bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of a Cotesia rubecula bracovirus gene expressed in the lepidopteran Pieris rapae
Authors: Glatz Richard , Schmidt Otto , Asgari Sassan
Published: September 2004

Polydnaviruses are endogenous particles that are crucial for the survival of endoparasitoid wasps, providing active suppression of the immune function of the lepidopteran host in which wasp larvae develop. The Cotesia rubecula bracovirus (CrBV) is unique in that only four gene products are detected in larval host (Pieris rapae) tissues and expression of CrBV genes is transient, occurring between 4 and 12 h post-parasitization. Two of the four genes, CrV1 and CrV3, have been characterized. CrV1 is a secreted glycoprotein that has been implicated in depolymerization of the actin cytoskeleton of host haemocytes, leading to haemocyte inactivation; CrV3 is a multimeric C-type lectin that shares homology with insect immune lectins. Here, a third CrBV-specific gene is described, CrV2, which is expressed in larval P. rapae tissues. CrV2, which is transcribed in haemocytes and fat body cells, has an ORF of 963 bp that produces a glycoprotein of approximately 40 kDa. CrV2 is secreted into haemolymph and appears to be internalized by host haemocytes. CrV2 has a coiled-coil region predicted at its C-terminus, which may be involved in the formation of putative CrV2 trimers that are detected in haemolymph of parasitized host larvae.
Dissection of measles virus V protein in relation to its ability to block alpha/beta interferon signal transduction
Authors: Ohno Shinji , Ono Nobuyuki , Takeda Makoto , Takeuchi Kaoru , Yanagi Yusuke
Published: September 2004

Interferon (IFN)-αand -βare the main cytokines for innate immune responses against viral infections. To replicate efficiently in the hosts, viruses have evolved various countermeasures to the IFN response. The V protein of measles virus (MV) has been shown to block IFN-α/βsignalling. Here, the wild-type IC-B strain of MV was shown to grow comparably in the presence and absence of IFN-α, whereas replication of the Edmonston tag strain recovered from cloned DNA was strongly suppressed in its presence. The V protein of the IC-B strain, but not the Edmonston tag strain, blocked IFN-αsignalling. The V protein of the Edmonston strain from the ATCC also inhibited IFN-αsignalling. There were three amino acid differences between the V proteins of the Edmonston ATCC and tag strains, and substitutions of both residues at positions 110 and 272 were required for the Edmonston ATCC V protein to lose IFN-antagonist activity. The P protein of the IC-B strain, which shares the N-terminal 231 aa residues with the V protein, also inhibited IFN-αsignalling. Indeed, fragments comprising only those 231 residues of the IC-B and Edmonston ATCC V proteins, but not the Edmonston tag V protein, were able to block IFN-αsignalling. However, the N-terminal region of the Edmonston tag V protein, when attached to the C-terminal region of the Edmonston ATCC V protein, inhibited IFN-αsignalling. Taken together, our results indicate that both the N- and C-terminal regions contribute to the IFN-antagonist activity of the MV V protein.
A β‐(1→4)‐xylosyltransferase involved in the synthesis of arabinoxylans in developing barley endosperms
Authors: Urahara Takeshi , Tsuchiya Kouji , Kotake Toshihisa , Tohno‐oka Takuji , Komae Kozo , Kawada Naoyuki , Tsumuraya Yoichi
Published: September 2004

A β‐(1→4)‐xylosyltransferase (XylTase; EC 2.4.2.24) participating in the synthesis of arabinoxylans was investigated using microsomal membranes prepared from developing barley (Hordeum vulgare L.) endosperms. The microsomal fraction transferred Xyl from uridine 5′‐diphosphoxylose (UDP‐Xyl) into exogenous β‐(1→4)‐xylooligosaccharides derivatized at their reducing ends with 2‐aminopyridine. HPLC analysis showed chain elongation of pyridylaminated β‐(1→4)‐xylotriose (Xyl3‐PA) by repeated attachment of one to five single xylosyl residues depending on the reaction time, leading to the formation of Xyl4−8‐PA. Methylation analysis and enzymatic digestions with β‐xylosidase (EC 3.2.1.37) and endo‐β‐(1→4)‐xylanase (EC 3.2.1.8) confirmed that the transfer of xylosyl residues into the newly synthesized products occurred through β‐(1→4)‐linkages. The activity of the XylTase was maximal at pH 6.8 and 20°C and most enhanced in the presence of 0.5% Triton X‐100 and 5 mM MnCl2. The apparent Michaelis constant and maximal velocity of the enzyme for Xyl3‐PA were 2.1 mM and 25 400 pmol min−1 mg protein−1, respectively. The enzyme also transferred [14C]Xyl from UDP‐[14C]Xyl into higher β‐(1→4)‐xylooligosaccharides and birchwood xylans through β‐(1→4)‐linkages. The enzyme activity varied according to the stage of development (7–35 days after flowering) of the endosperms. Maximal activity occurred at 13–16 days; no activity was detectable in mature seeds. A comparison of endosperms from 10 different cultivars of barley harvested 11–22 days after flowering showed no correlation between enzyme activity and the amount of Xyl in the cell walls.
Phenylalanine ammonia‐lyase‐induced freezing tolerance in jack pine (Pinus banksiana) seedlings treated with low, ambient levels of ultraviolet‐B radiation
Authors: Teklemariam Thomas A. , Blake Terence J.
Published: September 2004

