bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeastPichiapastoris
Authors: Choi Byung-Kwon , Bobrowicz Piotr , Davidson Robert C. , Hamilton Stephen R. , Kung David H. , Li Huijuan , Miele Robert G. , Nett Juergen H. , Wildt Stefan , Gerngross Tillman U.
Published: April 2003

The secretory pathway ofPichia pastoriswas genetically re-engineered to perform sequential glycosylation reactions that mimic early processing of N-glycans in humans and other higher mammals. After eliminating nonhuman glycosylation by deleting the initiating α-1,6-mannosyltransferase gene fromP. pastoris, several combinatorial genetic libraries were constructed to localize active α-1,2-mannosidase and human β-1,2-N-acetylglucosaminyltransferase I (GnTI) in the secretory pathway. First, >32 N-terminal leader sequences of fungal type II membrane proteins were cloned to generate a leader library. Two additional libraries encoding catalytic domains of α-1,2-mannosidases and GnTI from mammals, insects, amphibians, worms, and fungi were cloned to generate catalytic domain libraries. In-frame fusions of the respective leader and catalytic domain libraries resulted in several hundred chimeric fusions of fungal targeting domains and catalytic domains. Although the majority of strains transformed with the mannosidase/leader library displayed only modestin vivo[i.e., low levels of mannose (Man)5-(GlcNAc)2] activity, we were able to isolate several yeast strains that produce almost homogenous N-glycans of the (Man)5-(GlcNAc)2type. Transformation of these strains with a UDP-GlcNAc transporter and screening of a GnTI leader fusion library allowed for the isolation of strains that produce GlcNAc-(Man)5-(GlcNAc)2in high yield. Recombinant expression of a human reporter protein in these engineered strains led to the formation of a glycoprotein with GlcNAc-(Man)5-(GlcNAc)2as the primary N-glycan. Here we report a yeast able to synthesize hybrid glycans in high yield and open the door for engineering yeast to perform complex human-like glycosylation.
The nature of heme/iron-induced protein tyrosine nitration
Authors: Bian Ka , Gao Zhonghong , Weisbrodt Norman , Murad Ferid
Published: May 2003

Recently, substantial evidence has emerged that revealed a very close association between the formation of nitrotyrosine and the presence of activated granulocytes containing peroxidases, such as myeloperoxidase. Peroxidases share heme-containing homology and can use H
2
O
2
to oxidize substrates. Heme is a complex of iron with protoporphyrin IX, and the iron-containing structure of heme has been shown to be an oxidant in several model systems where the prooxidant effects of free iron, heme, and hemoproteins may be attributed to the formation of hypervalent states of the heme iron. In the current study, we have tested the hypothesis that free heme and iron play a crucial role in NO
2
-Tyr formation. The data from our study indicate that: (
i
) heme/iron catalyzes nitration of tyrosine residues by using hydrogen peroxide and nitrite, a reaction that revealed the mechanism underlying the protein nitration by peroxidase, H
2
O
2
, and NO

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; (
ii
) H
2
O
2
plays a key role in the protein oxidation that forms the basis for the protein nitration, whereas nitrite is an essential element that facilitates nitration by the heme(Fe), H
2
O
2
, and the NO

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system; (
iii
) the formation of a Fe(IV) hypervalent compound may be essential for heme(Fe)-catalyzed nitration, whereas O

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(ONOO
−
formation),
•
OH (Fenton reaction), and compound III are unlikely to contribute to the reaction; and (
iv
) hemoprotein-rich tissues such as cardiac muscle are vulnerable to protein nitration in pathological conditions characterized by the overproduction of H
2
O
2
and NO

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, or nitric oxide.
Cancer-related changes in prostate DNA as men age and early identification of metastasis in primary prostate tumors
Authors: Malins Donald C. , Johnson Paul M. , Barker Edward A. , Polissar Nayak L. , Wheeler Thomas M. , Anderson Katie M.
Published: April 2003

Using statistical analyses of Fourier transform-IR spectra, we show that DNA of the histologically normal prostates of men 16–80 years old undergoes structural changes in the bases and backbone with increasing age. Of the older men (ages 55–80), 42% exhibited a DNA phenotype mimicking that of primary prostate tumors from a comparable age group. This cancer-like phenotype, which was not found in the younger men (ages 16–36), appears to arise from progressive age-related damage to DNA. The mean concentrations of 8-hydroxypurine lesions (e.g., 8-hydroxyguanine) were substantially higher for the older men than for the younger men. This finding suggests that the hydroxyl radical contributed to the structural changes that characterize the cancer-like phenotype. Strikingly, we were additionally able to discriminate between the DNA of primary prostate tumors and the DNA of primary prostate tumors from which distant metastases had been identified. Moreover, logistic regression analysis was able to predict the probability that a tumor had metastasized with ≈90% sensitivity and specificity. Collectively, these findings are particularly promising for identifying men at risk for developing prostate cancer, as well as for the early determination of whether a primary tumor has progressed to the metastatic state. This is highly important because the prognosis of histologically similar prostate carcinomas varies, thus creating a need to predict which cancers are most likely metastatic.
Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system
Authors: Lin Li , Liu Yao-Guang , Xu Xinping , Li Baojian
Published: May 2003

