cell division and axiality of division unaltered, we observe that the frequencies of files with even and uneven cell numbers are equalized. Our findings are discussed in the context of auxin transport as synchronizing signal in cell patterning.
Brassinosteroids Promote Root Growth in Arabidopsis
Authors: Müssig Carsten , Shin Ga-Hee , Altmann Thomas
Published: November 2003

Although brassinosteroids (BRs) are known to regulate shoot growth, their role in the regulation of root growth is less clear. We show that low concentrations of BRs such as 24-epicastasterone and 24-epibrassinolide promote root elongation in Arabidopsis wild-type plants up to 50% and in BR-deficient mutants such as dwf1-6 (cbb1) and cbb3 (which is allelic to cpd) up to 150%. The growth-stimulating effect of exogenous BRs is not reduced by the auxin transport inhibitor 2,3,5-triidobenzoic acid. BR-deficient mutants show normal gravitropism, and 2,3,5-triidobenzoic acid or higher concentrations of 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid inhibit root growth in the mutants to the same extent as in wild-type plants. Simultaneous administration of 24-epibrassinolide and 2,4-dichlorophenoxyacetic acid results in largely additive effects. Exogenous gibberellins do not promote root elongation in the BR-deficient mutants, and the sensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid is not altered. Thus, the root growth-stimulating effect of BRs appears to be largely independent of auxin and gibberellin action. Furthermore, we analyzed BR interactions with other phytohormones on the gene expression level. Only a limited set of auxin- and ethylene-related genes showed altered expression levels. Genes related to other phytohormones barely showed changes, providing further evidence for an autonomous stimulatory effect of BR on root growth.
Recognition of the Agrobacterium tumefaciens VirE2 Translocation Signal by the VirB/D4 Transport System Does Not Require VirE1
Authors: Vergunst Annette C. , van Lier Miranda C.M. , den Dulk-Ras Amke , Hooykaas Paul J.J.
Published: November 2003

Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells. The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system. VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell. We analyzed whether the VirE1 chaperone is also essential for transport recognition of VirE2 by the VirB/D4 encoded type IV secretion system. In addition, we assayed whether translocation of VirF and VirE3, which also forms part of the virE operon, is affected by the absence of VirE1. We employed the earlier developed CRAFT (Cre recombinase Reporter Assay For Translocation) assay to detect transfer of Cre::Vir fusion proteins from A. tumefaciens into plants, monitored by stable reconstitution of a kanamycin resistance marker, and into yeast, screened by loss of the URA3 gene. We show that the C-terminal 50 amino acids of VirE2 and VirE3 are sufficient to mediate Cre translocation into host cells, confirming earlier indications of a C-terminal transport signal. This transfer was independent of the presence or absence of VirE1. Besides, the translocation efficiency of VirF is not altered in a virE1 mutant. The results unambiguously show that the VirE1 chaperone is not essential for the recognition of the VirE2 transport signal by the transport system and the subsequent translocation across the bacterial envelope into host cells.
Focal aggregation of voltage‐gated, Kv2.1 subunit‐containing, potassium channels at synaptic sites in rat spinal motoneurones
Authors: Muennich Elizabeth A. L. , Fyffe R. E. W.
Published: November 2003