Phenylalanine ammonia‐lyase (PAL) induction in UVB‐exposed plants leads to an increased synthesis of UV‐absorbing phenols. As phenols, including anthocycanins, are linked to many protective mechanisms in plants, we tested the hypothesis that UVB‐induced phenol accumulation, mediated by PAL, may confer freezing tolerance in jack pine (Pinus banksiana Lamb) seedlings. The hypothesis was tested by applying UVB in the presence and absence of the PAL‐inhibitor, 2‐aminoindan‐2‐phosphonic acid (AIP). Jack pine seedlings were grown for 3 weeks with and without 10 µM aqueous AIP. Each treatment was then divided into two groups. One group received near‐ambient UVB (5.5 kJ m−2 day−1of biologically effective radiation) for up to 30 h. A second, control group of seedlings received no UVB. Anthocyanin concentration declined by > 99% in PAL‐inhibited seedlings and other methanol‐extractable UV‐absorbing phenols declined by > 48%, relative to the controls. A 20‐h exposure to UVB increased seedling freezing (−15°C) tolerance in the absence of the PAL‐inhibitor, as shown by a 30% reduction in membrane injury, determined by electrolyte leakage measurements. In PAL‐inhibited seedlings, by contrast, the same UVB pre‐treatment increased freezing injury by 48%. A longer (30 h) UVB exposure was damaging to both AIP‐treated and untreated seedlings. Root feeding with 10 µM AIP during a 3‐week exposure of older (6‐month‐old) seedlings similarly reduced phenol accumulation in UVB‐exposed seedlings. The decline in phenol production in PAL‐inhibited seedlings correlated with increased freezing injury. These results suggest a role for ambient UVB in seedling frost hardiness, mediated by a PAL‐induced production of phenolic compounds.
Activity of membrane‐associated sucrose synthase is regulated by its phosphorylation status in cultured cells of sycamore (Acer pseudoplatanus)
Authors: Pozueta‐Romero Javier , Pozueta‐Romero Diego , Gonzalez Pedro , Etxeberria Ed
Published: September 2004

Changes in sucrose synthase (SuSy) activity, protein level and degree of phosphorylation were investigated in plasmalemma and tonoplast of sycamore cells cultured either in the presence of sucrose or after 24 h of starvation. SuSy activity was shown to be higher in the plasmalemma than in the tonoplast of cells cultured in the presence of sucrose. In clear contrast, SuSy was shown to be more active in the tonoplast than in the plasmalemma of starved cells. Western blot analyses on both membrane types did not show noticeable differences in SuSy protein levels under the two different regimes. However, phosphorylation state at the serine moieties of the enzyme was shown to be different in the presence or in the absence of sucrose. Plasmalemma‐associated SuSy is not phosphorylated in the presence of sucrose, whereas tonoplast‐associated SuSy is phosphorylated under similar conditions. Starvation brought about a reverse in phosphorylation state of membrane‐bound SuSy. Whereas plasmalemma‐associated SuSy became phosphorylated, tonoplast‐associated SuSy was completely de‐phosphorylated. Together, the data demonstrate that SuSy is simultaneously present in various cell membranes and also demonstrate a lack of direct relationship between membrane type location, and degree of phosphorylation, but substantiate the relevance of phosphorylation to enzymatic activity.
The role of BMP signalling in the control of ID3 expression in the hair follicle
Authors: O'Shaughnessy Ryan F. L. , Christiano Angela M. , Jahoda Colin A. B.
Published: September 2004

Abstract:  Both the production of the hair shaft in anagen and the initiation of a new hair cycle at telogen are the result of reciprocal interactions between the dermal papilla and the overlying epithelial cells. Secreted factors, such as those of the bone morphogenetic protein (BMP) family, play a crucial role in moderating these interactions. Analysis of hair follicles in different stages of the hair cycle showed that BMP signalling was only active during anagen and again during telogen. During catagen, no BMP signalling occurred in the dermal papilla. ID3, a gene expressed in the dermal papilla of both vibrissa and pelage follicles, is a BMP target, and as such, we found that ID3 was expressed from the earliest stages of morphogenesis. During the hair cycle, ID3 was only expressed in the dermal papilla at middle anagen and telogen. To test the significance of ID3 expression in the dermal papilla, we cultured dermal papilla cells and found that ID3 expression fell significantly after a single passage. ID3 expression was returned to in vivo levels in low‐ and high‐passage cells by culturing to high confluence or by the addition of BMP4. These studies reinforce the requirement for active BMP signalling and cell–cell contacts in the dermal papilla during specific stages in the hair cycle.
Limitations of human occipital scalp hair follicle organ culture for studying the effects of minoxidil as a hair growth enhancer
Authors: Magerl Markus , Paus Ralf , Farjo Nilofer , Müller‐Röver Sven , Peters Eva M. J. , Foitzik Kerstin , Tobin Desmond J.
Published: September 2004