Introduction and expression of multiple transgenes is frequently required for basic and applied studies. However, at present, multigene transformation is very difficult due to technical limitations of existing methods. Here, we describe a vector system for efficient multigene assembly and transformation. The system consists of a transformation-competent artificial chromosome (TAC)-based acceptor vector together with two donor vectors. By exploiting the Cre/
loxP
recombination system and homing endonucleases, multiple rounds of gene assembly cycling were carried out with alternate use of the donor vectors, and multiple genes were sequentially delivered into the TAC vector. With this system, we created constructs containing as many as 10 foreign DNA fragments. Multiple genes, including six resistant genes stacked in a construct, were transferred into rice genome by
Agrobacterium
-mediated transformation. This system extends the repertoire of molecular genetic studies and biotechnological endeavors by enabling simultaneous manipulation of multiple genes.
Different time courses for visual perception and action priming
Authors: Vorberg Dirk , Mattler Uwe , Heinecke Armin , Schmidt Thomas , Schwarzbach Jens
Published: May 2003

Visual stimuli may remain invisible but nevertheless produce strong and reliable effects on subsequent actions. How well features of a masked prime are perceived depends crucially on its physical parameters and those of the mask. We manipulated the visibility of masked stimuli and contrasted it with their influence on the speed of motor actions, comparing the temporal dynamics of visual awareness in metacontrast masking with that of action priming under the same conditions. We observed priming with identical time course for reportable and invisible prime stimuli, despite qualitative changes in the masking time course. Our findings indicate that experimental variations that modify the subjective visual experience of masked stimuli have no effect on motor effects of those stimuli in early processing. We propose a model that provides a quantitative account of priming effects on response speed and accuracy.
Targeting therapeutics to an exposed and conserved binding element of the HIV-1 fusion protein
Authors: Root Michael J. , Hamer Dean H.
Published: April 2003

There is an urgent need for new drugs that can kill HIV type 1 (HIV-1)-infected cells. HIV-1 glycoprotein Env, which promotes viral membrane fusion through receptor-mediated conformational changes, is an attractive target for such agents because it is expressed on the surface of both virions and infected cells. Unfortunately, conserved binding elements on this protein frequently are buried under a canopy of flexible, glycosylated peptide loops or exposed only transiently during the fusion process. Here, we investigate the exposure of the C-terminal region of the Env ectodomain outside the context of membrane fusion. This binding element is the target of the 5-Helix protein, a designed entry inhibitor that disrupts conformational changes in Env subunit gp41, essential for the fusion process. We show that 5-Helix is capable of interacting with HIV-1 Env in a receptor-independent fashion and that a chimeric 5-Helix/Pseudomonasexotoxin protein recognizes cells expressing Env from a broad spectrum of HIV-1 strains including primary isolates from clades B, D, E, G, and H. This recombinant toxin selectively kills HIV-1-infected cells and blocks spreading infection while still maintaining potent inhibitory activity against membrane fusion. Our results demonstrate that the C-terminal region of the gp41 ectodomain is an accessible target on HIV-1-infected cells for the development of antiviral therapeutics and neutralizing antibodies.
Transgenic overexpression of galanin in the dorsal root ganglia modulates pain-related behavior
Authors: Holmes Fiona E. , Bacon Andrea , Pope Robert J. P. , Vanderplank Penny A. , Kerr Niall C. H. , Sukumaran Madhu , Pachnis Vassilis , Wynick David
Published: May 2003

The neuropeptide galanin is expressed in the dorsal root ganglia (DRG) and spinal cord and is thought to be involved in the modulation of pain processing. However, its mechanisms of action are complex and poorly understood, as both facilitatory and inhibitory effects have been described. To understand further the role played by galanin in nociception, we have generated two transgenic lines that overexpress galanin in specific populations of primary afferent DRG neurons in either an inducible or constitutive manner. In the first line, a previously defined enhancer region from the galanin locus was used to target galanin to the DRG (Gal-OE). Transgene expression recapitulates the spatial endogenous galanin distribution pattern in DRG neurons and markedly overexpresses the peptide in the DRG after nerve injury but not in the uninjured state. In the second line, an enhancer region of the c-
Ret
gene was used to constitutively and ectopically target galanin overexpression to the DRG (Ret-OE). The expression of this second transgene does not alter significantly after nerve injury. Here, we report that intact Ret-OE, but not Gal-OE, animals have significantly elevated mechanical and thermal thresholds. After nerve damage, using a spared nerve-injury model, mechanical allodynia is attenuated markedly in both the Gal-OE and Ret-OE mice compared with WT controls. These results support an inhibitory role for galanin in the modulation of nociception both in intact animals and in neuropathic pain states.
Positive inotropic and lusitropic effects of HNO/NO
−
in failing hearts: Independence from β-adrenergic signaling
Authors: Paolocci Nazareno , Katori Tatsuo , Champion Hunter C. , St. John Marcus E. , Miranda Katrina M. , Fukuto Jon M. , Wink David A. , Kass David A.
Published: April 2003