Delayed rectifier K+ currents are involved in the control of α‐motoneurone excitability, but the precise spatial distribution and organization of the membrane ion channels that contribute to these currents have not been defined. Voltage‐activated Kv2.1 channels have properties commensurate with a contribution to delayed rectifier currents and are expressed in neurones throughout the mammalian central nervous system. A specific antibody against Kv2.1 channel subunits was used to determine the surface distribution and clustering of Kv2.1 subunit‐containing channels in the cell membrane of α‐motoneurones and other spinal cord neurones. In α‐motoneurones, Kv2.1 immunoreactivity (‐IR) was abundant in the surface membrane of the soma and large proximal dendrites, and was present also in smaller diameter distal dendrites. Plasma membrane‐associated Kv2.1‐IR in α‐motoneurones was distributed in a mosaic of small irregularly shaped, and large disc‐like, clusters. However, only small to medium clusters of Kv2.1‐IR were observed in spinal interneurones and projection neurones, and some interneurones, including Renshaw cells, lacked demonstrable Kv2.1‐IR. In α‐motoneurones, dual immunostaining procedures revealed that the prominent disc‐like domains of Kv2.1‐IR are invariably apposed to presynaptic cholinergic C‐terminals. Further, Kv2.1‐IR colocalizes with immunoreactivity against postsynaptic muscarinic (m2) receptors at these locations. Ultrastructural examination confirmed the postsynaptic localization of Kv2.1‐IR at C‐terminal synapses, and revealed clusters of Kv2.1‐IR at a majority of S‐type, presumed excitatory, synapses. Kv2.1‐IR in α‐motoneurones is not directly associated with presumed inhibitory (F‐type) synapses, nor is it present in presynaptic structures apposed to the motoneurone. Occasionally, small patches of extrasynaptic Kv2.1‐IR labelling were observed in surface membrane apposed by glial processes. Voltage‐gated potassium channels responsible for the delayed rectifier current, including Kv2.1, are usually assigned roles in the repolarization of the action potential. However, the strategic localization of Kv2.1 subunit‐containing channels at specific postsynaptic sites suggests that this family of voltage‐activated K+ channels may have additional roles and/or regulatory components.
Terminal care in paediatrics: where we are now
Authors: Hutchinson F , King N , Hain R D W
Published: November 2003

As in adults, palliative care in children is a comprehensive multidisciplinary approach to care that seeks to enhance the life of children and families living with life limiting conditions. It involves a holistic approach embracing symptom management, psychosocial/spiritual care, and bereavement support.
INVESTIGATING CONFLICTS IN COTS DECISION-MAKING
Authors: ALVES CARINA , FINKELSTEIN ANTHONY
Published: November 2003

The development of COTS-based systems largely depends on the success of the selection process. This activity encompasses the evaluation of COTS packages against customer requirements, so that it is necessary to perform a complex decision-making process to select the most suitable package. We argue that analysing the matching between features and requirements is a core task of COTS decision-making. In particular, it is very likely that mismatches occur, as COTS are designed to meet very general requirements rather than specific needs. In this paper, we investigate the different types of conflicts that can arise from these mismatches. We propose a goal driven framework to deal with such conflicts. We demonstrate the approach with the mail server case study.
Liposomal Alendronate Inhibits Systemic Innate Immunity and Reduces In-Stent Neointimal Hyperplasia in Rabbits
Authors: Danenberg Haim D. , Golomb Gershon , Groothuis Adam , Gao Jianchuan , Epstein Hila , Swaminathan Rajesh V. , Seifert Philip , Edelman Elazer R.
Published: November 2003

Background—

Innate immunity is of major importance in vascular repair. The present study evaluated whether systemic and transient depletion of monocytes and macrophages with liposome-encapsulated bisphosphonates inhibits experimental in-stent neointimal formation.

Methods and Results—

Rabbits fed on a hypercholesterolemic diet underwent bilateral iliac artery balloon denudation and stent deployment. Liposomal alendronate (3 or 6 mg/kg) was given concurrently with stenting. Monocyte counts were reduced by >90% 24 to 48 hours after a single injection of liposomal alendronate, returning to basal levels at 6 days. This treatment significantly reduced intimal area at 28 days, from 3.88±0.93 to 2.08±0.58 and 2.16±0.62 mm
2
. Lumen area was increased from 2.87±0.44 to 3.57±0.65 and 3.45±0.58 mm
2
, and arterial stenosis was reduced from 58±11% to 37±8% and 38±7% in controls, rabbits treated with 3 mg/kg, and rabbits treated with 6 mg/kg, respectively (mean±SD, n=8 rabbits/group,
P
<0.01 for all 3 parameters). No drug-related adverse effects were observed. Reduction in neointimal formation was associated with reduced arterial macrophage infiltration and proliferation at 6 days and with an equal reduction in intimal macrophage and smooth muscle cell content at 28 days after injury. Conversely, drug regimens ineffective in reducing monocyte levels did not inhibit neointimal formation.