Abstract:  Minoxidil induces new hair growth in approximately one‐third of patients with androgenetic alopecia after 1 year of treatment. With several conflicting reports in the literature based on small‐scale studies, the current study aimed to clarify whether organ culture of human scalp anagen VI hair follicles is a suitable in vitro test system for reproducing, and experimentally dissecting, the recognized in vivo hair‐growth‐promoting capacity of minoxidil. Hair shaft elongation was studied in terminal anagen VI hair follicles microdissected from the occipital scalp of 36 healthy adults. A total of 2300 hair follicles, approximately 65 per individual, were tested using modifications of a basic organ culture protocol. It is shown here that minoxidil does not significantly increase hair shaft elongation or the duration of anagen VI in ex vivo culture despite several enhancements on the conventional methodology. This disparity to what is seen clinically in minoxidil responders may be explained by the following: (i) use of occipital (rather than frontotemporal or vertex) hair follicles; (ii) use of, already maximally growing, anagen VI hair follicles; (iii) a predominance of hair follicles from minoxidil unresponsive‐donors; (iv) use of minoxidil rather than its sulfate metabolite; and/or (v) use of a suboptimal minoxidil dosage. This disparity questions the usefulness of standard human hair follicle organ culture in minoxidil research. Unexpectedly, minoxidil even inhibited hair shaft elongation in the absence of insulin, which may indicate that the actual hair‐growth‐modulatory effects of minoxidil depend on the concomitant local presence/absence of other growth modulators.
Mutation spectra of epidermal p53 clones adjacent to basal cell carcinoma and squamous cell carcinoma
Authors: Bäckvall Helena , Strömberg Sara , Gustafsson Anna , Asplund Anna , Sivertsson Åsa , Lundeberg Joakim , Ponten Fredrik
Published: September 2004

Abstract:  Foci of normal keratinocytes overexpressing p53 protein are frequently found in normal human skin. Such epidermal p53 clones are common in chronically sun‐exposed skin and have been suggested to play a role in skin cancer development. In the present study, we have analyzed the prevalence of p53 mutations in epidermal p53 clones from normal skin surrounding basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Using laser‐assisted microdissection, 37 epidermal p53 clones adjacent to BCC (21) and SCC (16) were collected. Genetic analysis was performed using a multiplex/nested polymerase chain reaction followed by direct DNA sequencing of p53 exons 2–11. In total, 21 of 37 analyzed p53 clones consisted of p53‐mutated keratinocytes. The identified mutations were located in p53 exons 4–8, corresponding to the sequence‐specific DNA‐binding domain. All mutations were missense, and 78% displayed a typical ultraviolet signature. The frequency of p53 mutations was similar in skin adjacent to BCC compared to SCC. The presented data confirm and extend previous knowledge on the genetic background of epidermal p53 clones. The mutation spectra found in epidermal p53 clones resemble that of non‐melanoma skin cancer. Approximately, 40% of the epidermal p53 clones lacked an underlying p53 mutation, suggesting that other genetic events in genes up‐ or downstream of the p53 gene can generate foci of normal keratinocytes overexpressing p53 protein.
DNA microarray technology and its applications in dermatology
Authors: Kunz M. , Ibrahim S. M. , Koczan D. , Scheid S. , Thiesen H. J. , Gross G.
Published: September 2004

Abstract: The use of DNA microarray technology in biomedical research has dramatically increased during the past years. In the present report, we provide an overview on the basic DNA microarray technology and biostatistical methods for gene expression analysis. A focus is then put on its applications in dermatological research. In recent years, a series of gene expression studies have been performed for various dermatological diseases, such as malignant melanoma, psoriasis and lupus erythematosus. These analyses have identified interesting target genes as well as putative disease susceptibility loci. However, further functional studies will be needed for a more complete understanding of the pathogenesis of these diseases. This may be performed by means of the recently developed RNA interference technology. Besides its role in large‐scale gene expression studies, DNA microarray technology has proved to be a valuable tool for genomic screens of genetic alterations, e.g. single nucleotide polymorphisms. These play a role in tumour development and progression, and also function as genetic markers for disease susceptibility. Taken together, DNA microarray technology opens enormous perspectives for dermatologists. It may help us understand the complex pathogenesis of a wide variety of dermatologic diseases and identify their genetic background.
Neural Evidence That Vivid Imagining Can Lead to False Remembering
Authors: Gonsalves Brian , Reber Paul J. , Gitelman Darren R. , Parrish Todd B. , Mesulam M.-Marsel , Paller Ken A.
Published: September 2004

The imperfect nature of memory is highlighted by the regularity with which people fail to remember, or worse, remember something that never happened. We investigated the formation of a particular type of erroneous memory by monitoring brain activity using functional magnetic resonance imaging during the presentation of words and photos. Participants generated a visual image of a common object in response to each word. Subsequently, they sometimes claimed to have seen photos of specific objects they had imagined but not actually seen. In precuneus and inferior parietal regions of the cerebral cortex, activations in response to words were greater when participants subsequently claimed to have seen the corresponding object than when a false memory for that object was not subsequently produced. These findings indicate that brain activity reflecting the engagement of visual imagery can lead to falsely remembering something that was only imagined.
Intact Conceptual Priming in the Absence of Declarative Memory
Authors: Levy D.A. , Stark C.E.L. , Squire L.R.
Published: September 2004