Nitroxyl anion (HNO/NO
−
), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal
in vivo
circulation. Here we tested whether HNO/NO
−
augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts β-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/NO
−
generated by Angelis' salt (AS) was infused (10 μg/kg per min, i.v.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all
P
< 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)/NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed β-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted β-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose 3-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/NO
−
cardiotropic action. Thus, HNO/NO
−
has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to β-adrenergic stimulation and stimulates CGRP release. This suggests potential of HNO/NO
−
donors for the treatment of heart failure.
Positional cloning of the wheat vernalization gene
VRN1
Authors: Yan L. , Loukoianov A. , Tranquilli G. , Helguera M. , Fahima T. , Dubcovsky J.
Published: May 2003

Winter wheats require several weeks at low temperature to flower. This process, vernalization, is controlled mainly by the
VRN1
gene. Using 6,190 gametes, we found
VRN1
to be completely linked to MADS-box genes
AP1
and
AGLG1
in a 0.03-centimorgan interval flanked by genes
Cysteine
and
Cytochrome B5
. No additional genes were found between the last two genes in the 324-kb
Triticum monococcum
sequence or in the colinear regions in rice and sorghum. Wheat
AP1
and
AGLG1
genes were similar to
Arabidopsis
meristem identity genes
AP1
and
AGL2
, respectively.
AP1
transcription was regulated by vernalization in both apices and leaves, and the progressive increase of
AP1
transcription was consistent with the progressive effect of vernalization on flowering time. Vernalization was required for
AP1
transcription in apices and leaves in winter wheat but not in spring wheat.
AGLG1
transcripts were detected during spike differentiation but not in vernalized apices or leaves, suggesting that
AP1
acts upstream of
AGLG1
. No differences were detected between genotypes with different
VRN1
alleles in the
AP1
and
AGLG1
coding regions, but three independent deletions were found in the promoter region of
AP1
. These results suggest that
AP1
is a better candidate for
VRN1
than
AGLG1
. The epistatic interactions between vernalization genes
VRN1
and
VRN2
suggested a model in which
VRN2
would repress directly or indirectly the expression of
AP1
. A mutation in the promoter region of
AP1
would result in the lack of recognition of the repressor and in a dominant spring growth habit.
Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway
Authors: DeLisa Matthew P. , Tullman Danielle , Georgiou George
Published: May 2003

To examine the relationship between folding and export competence by the twin-arginine translocation (Tat) pathway we analyzed the subcellular localization of fusions between a set of eight putative Tat leader peptides and alkaline phosphatase in isogenic
Escherichia coli
strains that either allow or disfavor the formation of protein disulfide bonds in the cytoplasm. We show that export by the Tat translocator is observed only in strains that enable oxidative protein folding in the cytoplasm. Further, we show that other disulfide-containing proteins, namely single-chain Fv and heterodimeric F
AB
antibody fragments, are export-competent only in strains having an oxidizing cytoplasm. Functional, heterodimeric F
AB
protein was exported from the cytoplasm by means of a Tat leader peptide fused to the heavy chain alone, indicating that the formation of a disulfide-bonded dimer preceeds export. These results demonstrate that
in vivo
only proteins that have attained the native conformation are exported by the Tat translocator, indicating that a folding quality-control mechanism is intrinsic to the export process. The ability to export proteins with disulfide bonds and the folding proofing feature of the Tat pathway are of interest for biotechnology applications.
SPARC-null mice exhibit increased adiposity without significant differences in overall body weight
Authors: Bradshaw A. D. , Graves D. C. , Motamed K. , Sage E. H.
Published: May 2003