Conclusions—

Systemic transient depletion of monocytes and macrophages, by a single liposomal bisphosphonates injection concurrent with injury, reduces in-stent neointimal formation and arterial stenosis in hypercholesterolemic rabbits.
Diverse Origin and Function of Cells With Endothelial Phenotype Obtained From Adult Human Blood
Authors: Gulati Rajiv , Jevremovic Dragan , Peterson Timothy E. , Chatterjee Suvro , Shah Vijay , Vile Richard G. , Simari Robert D.
Published: November 2003

Cells with endothelial phenotype generated from adult peripheral blood have emerging diagnostic and therapeutic potential. This study examined the lineage relationship between, and angiogenic function of, early endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) in culture. Culture conditions were established to support the generation of both EPCs and OECs from the same starting population of peripheral blood mononuclear cells (PBMCs). Utilizing differences in expression of the surface endotoxin receptor CD14, it was determined that the vast majority of EPCs arose from a CD14
+
subpopulation of PBMCs but OECs developed exclusively from the CD14
−
fraction. Human OECs, but not EPCs, expressed key regulatory proteins endothelial nitric oxide synthase (eNOS) and caveolin-1. Moreover, OECs exhibited a markedly greater capacity for capillary morphogenesis in in vitro and in vivo matrigel models, tube formation by OECs being in part dependent on eNOS function. Collectively, these data indicate lineage and functional heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype and provide rationale for the investigation of new cell-therapeutic approaches to ischemic cardiovascular disease.
Safety Profile of Tissue Plasminogen Activator Treatment Among Stroke Patients Carrying a Common Polymorphism (C-1562T) in the Promoter Region of the Matrix Metalloproteinase-9 Gene
Authors: Montaner Joan , Fernández-Cadenas Israel , Molina Carlos A. , Monasterio Jasone , Arenillas Juan F. , Ribó Marc , Quintana Manolo , Chacón Pilar , Andreu Antoni L. , Alvarez-Sabín José
Published: November 2003

Background and Purpose—

Matrix metalloproteinase-9 (MMP-9) expression, related to blood-brain barrier disruption, has been implicated in the appearance of hemorrhagic transformation (HT) after tissue plasminogen activator (tPA) treatment in stroke patients. Because an in vitro functional polymorphism of the promoter region of MMP-9 gene (C-1562T) has been described, we hypothesize that patients carrying this mutation might have higher MMP-9 levels and greater susceptibility to developing HT when receiving tPA.

Methods—

We studied strokes involving the middle cerebral artery territory of 61 patients who received tPA <3 hours after stroke onset. Blood samples were obtained before tPA administration. Plasmatic MMP-9 determinations were performed (enzyme-linked immunosorbent assay, ng/mL), and C-1562T genotype was determined by polymerase chain reaction. Healthy age-matched control subjects were used to study allele distribution (n=59). Hemorrhagic events were classified according to CT criteria (petechial hemorrhagic infarctions [HI,1 to 2] and large parenchymal hemorrhages [PH,1 to 2]).

Results—

Allele distribution was similar in patients and control subjects (CC/CT/TT: 72.3/27.7/0% versus 79.7/20.3/0%, respectively;
P
=0.37). Among patients, mutation carriers (CT/TT alleles) had similar rates of HT and PH than noncarriers (HT: 23.1% versus 38.2%,
P
=0.49; PH: 15.4% versus 17.6%,
P
=1.0). Although the highest MMP-9 level corresponded to patients who later developed a PH (PH, 191.4 ng/mL; non-PH, 68.05 ng/mL;
P
=0.022), no relation between MMP-9 mutation presence and plasmatic levels was found (CC, 127.12 ng/mL; CT/TT, 46.31 ng/mL;
P
=0.11).