Priming is an unconscious (nondeclarative) form of memory whereby identification or production of an item is improved by an earlier encounter. It has been proposed that declarative memory and priming might be related---for example, that conceptual priming results in more fluent processing, thereby providing a basis for familiarity judgments. In two experiments, we assessed conceptual priming and recognition memory across a 5-min interval in 5 memory-impaired patients. All patients exhibited fully intact priming in tests of both free association (study tent; at test, provide an association to canvas) and category verification (study lemon; at test, decide: Is lemon a type of fruit?). Yet the 2 most severely amnesic patients performed at chance on matched tests of recognition memory. These findings count against the notion that conceptual priming provides feelings of familiarity that can support accurate recognition judgments. We suggest that priming is inaccessible to conscious awareness and does not influence declarative memory.
Trust in Testimony: Children's Use of True and False Statements
Authors: Koenig Melissa A. , Clément Fabrice , Harris Paul L.
Published: September 2004

The extent to which young children monitor and use the truth of assertions to gauge the reliability of subsequent testimony was examined. Three- and 4-year-old children were presented with two informants, an accurate labeler and an inaccurate labeler. They were then invited to learn names for novel objects from these informants. The children correctly monitored and identified the informants on the basis of the truth of their prior labeling. Furthermore, children who explicitly identified the unreliable or reliable informant across two tasks went on to demonstrate selective trust in the novel information provided by the previously reliable informant. Children who did not consistently identify the unreliable or reliable informant proved indiscriminate.
Toddlers' Responsive Imitation Predicts Preschool-Age Conscience
Authors: Forman David R. , Aksan Nazan , Kochanska Grazyna
Published: September 2004

Imitation has long been considered a mechanism for the development of conscience. Despite the central role of imitation in theories of moral internalization, the prediction from imitation to moral behavior has not been tested using an individual differences approach. In a longitudinal design, we examined whether individual differences in toddlers' responsive imitation predict preschool-age conscience. Mothers modeled actions for their children to imitate, and both matching behavior and motivation were observed at 14 and 22 months of age. We also measured preschool conscience by observing children's internalized conduct and guilt at ages 33 and 45 months. Imitation measures consistently predicted conscience variables. These relations were strengthened by aggregation across measures and times of assessment. Motivation and matching behavior each contributed independent variance in predicting preschool-age conscience. Results are consistent with the claim that responsive imitation reflects a general receptive stance to parents' guidance, and with both neopsychoanalytic and social cognitive views of imitation's importance in early moral development.
A key role for ALD1 in activation of local and systemic defenses in Arabidopsis
Authors: Song Jong Tae , Lu Hua , McDowell John M. , Greenberg Jean T.
Published: September 2004

The Arabidopsis thaliana agd2‐like defense response protein1 (ald1) mutant was previously found to be hypersusceptible to the virulent bacterial pathogen Pseudomonas syringae and had reduced accumulation of the defense signal salicylic acid (SA). ALD1 was shown to possess aminotransferase activity in vitro, suggesting it generates an amino acid‐derived defense signal. We now find ALD1 to be a key defense component that acts in multiple contexts and partially requires the PHYTOALEXIN DEFICIENT4 (PAD4) defense regulatory gene for its expression in response to infection. ald1 plants have increased susceptibility to avirulent P. syringae strains, are unable to activate systemic acquired resistance and are compromised for resistance to the oomycete pathogen Peronospora parasitica in mutants with constitutively active defenses. ALD1 and PAD4 can act additively to control SA, PATHOGENESIS RELATED GENE1 (PR1) transcript and camalexin (an antimicrobial metabolite) accumulation as well as disease resistance. Finally, ALD1 and PAD4 can mutually affect each other's expression in a constitutive defense mutant, suggesting that these two genes can act in a signal amplification loop.
Expression of RPS4 in tobacco induces an AvrRps4‐independent HR that requires EDS1, SGT1 and HSP90
Authors: Zhang Yan , Dorey Stephan , Swiderski Michal , Jones Jonathan D. G.
Published: September 2004

The ArabidopsisRPS4 gene belongs to the Toll/interleukin‐1 receptor/nucleotide‐binding site/leucine‐rich repeat (TIR‐NB‐LRR) class of plant resistance (R) genes. It confers resistance to Pseudomonas syringae carrying the avirulence gene avrRps4. Transient expression of genomic RPS4 driven by the 35S promoter in tobacco leaves induces an AvrRps4‐independent hypersensitive response (HR). The same phenotype is seen after expression of a full‐length RPS4 cDNA. This indicates that alternative splicing of RPS4 is not involved in this HR. The extent of HR is correlated with RPS4 protein levels. Deletion analyses of RPS4 domains show the TIR domain is required for the HR phenotype. Mutations in the P‐loop motif of the NB domain abolish the HR. Using virus‐induced gene silencing, we found that the cell death resulting from RPS4 expression is dependent on the three plant signalling components EDS1, SGT1 and HSP90. All these data suggest that heterologous expression of an R gene can result in activation of cell death even in the absence of its cognate avirulence product, and provides a system for studying the RPS4 domains required for HR.
The composition of newly synthesized proteins in the endoplasmic reticulum determines the transport pathways of soybean seed storage proteins
Authors: Mori Takashi , Maruyama Nobuyuki , Nishizawa Keito , Higasa Takahiko , Yagasaki Kazuhiro , Ishimoto Masao , Utsumi Shigeru
Published: September 2004