Secreted protein acidic and rich in cysteine/osteonectin/BM-40 (SPARC) is a matrix-associated protein that elicits changes in cell shape, inhibits cell-cycle progression, and influences the synthesis of extracellular matrix (ECM). The absence of SPARC in mice gives rise to aberrations in the structure and composition of the ECM that result in generation of cataracts, development of severe osteopenia, and accelerated closure of dermal wounds. In this report we show that SPARC-null mice have greater deposits of s.c. fat and larger epididymal fat pads in comparison with wild-type mice. Similar to earlier studies of SPARC-null dermis, we observed a reduction in collagen I in SPARC-null fat pads in comparison with wild-type. Although elevated levels of serum leptin were observed in SPARC-null mice, their overall body weights were not significantly different from those of wild-type counterparts. The diameters of adipocytes from SPARC-null versus wild-type epididymal fat pads were 252 ± 61 and 161 ± 33 μm (means ± SD), respectively, and there was an increase in adipocyte number within SPARC-null fat pads in comparison with wild-type pads. Thus the absence of SPARC appears to result in an increase in the size of individual adipocytes as well as an increase in the number of adipocytes per fat pad. In fat pads isolated from wild-type mice, SPARC mRNA was associated with both the stromal/vascular and adipocyte fractions. We propose that SPARC limits the accumulation of adipose tissue in mice in part through its demonstrated effects on the regulation of cell shape and production of ECM.
The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives
Authors: Pavlović Davor , Neville David C. A. , Argaud Olivier , Blumberg Baruch , Dwek Raymond A. , Fischer Wolfgang B. , Zitzmann Nicole
Published: May 2003

We show that hepatitis C virus (HCV) p7 protein forms ion channels in black lipid membranes. HCV p7 ion channels are inhibited by long-alkyl-chain iminosugar derivatives, which have antiviral activity against the HCV surrogate bovine viral diarrhea virus. HCV p7 presents a potential target for antiviral therapy.
Public versus personal serotypes of a viral quasispecies
Authors: Hunziker Lukas , Ciurea Adrian , Recher Mike , Hengartner Hans , Zinkernagel Rolf M.
Published: May 2003

Noncytopathic RNA viruses persist in their natural hosts at various levels as highly mutating quasispecies. They exhibit only one known serotype. In most inbred DBA/2 mice infected with 2 × 104or 2 × 106plaque-forming units (pfu) of lymphocytic choriomeningitis virus (LCMV), the virus is transiently controlled below detectable levels measured with conventional assays (<1.7 pfu), but reemerges despite a common neutralizing Ab (nAb) response. Wild-type virus and cloned mutant viruses that had escaped polyclonal nAb responsesin vivoinduced nAb titers in new hosts that were usually cross-reactive; some sera were highly specific for certain mutants. The few mice that controlled LCMV infection for >170 days produced not only nAb against wild-type but also variably against many other mutants isolated from other mice with reemerging viremia. When DBA/2 mice were immunized and boosted with 200 pfu of a LCMV mutant, the neutralizing Ab response was limited to the immunizing “personal” clone. Thus, in contrast to classical serotype-defined cytopathic viruses (e.g., polio viruses) that induce strictly non-cross-reactive nAb titers, LCMV, a noncytopathic RNA virus, represents a dynamic multiplicity of personal serological submutants. Together, these mutants form a generally recognized “public” serotype. These findings may help to explain aspects of human infections and Ab responses against hepatitis B virus, hepatitis C virus, and HIV.
Caspase-mediated processing of the
Drosophila
NF-κB factor Relish
Authors: Stöven Svenja , Silverman Neal , Junell Anna , Hedengren-Olcott Marika , Erturk Deniz , Engström Ylva , Maniatis Tom , Hultmark Dan
Published: May 2003

The NF-κB-like transcription factor Relish plays a central role in the innate immune response of
Drosophila
. Unlike other NF-κB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IκB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IκB kinase complex and the caspase Dredd. Here we used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IκB-like domain, is the site of signal-dependent cleavage. We also show physical interaction between Relish and Dredd, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the
Drosophila
IκB kinase β. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation.
Functional Divergence of Two Zebrafish Midkine Growth Factors Following Fish-Specific Gene Duplication
Authors: Winkler Christoph , Schäfer Matthias , Duschl Jutta , Schartl Manfred , Volff Jean-Nicolas
Published: May 2003

In mammals, the uniquemidkine(mdk) gene encodes a secreted heparin-binding growth factor with neurotrophic activity. Here, we show the presence of two functionalmdkgenes namedmdkaandmdkbin zebrafish and rainbow trout. Both midkine proteins are clearly different from the related pleiotrophin, which was also identified in zebrafish and other fishes. Zebrafishmdkaandmdkbgenes map to linkage groups LG7 and LG25, respectively, both presenting synteny to human chromosome 11, in which the unique human orthologmdkis located. At least four other genes unique in mammals are also present as duplicates on LG7 and LG25. Phylogenetic and divergence analyses suggested that LG7/LG25 paralogs includingmdkaandmdkbhave been formed at approximately the same time, early during the evolution of the fish lineage. Hence, zebrafish and rainbow troutmdkaandmdkbmight have been generated by an ancient block duplication, and might be remnants of the proposed fish-specific whole-genome duplication. In contrast to the ubiquitous expression of their mammalian counterpart, zebrafishmdkaandmdkbare expressed in spatially restricted, mostly nonoverlapping patterns during embryonic development and strongly in distinct domains in the adult brain. Ectopic ubiquitous expression of bothmdkgenes in early zebrafish embryos caused completely distinct effects on neural crest and floorplate development. These data indicate thatmdkaandmdkbunderwent functional divergence after duplication. This provides an outstanding model to analyze the molecular mechanisms that lead to differences in pathways regulating the formation of homologous embryonic structures in different vertebrates.
Apoplasmic Barriers and Oxygen Transport Properties of Hypodermal Cell Walls in Roots from Four Amazonian Tree Species
Authors: De Simone Oliviero , Haase Karen , Müller Ewald , Junk Wolfgang J. , Hartmann Klaus , Schreiber Lukas , Schmidt Wolfgang
Published: May 2003

The formation of suberized and lignified barriers in the exodermis is suggested to be part of a suite of adaptations to flooded or waterlogged conditions, adjusting transport of solutes and gases in and out of roots. In this study, the composition of apoplasmic barriers in hypodermal cell walls and oxygen profiles in roots and the surrounding medium of four Amazon tree species that are subjected to long-term flooding at their habitat was analyzed. In hypodermal cell walls of the deciduous tree Crateva benthami, suberization is very weak and dominated by monoacids, 2-hydroxy acids, and ω-hydroxycarboxylic acids. This species does not show any morphological adaptations to flooding and overcomes the aquatic period in a dormant state. Hypodermal cells of Tabernaemontana juruana, a tree which is able to maintain its leaf system during the aquatic phase, are characterized by extensively suberized walls, incrusted mainly by the unsaturated C18ω-hydroxycarboxylic acid and the α,ω-dicarboxylic acid analogon, known as typical suberin markers. Two other evergreen species,Laetia corymbulosa and Salix martiana, contained 3- to 4-fold less aliphatic suberin in the exodermis, but more than 85% of the aromatic moiety of suberin are composed ofpara-hydroxybenzoic acid, suggesting a function of suberin in pathogen defense. No major differences in the lignin content among the species were observed. Determination of oxygen distribution in the roots and rhizosphere of the four species revealed that radial loss of oxygen can be effectively restricted by the formation of suberized barriers but not by lignification of exodermal cell walls.
Expression Profiling and Bioinformatic Analyses of a Novel Stress-Regulated Multispanning Transmembrane Protein Family from Cereals and Arabidopsis,
Authors: Breton Ghislain , Danyluk Jean , Charron Jean-Benoı̂t Frenette , Sarhan Fathey
Published: May 2003

Cold acclimation is a multigenic trait that allows hardy plants to develop efficient tolerance mechanisms needed for winter survival. To determine the genetic nature of these mechanisms, several cold-responsive genes of unknown function were identified from cold-acclimated wheat (Triticum aestivum). To identify the putative functions and structural features of these new genes, integrated genomic approaches of data mining, expression profiling, and bioinformatic predictions were used. The analyses revealed that one of these genes is a member of a small family that encodes two distinct groups of multispanning transmembrane proteins. The cold-regulated (COR)413-plasma membrane and COR413-thylakoid membrane groups are potentially targeted to the plasma membrane and thylakoid membrane, respectively. Further sequence analysis of the two groups from different plant species revealed the presence of a highly conserved phosphorylation site and a glycosylphosphatidylinositol-anchoring site at the C-terminal end. No homologous sequences were found in other organisms suggesting that this family is specific to the plant kingdom. Intraspecies and interspecies comparative gene expression profiling shows that the expression of this gene family is correlated with the development of freezing tolerance in cereals and Arabidopsis. In addition, several members of the family are regulated by water stress, light, and abscisic acid. Structure predictions and comparative genome analyses allow us to propose that the cor413 genes encode putative G-protein-coupled receptors.
Chimeric RNA/DNA Oligonucleotide-Based Site-Specific Modification of the Tobacco Acetolactate Syntase Gene
Authors: Kochevenko Andrej , Willmitzer Lothar
Published: May 2003

Single amino acid substitutions at either of two crucial positions in acetolactate synthase (ALS) result in a chlorsulfuron-insensitive form of this enzyme and, as a consequence, a herbicide-resistant phenotype. Here, we describe the successful in vivo targeting of endogenous tobacco (Nicotiana tabacum) ALS genes using chimeric RNA/DNA and all-DNA oligonucleotides at two different locations. Similar number of conversion events with two different chimeras indicates the absence of restricting influence of genomic target sequence on the gene repair in tobacco. Chlorsulfuron-resistant plants were regenerated from calli after mesophyll protoplast electroporation or leaf tissue particle bombardment with these specifically constructed chimeras. Sequence analysis and enzyme assays proved the resulting alterations to ALS at both DNA and protein levels. Furthermore, foliar application of chlorsulfuron confirmed the development of resistant phenotypes. Lines with proline-196-alanine, threonine, glutamine, or serine substitutions or with tryptophan-573-leucine substitutions were highly resistant at both cellular and whole plant levels, whereas lines with proline-196-leucine substitutions were less resistant. The stability of these modifications was demonstrated by the continuous growth of calli on chlorsulfuron-containing medium and by the transmission of herbicide resistance to progeny in a Mendelian manner. Ability of haploid state to promote chimera-mediated conversions is discussed.
A Role for Phosphatidylinositol 3-Phosphate in Abscisic Acid-Induced Reactive Oxygen Species Generation in Guard Cells
Authors: Park Ki-Youb , Jung Ji-Yul , Park Jumok , Hwang Jae-Ung , Kim Yong-Woo , Hwang Inhwan , Lee Youngsook
Published: May 2003

Guard cells generate reactive oxygen species (ROS) in response to abscisic acid (ABA), which leads to stomatal closing. The upstream steps of the ABA-induced ROS generation pathway remain largely unknown. In animal cells, ROS generation in neutrophils is activated by phosphatidylinositol 3-phosphate (PI3P). Stomatal guard cells contain PI3P and PI 3-kinase activity. In this study, we tested whether PI3P has a role in ROS generation in guard cells exposed to ABA. We found that PI 3-kinase inhibitors wortmannin or LY294002 inhibited ABA-induced ROS generation and stomatal closing. Endosome-binding domain (of human EEA1), which specifically binds to PI3P, also inhibited ABA-induced ROS generation and stomatal closing when overexpressed in guard cells. Hydrogen peroxide partially reversed the effects of wortmannin or LY294002 on ABA-induced stomatal closing. These results support a role for PI3P in ABA-induced ROS generation and stomatal closing movement.
Laser Capture Microdissection of Cells from Plant Tissues
Authors: Kerk Nancy M. , Ceserani Teresa , Tausta S. Lorraine , Sussex Ian M. , Nelson Timothy M.
Published: May 2003

Laser capture microdissection (LCM) is a technique by which individual cells can be harvested from tissue sections while they are viewed under the microscope, by tacking selected cells to an adhesive film with a laser beam. Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein spectra from individual cell types. We have optimized LCM for a variety of plant tissues and species, permitting the harvesting of cells from paraffin sections that maintain histological detail. We show that RNA can be extracted from LCM-harvested plant cells in amount and quality that are sufficient for the comparison of RNAs among individual cell types. The linear amplification of LCM-captured RNA should permit the expression profiling of plant cell types.
Generation and Analysis of an Artificial Gene Dosage Series in Tomato to Study the Mechanisms by Which the Cloned Quantitative Trait Locus fw2.2 Controls Fruit Size
Authors: Liu Jiping , Cong Bin , Tanksley Steven D.
Published: May 2003

It has been proposed that fw2.2 encodes a negative fruit-growth regulator that underlies natural fruit-size variation in tomato (Lycopersicon spp.) via heterochronic allelic variation of fw2.2 expression, rather than by variation in the structural protein itself. To further test the negative regulator and the transcriptional control hypotheses, a gene dosage series was constructed, which produced a wider range offw2.2 transcript accumulation than can be found in natural tomato populations. Fruit developmental analyses revealed thatfw2.2 transcript levels were highly correlated (negatively) with fruit mass, supporting the negative regulator and transcriptional regulation hypotheses. Further, the effect offw2.2 on fruit mass was mediated by repressing three- and two-dimensional cell division in placental and pericarp tissues, respectively. Finally, fw2.2 had little effect on fertility and seed size/number, indicating that fruit size effects offw2.2 are due largely to expression in the maternal tissues of developing fruit and not mediated through fertility or seed-setting-related processes.
A Novel Family in Medicago truncatula Consisting of More Than 300 Nodule-Specific Genes Coding for Small, Secreted Polypeptides with Conserved Cysteine Motifs,
Authors: Mergaert Peter , Nikovics Krisztina , Kelemen Zsolt , Maunoury Nicolas , Vaubert Danièle , Kondorosi Adam , Kondorosi Eva
Published: May 2003

Transcriptome analysis of Medicago truncatula nodules has led to the discovery of a gene family named NCR (nodule-specific cysteine rich) with more than 300 members. The encoded polypeptides were short (60–90 amino acids), carried a conserved signal peptide, and, except for a conserved cysteine motif, displayed otherwise extensive sequence divergence. Family members were found in pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), and Galega orientalis but not in other plants, including other legumes, suggesting that the family might be specific for galegoid legumes forming indeterminate nodules. Gene expression of all family members was restricted to nodules except for two, also expressed in mycorrhizal roots. NCR genes exhibited distinct temporal and spatial expression patterns in nodules and, thus, were coupled to different stages of development. The signal peptide targeted the polypeptides in the secretory pathway, as shown by green fluorescent protein fusions expressed in onion (Allium cepa) epidermal cells. Coregulation of certain NCR genes with genes coding for a potentially secreted calmodulin-like protein and for a signal peptide peptidase suggests a concerted action in nodule development. Potential functions of the NCR polypeptides in cell-to-cell signaling and creation of a defense system are discussed.
Role of the Reversible Xanthophyll Cycle in the Photosystem II Damage and Repair Cycle in Dunaliella salina
Authors: Jin EonSeon , Yokthongwattana Kittisak , Polle Juergen E.W. , Melis Anastasios
Published: May 2003

The Dunaliella salina photosynthetic apparatus organization and function was investigated in wild type (WT) and a mutant (zea1) lacking all β,β-epoxycarotenoids derived from zeaxanthin (Z). The zea1 mutant lacked antheraxanthin, violaxanthin, and neoxanthin from its thylakoid membranes but constitutively accumulated Z instead. It also lacked the so-called xanthophyll cycle, which, upon irradiance stress, reversibly converts violaxanthin to Z via a de-epoxidation reaction. Despite the pronounced difference observed in the composition of β,β-epoxycarotenoids between WT and zea1, no discernible difference could be observed between the two strains in terms of growth, photosynthesis, organization of the photosynthetic apparatus, photo-acclimation, sensitivity to photodamage, or recovery from photo-inhibition. WT and zea1 were probed for the above parameters over a broad range of growth irradiance and upon light shift experiments (low light to high light shift and vice versa). A constitutive accumulation of Z in the zea1 strain did not affect the acclimation of the photosynthetic apparatus to irradiance, as evidenced by indistinguishable irradiance-dependent adjustments in the chlorophyll antenna size and photosystem content of WT and zea1 strain. In addition, a constitutive accumulation of Z in the zea1 strain did not affect rates of photodamage or the recovery of the photosynthetic apparatus from photo-inhibition. However, Z in the WT accumulated in parallel with the accumulation of photodamaged PSII centers in the chloroplast thylakoids and decayed in tandem with a chloroplast recovery from photo-inhibition. These results suggest a role for Z in the protection of photodamaged and disassembled PSII reaction centers, apparently needed while PSII is in the process of degradation and replacement of the D1/32-kD reaction center protein.
Clustering of Microarray Data Reveals Transcript Patterns Associated with Somatic Embryogenesis in Soybean,
Authors: Thibaud-Nissen Françoise , Shealy Robin T. , Khanna Anupama , Vodkin Lila O.
Published: May 2003

Globular somatic embryos can be induced from immature cotyledons of soybean (Glycine max L. Merr. cv Jack) placed on high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, we have compared steady-state RNA from the adaxial side from which embryos develop and from the abaxial callus at five time points over the course of the 4 weeks necessary for the development of globular embryos. In a second set of experiments, we have profiled the expression of each clone in the adaxial side during the same period. A total of 495 genes differentially expressed in at least one of these experiments were grouped according to the similarity of their expression profiles using a nonhierarchical clustering algorithm. Our results indicate that the appearance of somatic embryos is preceded by dedifferentiation of the cotyledon during the first 2 weeks on auxin. Changes in mRNA abundance of genes characteristic of oxidative stress and genes indicative of cell division in the adaxial side of the cotyledons suggest that the arrangement of the new cells into organized structures might depend on a genetically controlled balance between cell proliferation and cell death. Our data also suggest that the formation of somatic globular embryos is accompanied by the transcription of storage proteins and the synthesis of gibberellic acid.
Evaluation of hysteresis loss using variable pinning parameter
Authors: Wlodarski Zdzislaw , Wlodarska Jadwiga
Published: April 2003

The Jiles‐Atherton model of magnetization gives a general description of magnetic induction and hysteresis loss against magnetic field and corresponding current. At wider regions of magnetization, however, the differences between calculated and empirical values become noticeable, especially in the case of hysteresis loss. It seems that these inaccuracies are caused to a large degree by the variation of the optimum values of model parameters in the successive regions of magnetization, and in particular by the change of the pinning coefficient. The application of a simple linear dependence of this parameter on the amplitude of magnetization has substantially improved the accuracy of the model.
Field‐circuit analysis of construction modifications of a torus‐type PMDC motor
Authors: Lukaniszyn Marian , Wrobe Rafal , Jagiela Mariusz
Published: April 2003

This paper presents the field‐circuit analysis of a disc‐type torus DC motor with permanent magnets. Calculations of the magnetic field are carried out using the finite element method (FEM) in the 3D space. The integral quantities like the ripple‐cogging torque, back electromotive force, flux linkage, self and mutual inductances of the winding are analyzed. The electromagnetic torque is comparatively determined from the Maxwell stress tensor and co‐energy methods. Based on the 3D magnetic field calculations, the lumped‐parameter model of the tested motor is constructed, taking into account an electronic power converter as well. For comparison, various permanent magnet widths and teeth thicknesses of the stator core are considered. The torque pulsations are shown in simulations to be effectively reduced by an appropriate selection of a permanent magnet width on the pole pitch. Additionally, the efficiency of the tested motor can be significantly improved by a proper selection of the teeth thickness. The simulation results are verified with experimental data obtained from the slotless version of the motor prototype.
Stability analysis of the doubly fed machine with emphasis on saturation effects
Authors: Masmoudi Ahmed
Published: April 2003

The doubly fed machine (DFM) is presently given an increasing attention in high power variable speed drives and in wind power generation systems, where it exhibits high performances. This has been gained thanks to the stator flux oriented control. Nevertheless, beyond the effect of heating, the robustness of such control strategy is affected by saturation especially the main magnetic one. Accounting for the effect of the magnetizing branch saturation in steady‐state stability analysis, considering the case of a voltage‐controlled DFM and the case of a current‐controlled one, represents the aim of the study. To this end, a numerical procedure based on a combination of the eigenvalue and the fixed point methods has been developed. It has been found that, in both cases, accounting for saturation yields a stabilization effect which is more or less significant depending on the rotor supply parameters.
Anorexia Nervosa and Refusal of Naso‐Gastric Treatment: A Response to Heather Draper
Authors: Giordano Simona
Published: May 2003

Imposing artificial feeding on people with anorexia nervosa may be unethical. This seems to be Heather Draper's suggestion in her article, ‘Anorexia Nervosa and Respecting a Refusal of Life‐Prolonging Therapy: A Limited Justification.’ Although this is an important point, I shall show that the arguments supporting this point are flawed. Draper should have made a brave claim: she should have claimed that people with anorexia nervosa, who competently decide not to be artificially fed, should be respected because everybody is entitled to exercise their autonomy, not only ‘in the middle’ of their life, but also at the end of it, or when their own life is at stake, because autonomy also extends to the most difficult moments of our life, and, ultimately, ‘stretches [.*T*.*T*.] far out into the distance’ at the end of it. I explain why Draper should have made the brave claim, and why she has not made it. I conclude that a defence of people's entitlement to competently refuse artificial feeding cannot rest upon the arguments developed by Draper. Whether or not we should respect competent refusal of artificial feeding depends on the normative strength that we are ready to ascribe to the principle of autonomy, to the moral relevance that we ascribe to the circumstances in which a person's autonomy is exercised, and, perhaps, eventually, on our sense of compassion.
Heteromeric HCN1–HCN4 Channels: A Comparison with Native Pacemaker Channels from the Rabbit Sinoatrial Node
Authors: Altomare Claudia , Terragni Benedetta , Brioschi Chiara , Milanesi Raffaella , Pagliuca Cinzia , Viscomi Carlo , Moroni Anna , Baruscotti Mirko , DiFrancesco Dario
Published: April 2003

‘Funny‐’ (f‐) channels of cardiac sino‐atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f‐channels are the hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f‐channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native If in SAN cells by co‐assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co‐transfected (HCN4 + HCN1) and concatenated (HCN4‐HCN1 (4–1) tandem or HCN1‐HCN4 (1–4) tandem) rabbit constructs and compared them with those of the native f‐current from rabbit SAN. 4–1 tandem, but not co‐transfected, currents had activation kinetics approaching those of If; however, the activation range of 4–1 tandem channels was more negative than that of the f‐channel and their cAMP sensitivity were poorer (although that of 1–4 tandem channels was normal). Co‐transfection of 4–1 tandem channels with minK‐related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f‐channels, but a ‘context’‐dependent mechanism is also likely to modulate the channel properties in native tissues.
The Case for an Internal Dynamics Model versus Equilibrium Point Control in Human Movement
Authors: Hinder Mark R. , Milner Theodore E.
Published: April 2003

The equilibrium point hypothesis (EPH) was conceived as a means whereby the central nervous system could control limb movements by a relatively simple shift in equilibrium position without the need to explicitly compensate for task dynamics. Many recent studies have questioned this view with results that suggest the formation of an internal dynamics model of the specific task. However, supporters of the EPH have argued that these results are not incompatible with the EPH and that there is no reason to abandon it. In this study, we have tested one of the fundamental predictions of the EPH, namely, equifinality. Subjects learned to perform goal‐directed wrist flexion movements while a motor provided assistance in proportion to the instantaneous velocity. It was found that the subjects stopped short of the target on the trials where the magnitude of the assistance was randomly decreased, compared to the preceding control trials (P= 0.003), i.e. equifinality was not achieved. This is contrary to the EPH, which predicts that final position should not be affected by external loads that depend purely on velocity. However, such effects are entirely consistent with predictions based on the formation of an internal dynamics model.