Conclusions—

Although MMP-9 level predicts PH appearance after tPA treatment, no relationship exists with the C-1562T polymorphism, probably because this mutation is not functional in response to cerebral ischemia in vivo.
Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow
Authors: Ratajczak Janina , Reca Ryan , Kucia Magda , Majka Marcin , Allendorf Daniel J. , Baran Jarek T. , Janowska-Wieczorek Anna , Wetsel Rick A. , Ross Gordon D. , Ratajczak Mariusz Z.
Published: November 2003

The mechanisms regulating the homing/mobilization of hematopoietic stem/progenitor cells (HSPCs) are not fully understood. In our previous studies we showed that the complement C3 activation peptide, C3a, sensitizes responses of HSPCs to stromal-derived factor 1 (SDF-1). In this study, mobilization was induced with granulocyte colony-stimulating factor (G-CSF) in both C3-deficient (C3–/–) and C3a receptor–deficient (C3aR–/–) mice as well as in wild-type (wt) mice in the presence or absence of a C3aR antagonist, SB 290157. The data indicated (1) significantly increased G-CSF–induced mobilization in C3–/– and C3aR–/– mice compared with wt mice, (2) significantly accelerated and enhanced G-CSF–induced mobilization in wt, but not in C3–/– or C3aR–/–, mice treated with SB 290157, and (3) deposition of C3b/iC3b fragments onto the viable bone marrow (BM) cells of G-CSF–treated animals. Furthermore, mobilization studies performed in chimeric mice revealed that wt mice reconstituted with C3aR–/– BM cells, but not C3aR–/– mice reconstituted with wt BM cells, are more sensitive to G-CSF–induced mobilization, suggesting that C3aR deficiency on graft-derived cells is responsible for this increased mobilization. Hence we suggest that C3 is activated in mobilized BM into C3a and C3b, and that the C3a-C3aR axis plays an important and novel role in retention of HSPCs (by counteracting mobilization) by increasing their responsiveness to SDF-1, the concentration of which is reduced in BM during mobilization. The C3a-C3aR axis may prevent an uncontrolled release of HSPCs into peripheral blood. These data further suggest that the C3aR antagonist SB 290157 could be developed as a drug to mobilize HSPCs for transplantation.
Loss of circulating CD27+ memory B cells and CCR4+ T cells occurring in association with elevated EBV loads in XLP patients surviving primary EBV infection
Authors: Malbran Alejandro , Belmonte Liliana , Ruibal-Ares Beatriz , Baré Patricia , Massud Ivana , Parodi Cecilia , Felippo Marta , Hodinka Richard , Haines Kathleen , Nichols Kim E. , de Bracco María M.
Published: November 2003

Detailed longitudinal studies of patients with X-linked lymphoproliferative disease (XLP) may increase our understanding of the immunologic defects that contribute to the development of lymphoma and hypogammaglobulinemia in XLP. We describe progressive changes observed in immunoglobulin concentrations, lymphocyte subsets, and Epstein-Barr virus (EBV) loads occurring in a 2-year period in a newly infected, but otherwise healthy, carrier (patient 9). We compare these findings with those observed in the patient's brother, who had hypogammaglobulinemia and XLP (patient 4). Immunoglobulin G (IgG), IgM, and IgA concentrations increased in patient 9 during acute EBV infection, but thereafter they decreased steadily to concentrations consistent with hypogammaglobulinemia, reaching a plateau 5 months after infection. In both patients, CD19+ B-lymphocyte rates remained lower than 3%, with a contraction of the B-cell memory compartment (CD27+ CD19+/CD19+) to 20% (normal range, 32%-56%). T-lymphocyte subpopulations showed a reduction in CD4+ T-cell counts and a permanent CD8+ T-cell expansion. Interestingly, CXCR3 memory TH1 cells were expanded and CCR4+ TH2 lymphocytes were reduced, suggesting that abnormal skewing of memory T-cell subsets might contribute to reduced antibody synthesis. Despite an expanded number of CD3+CD8+ lymphocytes, increased EBV loads occurred in both patients without overt clinical symptoms of mononucleosis, lymphoproliferative disease, or lymphoma.
Involvement of Protein Kinase C-Dependent Mitogen-Activated Protein Kinase p44/42 Signaling Pathway for Cross-Talk between Estradiol and Transforming Growth Factor-β3 in Increasing Basic Fibroblast Growth Factor in Folliculostellate Cells
Authors: Chaturvedi Kirti , Sarkar Dipak K.
Published: November 2003