Glycinin (11S) and β‐conglycinin (7S) are major storage proteins in soybean (Glycine max L.) seeds and accumulate in the protein storage vacuole (PSV). These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the PSV by vesicles. Electron microscopic analysis of developing soybean cotyledons of the wild type and mutants with storage protein composition different from that of the wild type showed that there are two transport pathways: one is via the Golgi and the other bypasses it. Golgi‐derived vesicles were observed in all lines used in this study and formed smooth dense bodies with a diameter of 0.5 to several micrometers. ER‐derived protein bodies (PBs) with a diameter of 0.3–0.5 μm were observed at high frequency in the mutants containing higher amount of 11S group I subunit than the wild type, whereas they were hardly observed in the mutants lacking 11S group I subunit. These indicate that pro11S group I may affect the formation of PBs. Thus, the composition of newly synthesized proteins in the ER is important in the selection of the transport pathways.
Novel cell wall architecture of isoxaben‐habituated Arabidopsis suspension‐cultured cells: global transcript profiling and cellular analysis
Authors: Manfield Iain W. , Orfila Caroline , McCartney Lesley , Harholt Jesper , Bernal Adriana J. , Scheller Henrik Vibe , Gilmartin Philip M. , Mikkelsen Jørn D. , Paul Knox J. , Willats William G. T.
Published: September 2004

The herbicide isoxaben is a highly specific and potent inhibitor of cellulose synthesis in plants. Nevertheless, suspension‐cultured cells can be habituated to grow in high concentrations of isoxaben, and apparently compensate for the disruption of cellulose synthesis by the modulation of other cell wall components. We have habituated Arabidopsis cells to isoxaben and characterized the cellular and genetic consequences. Near whole‐genome transcript profiling implicated novel genes in cell wall assembly and extended our understanding of the activity of known cell wall‐related genes including glycosyltransferases involved in cellulose and pectin biosynthesis. Habituation does not appear to be mediated by stress response processes, nor by functional redundancy within the cellulose synthase (AtCesA) family. Uniquely, amongst the cellulose synthase superfamily, AtCslD5 was highly upregulated and may play a role in the biosynthesis of the novel walls of habituated cells. In silico analysis of differentially expressed genes with unknown functions identified a putative glycosyltransferase and collagen‐like putative cell wall protein.
Large‐scale analysis of the barley transcriptome based on expressed sequence tags
Authors: Zhang Hangning , Sreenivasulu Nese , Weschke Winfriede , Stein Nils , Rudd Stephen , Radchuk Volodymyr , Potokina Elena , Scholz Uwe , Schweizer Patrick , Zierold Uwe , Langridge Peter , Varshney Rajeev K. , Wobus Ulrich , Graner Andreas
Published: September 2004

To provide resources for barley genomics, 110 981 expressed sequence tags (ESTs) were generated from 22 cDNA libraries representing tissues at various developmental stages. This EST collection corresponds to approximately one‐third of the 380 000 publicly available barley ESTs. Clustering and assembly resulted in 14 151 tentative consensi (TCs) and 11 073 singletons, altogether representing 25 224 putatively unique sequences. Of these, 17.5% showed no significant similarity to other barley ESTs present in dbEST. More than 41% of all barley genes are supposed to belong to multigene families and approximately 4% of the barley genes undergo alternative splicing. Based on the functional annotation of the set of unique sequences, the functional category ‘Energy’ was further analysed to reveal tissue‐ and stage‐specific differences in gene expression. Hierarchical clustering of 362 differentially expressed TCs resulted in the identification of seven major clusters. The clusters reflect biochemical pathways predominantly activated in specific tissues and at various developmental stages. During seed germination glycolysis could be identified as the most predominant biochemical pathway. Germination‐specific glycolysis is characterized by the coordinated expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase, whose antagonistic actions possibly regulate the flux of amino acids into protein biosynthesis and gluconeogenesis respectively. The expression of defence‐related and antioxidant genes during germination might be controlled by the ethylene‐signalling pathway as concluded from the coordinated expression of those genes and the transcription factors (TF) EIN3 and EREBPG. Moreover, because of their predominant expression in germinating seeds, TF of the AP2 and MYB type are presumably major regulators of germination.
Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis
Authors: Samanani Nailish , Liscombe David K. , Facchini Peter J.
Published: September 2004

(S)‐Norcoclaurine synthase (NCS) (EC 4.2.1.78) catalyzes the condensation of 3,4‐dihydroxyphenylethylamine (dopamine) and 4‐hydroxyphenylacetaldehyde (4‐HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids such as morphine, sanguinarine, and berberine, in plants. A molecular clone encoding NCS was isolated from a meadow rue (Thalictrum flavum ssp. glaucum) cell suspension culture cDNA library. Heterologous expression of the NCS cDNA, truncated to remove a putative signal peptide, produced a recombinant protein with NCS activity. Recombinant NCS showed sigmoidal saturation kinetics for dopamine (Hill coefficient=1.98), hyperbolic saturation kinetics for 4‐HPAA (Km of 700 μm), and pH and temperature optima of 7.0 and 40°C, respectively, all similar to the purified, plant‐derived enzyme. NCS exhibits 28–38% identity, and putative structural homology, with the Bet v 1 allergen and pathogenesis‐related (PR)10 protein families. NCS also displays 35% identity with the enzyme (HYP1) responsible for hypericin biosynthesis in St John's wort (Hypericum perforatum). The novel catalytic functions of NCS and HYP1 define a new class of plant secondary metabolic enzymes within the Bet v 1 and PR10 protein families. Weaker homology was also detected between NCS and proteins identified in the latex of Papaver somniferum (opium poppy), and in Arabidopsis thaliana. A family of three to five NCS genes is abundantly expressed in the rhizome, followed by petioles and roots of T. flavum. NCS transcripts were localized to the immature endodermis and pericycle in roots, and the protoderm of leaf primordia in rhizomes; thus, the sites of NCS gene expression and berberine accumulation are temporally and spatially separated in roots and rhizomes respectively.
Organic acid exudation induced by phosphorus deficiency and/or aluminium toxicity in two contrasting soybean genotypes
Authors: Dong Dengfeng , Peng Xinxiang , Yan Xiaolong
Published: September 2004

Experiments in nutrient solution were conducted to investigate the exudation of organic acids (OAs) induced by phosphorus deficiency (–P) and/or aluminium toxicity (+Al) in two contrasting soybean genotypes as related to internal OA concentration and related enzyme activities. Baxi 10 (BX10), a known P‐efficient soybean (Glycine max[L] Merr.) genotype, was shown to be more resistant to +Al than a P‐inefficient genotype Bendi 2 (BD2), indicating the potential of selecting soybean cultivars with dual resistance to –P and +Al. The two contrasting genotypes were further characterized for root exudation and formation of oxalate, malate and citrate and their related enzyme activities in response to –P, +Al or both combined. –P significantly induced malate and oxalate exudation from both soybean genotypes, although the P‐efficient BX10 tended to excrete much more oxalate than the P‐inefficient BD2. The +Al treatment triggered citrate efflux from both genotypes, with BX10 having a much greater efflux rate than BD2. Interestingly, –P did not appear to induce citrate exudation, whereas +Al had no obvious effect on malate or oxalate exudation from the two genotypes. The exudation of OAs was generally diminished under the coupled stress of –P and +Al in comparison with either single stress, implying a possible antagonistic effect of the two stresses on OA exudation. Root malate content was negatively correlated with its exudation in BX10 but positively in BD2. A similar tendency was observed for oxalate content and exudation only with less magnitude. Determination of six related enzymes, phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate phosphatase (PEPP), malate enzyme (ME), isocitrate dehydrogenase (ICDH), malate dehydrogenase (MDH), and pyruvate kinase (PK), in the root tips showed that their activities were not significantly altered during the early stage of treatments (2 and 4 days) whereas at 14 days after stress imposition, the activities of PEPC, PEPP, ME and ICDH were generally enhanced for both genotypes. However, the activity of these enzymes did not appear to be correlated with OA exudation or formation. This study clearly demonstrates that OA exudation is differentially induced by –P and +Al in soybean plants, with specific induction of oxalate and malate by –P and citrate by +Al. The lack of a close relationship between OA exudation and internal concentration or enzyme activities may suggest that the regulation of OA formation and exudation by –P and/or +Al could be imposed at different stages.
Isolation and characterization of a BURP domain‐containing gene BnBDC1 from Brassica napus involved in abiotic and biotic stress
Authors: Shunwu Yu , Zhang Lida , Zuo Kaijing , Li Zhugang , Tang Kexuan
Published: September 2004

A new gene encoding BURP domain‐containing protein was isolated from oilseed rape (Brassica napus) following drought treatment. The cloned gene, designated as BnBDC1 (GenBank accession number AY293830), contains four major domains: an N‐terminal hydrophobic domain, a short conserved segment, a characteristic repeated region and a C‐terminal BURP domain. BnBDC1 is highly homologous to the dehydration‐responsive RD22 gene from Arabidopsis thaliana, and belongs to the same gene family as BNM2 from B. napus. Northern blot and semi‐quantitative reverse transcription polymerase chain reaction (RT‐PCR) analyses show that BnBDC1 is constitutively expressed in rape shoots, but is not detectable in the roots, indicating that the expression of BnBDC1 is tissue specific. The expression level of BnBDC1 transcript is upregulated by mannitol, NaCl and abscisic acid (ABA), downregulated by UV irradiation and salicylic acid, and unresponsive to H2O2 and cold treatment. The accumulation of BnBDC1 transcripts generally increases within 2 h after initiation of the promoting stimulus. These results suggest that BnBDC1 has similar functions to those of RD22 of Arabidopsis, and may play an important role in response to osmotic stress and/or plant pathogen infection in B. napus.
Different arabinogalactan proteins are present in carrot (Daucus carota) cell culture medium and in seeds
Authors: Immerzeel Peter , Schols Henk A. , Voragen Alphons G. J. , De Vries Sacco C.
Published: September 2004

Arabinogalactan proteins (AGPs) were isolated by Yariv phenylglycoside precipitation from the medium of carrot (Daucus carota L.) cell cultures and from carrot seeds. The isolates showed a different composition of AGPs. The medium AGPs contained an arabinose poor AGP fraction that had relatively high levels of glucuronic acid and rhamnose. In contrast the seed AGPs only contained arabinose and galactose‐rich AGP fractions that had low levels of glucuronic acid. Linkage analysis on all fractions showed that most of the arabinose residues were terminally linked and that almost all galactose was present in the 1,3‐, 1,6‐ and 1,3,6‐ form. The strongly branched type II arabinogalactans are characteristic of the carbohydrate part of AGPs. AGP characteristic amino acid residues as Hyp, Pro, Glx, Ser, Gly, Asx, Ala, Leu and Thr were detected in three different fractions.
Changes in chlorophyll and carotenoid contents in radish (Raphanus sativus) cotyledons show different time courses during senescence
Authors: Suzuki Yasuyo , Shioi Yuzo
Published: September 2004

Changes of chlorophylls and carotenoids from green to yellow cotyledons of the radish (Raphanus sativus) were simultaneously and systematically analysed by high‐performance liquid chromatography (HPLC) with photodiode array detection. Twenty‐one components, seven chlorophylls and 14 carotenoids, were detectable. Seven chlorophylls and five carotenoids were identified from the results of HPLC analyses. Most chlorophyll species degraded during senescence, whereas carotenoids showed different behaviour in their metabolism depending on pigment species. For instance, during senescence, the contents of lutein and violaxanthin changed only slightly, β‐carotene in 5‐day‐senescent cotyledons became 2.7 times higher than non‐senescent leaves. Carotenoids of radish cotyledons were classified into three groups by their changes in concentration during senescence (increased, degraded and constant) and their roles discussed.
Effects of corn processing on the glycaemic and insulinaemic responses in horses
Authors: Vervuert I. , Coenen M. , Bothe C.
Published: September 2004

This study was conducted to evaluate the effects of different corn processing techniques on the glycaemic and insulinaemic responses in horses. It was hypothesized that the changes in pre‐caecal starch digestibility caused by various types of corn processing would alter the post‐prandial glycaemic and/or insulinaemic response. Six horses were fed in random order: untreated, finely ground, steamed, micronized, steam‐flaked and popped corn. The total corn intake was adjusted to 630 g starch/horse/day (1.2–1.5 g starch/kg BW/day). During a stabilization period of 10 days, horses also received 6 kg grass hay/horse/day. At blood collection day horses were fed their test diet (exclusively corn), and blood samples were taken at defined times. Corn feeding resulted in a significant increase in mean plasma glucose and insulin concentration, but glucose and insulin peaks as well as areas under the curve (AUC) were not clearly influenced by corn processing. The glycaemic index (in which each test diet's plasma glucose AUC was expressed relative to untreated corn) varied between 91.4 ± 9.4% (steamed corn) and 108.4 ± 11.8% (popped corn, treatment n.s.), the insulinaemic index (in which each test diet's plasma insulin AUC was expressed relative to untreated corn) ranged between 98.2 ± 12.6% (steamed corn) and 121.0 ± 29.9% (micronized corn, treatment n.s.). However, the well‐established improvement in pre‐caecal starch digestibility was not reflected by differences in the glucose or insulin responses.
Effect of size on electrolyte apparent absorption rates and fermentative activity in dogs
Authors: Weber M. P. , Hernot D. , Nguyen P. G. , Biourge V. C. , Dumon H. J.
Published: September 2004

The objectives of the study were to assess overall electrolyte absorption capacity as well as fermentative activity in dogs varying in body size in order to verify whether these parameters can explain the poor faecal quality in large dogs. Four breeds of dogs were used: six Miniature Poodles (MP), six Standard Schnauzers (SS), six Giant Schnauzers (GS) and six Great Danes (GD). Net absorption rates of electrolytes were assessed by calculating the apparent digestibility of sodium and potassium and measuring their content in stool. Fermentative activity was calculated by measuring faecal concentrations of lactic acid and short‐chain fatty acids. The results showed higher faecal concentrations of fermentation products and electrolytes as well as poorer digestibility of electrolytes in Giant Schnauzers and Great Danes. Strong positive correlations were found between these different parameters and stool quality (scores and moisture). Lower overall electrolyte absorption and higher fermentative activity could thus be factors capable of explaining poorer faecal quality in these two large dog breeds.
Modulation of premotor mirror neuron activity during observation of unpredictable grasping movements
Authors: Gangitano Massimo , Mottaghy Felix M. , Pascual‐Leone Alvaro
Published: September 2004

Using transcranial magnetic stimulation, we explored the properties of premotor mirror neurons during the passive observation of a reaching–grasping movement in human subjects. Two different experiments were run using video‐clips as visual stimuli. Video‐clips showed a normally performed (control stimulus) or an anomalous reaching–grasping movement executed by delaying the time of the appearance of the maximal finger aperture (experiment 1), or substituting it with an unpredictable closure (experiment 2). Motor evoked potentials were recorded at different time‐points during the observation of the video‐clips. Profiles of cortical excitability were drawn and compared with the kinematic profiles of the corresponding movement. Passive observation of the natural movement evoked a profile of cortical excitability that is in concordance with the timing of the kinematic profile of the shown finger movements. Observation of the uncommon movements did not exert any modulation (experiment 1) or evoked an activity that matched, at the beginning, the modulation obtained with observation of the natural movement (experiment 2). Results show that the resonant motor plan is loaded as whole at the beginning of observation and once started tends to proceed to its completion regardless of changes to the visual cues. The results exclude the possibility of a temporal fragmentation of the resonant plan, because activation of different populations of mirror neurons for each phase of the ongoing action. They further support the notion of the role of the mirror system as neural substrate for the observing–execution matching system and extend the current knowledge regarding mechanisms that trigger the internal representation of an action.
Reduction in endogenous parkin levels renders glial cells sensitive to both caspase‐dependent and caspase‐independent cell death
Authors: MacCormac Luci P. , Muqit Miratul M. K. , Faulkes David J. , Wood Nicholas W. , Latchman David S.
Published: September 2004

Mutations in the parkin gene give rise to a familial form of Parkinson's disease, autosomal recessive juvenile Parkinsonism (AR‐JP). Although the exact mechanisms are unclear, it is thought that these ‘loss‐of‐function’ mutations contribute to the pathological process by interfering with parkin's E3 ubiquitin ligase activity. In order to mimic the in vivo loss‐of‐function, we produced tet‐inducible glial cell lines that, in the presence of doxycycline, were able either to under‐ or to over‐express the parkin protein. Using this cell‐culture system, we found that the induced alteration of parkin levels in glial cell lines caused different responses compared with their un‐induced counterparts under conditions of stress (staurosporine, hydrogen peroxide and dopamine). In particular, reduction in the levels of parkin within the transfected cells rendered them more susceptible to both apoptotic and necrotic cell death. Interestingly, blocking the cell death pathway with caspase inhibitors rescued the cells under‐expressing parkin from only some of the stress‐induced death. These findings implicate a pathogenic role of glial cells in the pathogenesis of AR‐JP caused by mutations in the parkin gene.
The contribution of activated phagocytes and myelin degeneration to axonal retraction/dieback following spinal cord injury
Authors: McPhail Lowell T. , Stirling David P. , Tetzlaff Wolfram , Kwiecien Jacek M. , Ramer Matt S.
Published: September 2004

Myelin‐derived molecules inhibit axonal regeneration in the CNS. The Long–Evans Shaker rat is a naturally occurring dysmyelinated mutant, which although able to express the components of myelin lacks functional myelin in adulthood. Given that myelin breakdown exposes axons to molecules that are inhibitory to regeneration, we sought to determine whether injured dorsal column axons in a Shaker rat would exhibit a regenerative response absent in normally myelinated Long–Evans (control) rats. Although Shaker rat axons did not regenerate beyond the lesion, they remained at the caudal end of the crush site. Control rat axons, in contrast, retracted and died back from the edge of the crush. The absence of retraction/dieback in Shaker rats was associated with a reduced phagocytic reaction to dorsal column crush around the caudal edge of the lesion. Systemic injection of minocycline, a tetracycline derivative, in control rats reduced both the macrophage response and axonal retraction/dieback following dorsal column injury. In contrast, increasing macrophage activation by spinal injection of the yeast particulate zymosan had no effect on axonal retraction/dieback in Shaker rats. Schwann cell invasion was reduced in minocycline‐treated control rats compared with untreated control rats, and was almost undetectable in Shaker rats, suggesting that like axonal retraction/dieback, spinal Schwann cell infiltration is dependent upon macrophage‐mediated myelin degeneration. These results indicate that following spinal cord injury the phagocyte‐mediated degeneration of myelin and subsequent exposure of inhibitory molecules to the injured axons contributes to their retraction/dieback.
GABAA receptors signal bidirectional reward transmission from the ventral tegmental area to the tegmental pedunculopontine nucleus as a function of opiate state
Authors: Laviolette Steven R. , Van Der Kooy Derek
Published: September 2004

The brainstem tegmental pedunculopontine nucleus (TPP) is involved in reward signalling and is functionally and anatomically linked to the VTA. We examined the possible role of the TPP as a reward transmission output for GABAA receptors in the VTA in rats not previously exposed to opiates vs. rats that were chronically exposed to and in withdrawal from opiates or in rats that had recovered from chronic opiate exposure. Bilateral lesions of the TPP blocked the rewarding effects of a GABAA antagonist but not the rewarding effects of a GABAA receptor agonist in rats previously unexposed to opiates. This functional pattern was reversed in rats that were dependent on opiates and in withdrawal. However, once rats had recovered from chronic opiate exposure the functional parameters of VTA GABAA receptor reward signalling reverted to the pattern observed in animals that had not been exposed to opiates. These findings suggest that GABAA receptors in the VTA can regulate differential reward signalling through separate neural systems during the transition from a drug‐naive to a drug‐dependent and withdrawn state.
Recovery of bimodal locomotion in the spinal‐transected salamander, Pleurodeles waltlii
Authors: Chevallier Stéphanie , Landry Marc , Nagy Frédéric , Cabelguen Jean‐Marie
Published: September 2004

Electromyographic (EMG) analysis was used to provide an assessment of the recovery of locomotion in spinal‐transected adult salamanders (Pleurodeles waltlii). EMG recordings were performed during swimming and overground stepping in the same animal before and at various times (up to 500 days) after a mid‐trunk spinalization. Two–three weeks after spinalization, locomotor EMG activity was limited to the forelimbs and the body rostral to the transection. Thereafter, there was a return of the locomotor EMG activity at progressively more caudal levels below the transection. The animals reached stable locomotor patterns 3–4 months post‐transection. Several locomotor parameters (cycle duration, burst duration, burst proportion, intersegmental phase lag, interlimb coupling) measured at various recovery times after spinalization were compared with those in intact animals. These comparisons revealed transient and long‐term alterations in the locomotor parameters both above and below the transection site. These alterations were much more pronounced for swimming than for stepping and revealed differences in adaptive plasticity between the two locomotor networks. Recovered locomotor activity was immediately abolished by retransection at the site of the original spinalization, suggesting that the spinal cord caudal to the transection was reinnervated by descending brain and/or propriospinal axons, and that this regeneration contributed to the restoration of locomotor activity. Anatomical studies conducted in parallel further demonstrated that some of the regenerated axons came from glutamatergic and serotoninergic immunoreactive cells within the reticular formation.