We have recently shown that TGF-β3, in the presence of estradiol, increases the release of basic fibroblast growth factor (bFGF) from folliculostellate (FS) cells in the pituitary. We determined the interactive effects of TGF-β3 and estradiol on bFGF production and release from FS cells, and the role of the MAPK pathway in TGF-β3 and estradiol interaction. We found that TGF-β3 and estradiol alone moderately increased cell content and release of bFGF from FS cells; but together, they markedly increased the peptide. Estradiol and TGF-β3 alone moderately activated MAPK p44/42; together they produced marked activation of MAPK p44/42. Pretreatment of FS cells with an MAPK kinase 1/2 inhibitor or with protein kinase C inhibitors suppressed the activation of MAPK p44/42, bFGF release, and protein level increases, all of which were induced by TGF-β3 and estradiol. Estradiol and TGF-β3, either alone or in combination, increased the levels of active Ras. Furthermore, bFGF induction by TGF-β3 and estradiol was blocked by overexpression of Ras N17, a dominant negative mutant of Ras p21. Estrogen receptor blocker ICI 182,780 failed to prevent estrogen’s and TGF-β3’s effects on bFGF. These data suggest that an estradiol receptor-independent protein kinase C- activated Ras-dependent MAPK pathway is involved in the cross-talk between TGF-β3 and estradiol to increase bFGF production and/or release from FS cells.
Alternobaric vertigo in sport SCUBA divers and the risk factors
Authors: Uzun Cem , Yagiz Recep , Tas Abdullah , Adali Mustafa K. , Inan Nurkan , Koten Muhsin , Karasalihoglu Ahmet R.
Published: November 2003

We investigated the eustachian tube function and the incidence of alternobaric vertigo (AV) in 29 sport self-contained underwater breathing apparatus (SCUBA) divers with, or without, some possible risk factors for AV. The divers had normal audiological and otoscopic findings at the pre-dive examination. We used the nine-step inflation/deflation tympanometric test and Toynbee test for evaluation of eustachian tube function, and the Valsalva manoeuvre for patency. Information on divers, their history, and their otolaryngologic examination were obtained in the pre-dive examination. Divers performed 1086 dives (mean 37, range: 3–100) during the observation period. Four divers (14 per cent) experienced AV during five dives (0.46 per cent), (one diver experienced AV two times). It was found that having an otitis media history or eustachian tube dysfunction determined with the nine-step inflation/deflation tympanometric test before diving, or difficulty in clearing ears during diving could be important risk factors for AV in sport SCUBA divers (p<.05). Divers with such findings seem to be more prone to AV and should pay rigorous attention to the precautions for prevention of AV.
Degloving of the inferior turbinates: pilot study to assess the effectiveness of a new technique in turbinate reduction
Authors: Chevretton E. B. , Hopkins C. , Black I. M. , Tierney P. , Smeeton N. C.
Published: November 2003

A pilot study to assess the safety and efficacy of ’degloving’ of the inferior turbinate is described. This prospective study reports on the effectiveness of the technique in 37 patients with nasal obstruction due to inferior turbinate hypertrophy. Both objective and subjective measurements of nasal patency were made pre-operatively, and repeated at six weeks, six months and two years post-operatively using nasal inspiratory peak flow rates, saccharin clearance time and an end referenced visual analogue scale to record nasal symptoms.

There was a significant improvement in the peak inspiratory flow at six weeks (96.4 to 151.2 l/min) and at six months (148.4 l/min), which was sustained at two years (117.1 l/min, p<0.001). There was an overall improvement in patient satisfaction with nasal symptoms (23.4 to 76.7, 76.8 and 66.8 at six weeks, six months and two years, p<0.001) and a decrease in the sensation of nasal obstruction (71.9 to 21.5, 32.9 and 29.8, p<0.001), which was also sustained. Rhinorrhoea was reduced, and sense of smell increased across the group, but the improvements did not reach statistical significance. The results for postnasal drip and saccharin clearance did not show a significant change over the period of the study. There were no haemorrhagic complications in the group studied.

This pilot study demonstrates a new surgical technique for reduction of the inferior turbinates, that yields significant improvement in nasal obstruction, an acceptably low complication rate and is well tolerated by patients. A randomized controlled trial is being planned.
Oxidant, vitamin A and angiogenic markers in laryngeal cancer patients
Authors: Kandpal N. , Shukla G. K. , Bhatia N. , Agarwal S. P. , Saha S. , Puri V. N.
Published: November 2003

In this study the status of oxidant stress, vitamin A and angiotensin-converting enzyme (ACE) levels were evaluated in cases of laryngeal carcinoma patients from Northern India. In control subjects the levels of malondialdehyde (MDA), vitamin A and ACE were 0.23 ± 0.07 nmole/ml, 2515 ± 84 IU, and 1.4 ± 0.8 U/ml respectively. Thirty laryngeal cancer patients were divided into three groups according to the TNM classification (American Joint Committee on Cancers). In laryngeal cancer patients according to tumour size, MDA and ACE levels increased to 0.32 ± 0.04 nmole/ml and 4.7 ± 0.5 U/ml respectively and the effect was statistically significant (p<0.01). The correlation coefficient between different subgroups was also highly significant (r =0.96, p<0.01). However, serum vitamin A levels decreased to 621 ± 20 IU and the effect was statistically significant (p<0.01). In another two groups of laryngeal cancer patients, a similar pattern of various markers was obtained. Thirty patients with laryngeal carcinoma were divided into four different groups according to nodal involvement and it was observed that in laryngeal cancer patients with no nodal involvement, ACE levels were low 3.6 ± 1.4 U/ml while patients with maximum nodal involvement had the highest levels of ACE 7.1 ± 0.18 U/ml. The correlation coefficient between different groups is highly statistically significant (r = 0.95, p<0.01). In patients with laryngeal cancer the serum MDA and vitamin A levels correlation coefficient between different groups was not significant. It is thus concluded that serum ACE might be a specific test marker for laryngeal cancer disease burden. The use of this marker enzyme for therapeutics is being planned.
Mucous membrane pemphigoid: nasal and laryngeal manifestations
Authors: Whiteside O. J. H. , Martinez Devesa P. , Ali I. , Capper J. W. R.
Published: November 2003

Mucous membrane pemphigoid (MMP) is a sub-epithelial blistering disease that primarily involves mucosal surfaces but may also involve the skin. Clinically, it appears as vesiculobullous lesions of the oral cavity and eyes, but other tissues such as the nasopharyngeal and laryngeal mucosa can also be affected. Ultimately, scarring and airway stenosis may occur. The condition should be managed by a multidisciplinary team led by a dermatologist. Immunosuppressive therapy forms the mainstay of treatment, with surgery having both a diagnostic role and a use in the treatment of complications such as airway obstruction. There must be a low threshold of suspicion for laryngeal involvement in this group of patients, so that prompt action can be taken. Once laryngeal stenosis has occurred repeated endoscopic laser excision of scar tissue can be used to maintain an adequate airway. Adjuvant use of mitomycin-C can be used beneficially in the treatment of laryngeal complications of MMP.
Orbitofrontal cholesterol granuloma: percutaneous endoscopic-assisted curettage
Authors: Selva Dinesh , Lai Tze , Krishnan Suren
Published: November 2003

This paper describes the use of endoscopic visualization in curettage of orbital cholesterol granuloma (OCG). Two males aged 54 and 50 years presented with orbitofrontal cholesterol granulomas arising in the superolateral frontal bone and abutting the dura. The granulomas were approached via a superior eyelid crease incision and a 70 degree rigid endoscope was used to visualize curettage of the granuloma from the inner surface of the frontal bone and the dura. Both patients made an uncomplicated recovery and there was no recurrence at eight months and two years follow up.

Percutaneous endoscopic curettage is an alternative to blind curettage, lateral orbitotomy or frontal craniotomy for OCG.
Fragmentation of extracellular matrix by hypochlorous acid
Authors: WOODS Alan A. , DAVIES Michael J.
Published: November 2003

The interaction of extracellular matrix with cells regulates their adhesion, migration and proliferation, and it is believed that damage to vascular matrix components is a factor in the development of atherosclerosis. Evidence has been provided for a role for the haem enzyme MPO (myeloperoxidase), released by activated monocytes (and possibly macrophages), in oxidative events within the artery wall. As MPO is released extracellularly, and is highly basic, it might be expected to associate with poly-anionic matrix components thereby localizing damage to these materials. In this study the reaction of the MPO-derived oxidant hypochlorous acid (HOCl) with extracellular matrix from vascular smooth muscle cells and healthy pig arteries has been examined. HOCl is rapidly consumed by such matrix samples, with the formation of matrix-derived chloramines or chloramides. The yield of these intermediates increases with HOCl dose. These materials undergo a time- and temperature-dependent decay, which parallels the release of sugar and protein components from the treated matrix, consistent with these species being important intermediates. Matrix damage is enhanced by species that increase chloramine/chloramide decomposition, with copper and iron ions being effective catalysts, and decreased by compounds which scavenge chloramines/chloramides, or species derived from them. The effect of such matrix modifications on cellular behaviour is poorly understood, though it is known that changes in matrix materials can have profound effects on cell adhesion, proliferation, growth and phenotype. The observed matrix modifications reported here may therefore modulate cellular behaviour in diseases such as atherosclerosis where MPO-derived oxidants are generated.
Glutathione transferase P1-1: self-preservation of an anti-cancer enzyme
Authors: RICCI Giorgio , CACCURI Anna Maria , LO BELLO Mario , PARKER Michael W. , NUCCETELLI Marzia , TURELLA Paola , STELLA Lorenzo , DI IORIO Ernesto E. , FEDERICI Giorgio
Published: November 2003

Self-preservation is a typical property of living organisms, observed in the simplest prokaryotic cell as well as in the more complex pluricellular organisms. Surprisingly we found a self-preservation mechanism operating at the level of a single enzyme. Human glutathione transferase P1-1 operates in such a way towards either killer compounds (competitive and irreversible inhibitors) or physical factors (temperature and UV-rays), which could suppress its detoxicating and anti-cancer activity in the cell. This property, here termed ‘co-operative self-preservation’, is based on a structural intersubunit communication, by which one subunit, as a consequence of an inactivating modification, triggers a defence arrangement in the other subunit. Paradoxically this ability, developed during evolution for the survival of the cell, may not always be advantageous for us. In fact, glutathione transferase P1-1 is overexpressed in most tumour cells and pharmacological attempts to inhibit this enzyme in vivo, to prevent the drug resistance phenomenon during chemotherapy, may be thwarted by such self-preservation.
Role of the glutamate dehydrogenase reaction in furnishing aspartate nitrogen for urea synthesis: studies in perfused rat liver with 15N
Authors: NISSIM Itzhak , HORYN Oksana , LUHOVYY Bohdan , LAZAROW Adam , DAIKHIN Yevgeny , NISSIM Ilana , YUDKOFF Marc
Published: November 2003

The present study was designed to determine: (i) the role of the reductive amination of α-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of α-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N-acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.
Polyester synthases: natural catalysts for plastics
Authors: REHM Bernd H. A.
Published: November 2003

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8–96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to α/β-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the α/β-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism.