bluuebunny/crossref_metadata_2025 huggingface dataset:
Fetoscopy and fetal endoscopic surgery: review of the literature
Authors: De Keersmaecker B , Ville Y
Published: July 2002

The first attempt to visualise the fetus with a panendoscope was performed in 1954. Embryoscopy and fetoscopy were developed in the 1970's. They were introduced as a diagnostic tool to visualise external malformations of the fetus in the first, second and third trimesters of pregnancy as well as to obtain fetal tissue for diagnostic purposes and perform fetal intravascular transfusion.

Diagnostic fetoscopy during the second and third trimester was abandoned in the late 80's due to the invasive nature of the technique and the development of high resolution ultrasound imaging. Percutaneous access to fetal blood and tissue sampling under continuous ultrasound guidance also became possible in the late eighties. Recently there has been renewed interest in embryo-fetoscopy due to the increasing use of ultrasound in the first trimester together with the development of new equipment, especially miniature fiberscopes. These have improved image resolution while being less invasive. Anomalies suspected in the first trimester often call for a rapid and complete fetal evaluation and microendoscopy allows for a sono-endoscopic assessment of fetal anatomy. This may help a couple to decide whether to carry on with the pregnancy or to opt for early termination of pregnancy (TOP). More recently, embryoscopy and fetoscopy have been used to operate on the placenta and membranes and also on the fetus itself.
Immunohistochemical localisation of growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I) and type I IGF-I receptor, and gene expression of GH and GHR in rat pre-antral follicles
Authors: Zhao J. , Taverne M.A.M. , van der Weijden G.C. , Bevers M.M. , van den Hurk R.
Published: July 2002

We previously demonstrated that the development of cultured rat pre-antral follicles is stimulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I) and that the mRNA of IGF-I and type I IGF receptor (IGFR) is present in the oocyte and wall of these follicles. To gain a closer insight into the regulation of early folliculogenesis by GH and IGF-I, the present study investigated the gene expression of GH and GHR mRNA in isolated oocytes and follicular wall cells of pre-antral follicles, using reverse transcriptase polymerase chain reaction, and the localisation of immunoreactive IGF-I, IGFR, GH and GHR proteins in ovarian sections of 10-day-old rats. GH was detected in oocytes and follicular wall tissue of pre-antral follicles, whereas expression of the GH mRNA was absent. The GHR mRNA was present in follicular wall tissue and not in the oocyte, while positive immunostaining for GHR was observed in all cells of the pre-antral follicles. Immunoreactive IGF-I and IGFR was also visible in the pre-antral follicles, especially in the oocytes. In conclusion, the data show that the previously demonstrated local gene expression of IGF-I and IGFR in oocytes and their enveloping follicular cells also leads to translation, which points to the involvement of intrafollicular IGF-I in early follicular development. The presence of the GHR mRNA and the GHR and GH proteins in pre-antral follicles in the absence of ovarian GH mRNA suggest a direct effect of systemic GH on early follicular development.
Fertilisability and developmental ability of mouse oocytes with reduced amounts of cytoplasm
Authors: Wakayama T. , Yanagimachi R.
Published: July 2002

To determine the minimal amount of cytoplasm necessary to support normal development of mouse oocytes, mature unfertilised oocytes were reduced in size into approximately 1/2, 1/4 and 1/8 by removing the cytoplasm, then inseminated. More than 80% of 1/2-size oocyte were fertilised normally and almost all fertilised eggs developed to blastocysts. When transferred to foster females, 31% of the blastocysts developed into normal offspring. In contrast, 1/4- and 1/8-size oocytes, although they were penetrable by spermatozoa and extruded the second polar bodies, could not reach the 2-cell stage. In these oocytes, sperm nuclei did not develop into full-sized pronuclei. These results suggest that an oocyte can develop to full term after losing about half its cytoplasm, but not more.
THE FIX OF FORM: AN OPEN LETTER
Authors: Tucker Herbert
Published: July 2002

Charlottesville Fin de siècle XX

O thou that after toil and storm Mayst seem to have reached a purer air, Whose faith has centre everywhere, Nor cares to fix itself to form, or — as the undersigned trusts to find you still —

Dear Reader,

THERE WAS A TIME when the New Criticism taught you to pay attention to literary form as the embodied elaboration of meaning. You knew that authors had a design on you, but you learned to bracket questions of intention and affect so as to concentrate on the design itself, where creative purpose got built out in verbal deed. You dealt, by and large, with the concrete particular, anatomizing complexities for the sake of an analytically earned appreciation of distinct instances of formal unity. While every instance was unique, what you found to say about the unity of this poem or that novel did evince a family resemblance among literary works, and among critical interpretations, that linked them to each other and, presumably, to a family of wholesome universals. Or did it just seem to? If you couldn’t be sure then, and can’t be sure now either, that’s because these larger issues were somehow off in the background and beside the point, which was to practice criticism as a cognition of integral form.
IR‐based satellite and radar rainfall estimates of convective storms over northern Italy
Authors: Amorati R , Alberoni P P , Levizzani V , Nanni S
Published: July 2002

Convective precipitation events in northern Italy during 1996 and 1997 are analysed using two infrared‐based geosynchronous satellite rainfall estimation methods to verify the level of applicability of the techniques for operational applications in the area, their quantitative results, and relative performances. The Negri–Adler–Wetzel (NAW) and the convective stratiform technique (CST) are applied to METEOSAT's thermal infrared (IR) data. C‐band radar reflectivity fields detail the vertical and horizontal structure of the cloud systems, and radar rainfall data are retrieved. Satellite rain areas are checked against simultaneous radar rainfall retrievals through a contingency analysis procedure. A semi‐quantitative analysis is presented. Positive brightness temperature differences between water vapour and thermal IR channels are also examined and related to the storms' development stage and rainrate. Results show that NAW and CST perform reasonably in delimiting rain areas during active convection and care should be used in the initial and final development stage when statistical parameters lose most of their significance. NAW tends to overestimate rainfall while CST approaches more closely radar measurements. Most common errors arise from considering only portions of the storm, contamination from cold non‐precipitating cloud, and merging of two or more cloud masses of independent origin. Operational applications, though not completely quantitative, are also possible, including positive values of the difference between water vapour and IR brightness temperature. Copyright © 2000 Royal Meteorological Society
Two cases of severe weather in Catalonia (Spain): an observational study
Authors: Ramis Clemente , Arús Joan , López José Manuel , Mestres Antoni M.
Published: July 2002

Surface observations, satellite and radar imagery and cloud‐to‐ground lightning data are used in an observational study of two cases that produced severe weather in Catalonia (Spain). The first one occurred on 24 August 1993; a squall line crossed Catalonia from west to east producing heavy rain with rates of up to 100 mm h−1 and hail of 7 cm diameter. The observational information provided is a good tool for monitoring the event and issuing a reasonable nowcast. The second case, which occurred on 31 August 1994, was associated with the development of a tornado (F1 in the Fujita scale) as well as hail of up to 5 cm diameter. In this case the convection was almost stationary and no clear signatures of severe weather can be identified from available satellite and radar imagery. Copyright © 1997 Royal Meteorological Society
MR spectroscopy in HIV and stimulant dependence
Authors: TAYLOR MICHAEL J. , ALHASSOON OMAR M. , SCHWEINSBURG BRIAN C. , VIDEEN JOHN S. , GRANT IGOR ,   THE HNRC GROUP
Published: July 2002

HIV infection and abuse of central nervous system (CNS)
stimulants are both associated with brain damage and
dysfunction. CNS stimulant overdose can lead to microinfarction,
hemorrhagic lesions, and vasculitis (Bostwick, 1981; Cahill
et al., 1981), and may impact frontostriatal systems. Investigations
of HIV-infected (HIV+) individuals have demonstrated deficits
in attention, speed of information processing, motor functioning,
executive functioning, and learning efficiency. These deficits
are consistent with frontostriatal involvement (Heaton
et al., 1995; Martin, 1994). Given the rise in AIDS cases
attributable to drug use at a time when the number of AIDS
cases due to sexual transmission is stable or declining,
it is critical to determine if drug use, especially CNS
stimulants, potentiates HIV-related neuronal injury.
Competing harvesting strategies in a simulated population under uncertainty
Authors: Milner‐Gulland E. J. , Shea K. , Possingham H. , Coulson T. , Wilcox C.
Published: July 2002

We present a case study of the use of simulation modelling to develop and test strategies for managing populations under uncertainty. Strategies that meet a stock conservation criterion under a base case scenario are subjected to a set of robustness trials, including biased and highly variable abundance estimates and poaching. Strategy performance is assessed with respect to a conservation criterion, the revenues achieved and their variability. Strategies that harvest heavily, even when the population is apparently very large, perform badly in the robustness trials. Setting a threshold below which harvesting does not take place, and above which all individuals are harvested, does not provide effective protection against over‐harvesting. Strategies that rely on population growth rates rather than estimates of population size are more robust to biased estimates. The strategies that are most robust to uncertainty are simple, involving harvesting a relatively small proportion of the population each year. The simulation modelling approach to exploring harvesting strategies is suggested as a useful tool for the assessment of the performance of competing strategies under uncertainty.
Functional antagonism between E2F family members
Authors: Frolov Maxim V. , Huen David S. , Stevaux Olivier , Dimova Dessislava , Balczarek-Strang Kristi , Elsdon Mark , Dyson Nicholas J.
Published: July 2002

E2F is a heterogenous transcription factor and its role in cell cycle control results from the integrated activities of many different E2F family members. Unlike mammalian cells, that have a large number of E2F-related genes, the Drosophila genome encodes just two E2F genes, de2f1 and de2f2. Here we show that de2f1and de2f2 provide different elements of E2F regulation and that they have opposing functions during Drosophila development. dE2F1 and dE2F2 both heterodimerize with dDP and bind to the promoters of E2F-regulated genes in vivo. dE2F1 is a potent activator of transcription, and the loss of de2f1 results in the reduced expression of E2F-regulated genes. In contrast, dE2F2 represses the transcription of E2F reporters and the loss of de2f2 function results in increased and expanded patterns of gene expression. The loss of de2f1 function has previously been reported to compromise cell proliferation. de2f1 mutant embryos have reduced expression of E2F-regulated genes, low levels of DNA synthesis, and hatch to give slow-growing larvae. We find that these defects are due in large part to the unchecked activity of dE2F2, since they can be suppressed by mutation of de2f2. Examination of eye discs fromde2f1; de2f2 double-mutant animals reveals that relatively normal patterns of DNA synthesis can occur in the absence of both E2F proteins. This study shows how repressor and activator E2Fs are used to pattern transcription and how the net effect of E2F on cell proliferation results from the interplay between two types of E2F complexes that have antagonistic functions.
Two Time Windows of Anisomycin-Induced Amnesia for Inhibitory Avoidance Training in Rats: Protection from Amnesia by Pretraining but not Pre-exposure to the Task Apparatus
Authors: Quevedo João , Vianna Monica R.M. , Roesler Rafael , de-Paris Fernanda , Izquierdo Ivan , Rose Steven P.R.
Published: July 2002

We have studied the effect of training conditions on hippocampal protein synthesis-dependent processes in consolidation of the inhibitory avoidance task. Adult male Wistar rats were trained and tested in a step-down inhibitory avoidance task (0.4 mA foot shock, 24 hr training–test interval). Fifteen minutes before or 0, 3, or 6 hr after training, animals received a 0.8-μl intrahippocampal infusion of the protein-synthesis inhibitor anisomycin (80 μg) or vehicle (PBS, pH 7.4). The infusion of anisomycin impaired retention test performance in animals injected 15 min before and 3 hr after the training session, but not at 0 or 6 h post-training. Pretraining with a low foot shock intensity (0.2 mA) 24 hr before training, prevented the amnestic effect of anisomycin injected at 15 min before or 3 hr after training. However, simple pre-exposure to the inhibitory avoidance apparatus did not alter the amestic effects of anisomycin. The results suggest that hippocampal protein synthesis is critical in two periods, around the time of, and 3 hr after training. A prior weak training session, however, which does not itself alter step-down latencies, is sufficient to prevent the amnestic effect of anisomycin, suggesting that even if not behaviorally detectable, weak training must be sufficient to produce some lasting cellular expression of the experience.
The Operant and the Classical in Conditioned Orientation of Drosophila melanogaster at the Flight Simulator
Authors: Brembs Björn , Heisenberg Martin
Published: July 2002

Ever since learning and memory have been studied experimentally, the relationship between operant and classical conditioning has been controversial. Operant conditioning is any form of conditioning that essentially depends on the animal's behavior. It relies on operant behavior. A motor output is called operant if it controls a sensory variable. The Drosophila flight simulator, in which the relevant behavior is a single motor variable (yaw torque), fully separates the operant and classical components of a complex conditioning task. In this paradigm a tethered fly learns, operantly or classically, to prefer and avoid certain flight orientations in relation to the surrounding panorama. Yaw torque is recorded and, in the operant mode, controls the panorama. Using a yoked control, we show that classical pattern learning necessitates more extensive training than operant pattern learning. We compare in detail the microstructure of yaw torque after classical and operant training but find no evidence for acquired behavioral traits after operant conditioning that might explain this difference. We therefore conclude that the operant behavior has a facilitating effect on the classical training. In addition, we show that an operantly learned stimulus is successfully transferred from the behavior of the training to a different behavior. This result unequivocally demonstrates that during operant conditioning classical associations can be formed.
Isolation and Characterization of Two Germacrene A Synthase cDNA Clones from Chicory
Authors: Bouwmeester Harro J. , Kodde Jan , Verstappen Francel W.A. , Altug Iris G. , de Kraker Jan-Willem , Wallaart T. Eelco
Published: July 2002

Chicory (Cichorium intybus) sesquiterpene lactones were recently shown to be derived from a common sesquiterpene intermediate, (+)-germacrene A. Germacrene A is of interest because of its key role in sesquiterpene lactone biosynthesis and because it is an enzyme-bound intermediate in the biosynthesis of a number of phytoalexins. Using polymerase chain reaction with degenerate primers, we have isolated two sesquiterpene synthases from chicory that exhibited 72% amino acid identity. Heterologous expression of the genes in Escherichia coli has shown that they both catalyze exclusively the formation of (+)-germacrene A, making this the first report, to our knowledge, on the isolation of (+)-germacrene A synthase (GAS)-encoding genes. Northern analysis demonstrated that both genes were expressed in all chicory tissues tested albeit at varying levels. Protein isolation and partial purification from chicory heads demonstrated the presence of two GAS proteins. On MonoQ, these proteins co-eluted with the two heterologously produced proteins. TheK  m value, pH optimum, and MonoQ elution volume of one of the proteins produced in E. coli were similar to the values reported for the GAS protein that was recently purified from chicory roots. Finally, the two deduced amino acid sequences were modeled, and the resulting protein models were compared with the crystal structure of tobacco (Nicotiana tabacum) 5-epi-aristolochene synthase, which forms germacrene A as an enzyme-bound intermediate en route to 5-epi-aristolochene. The possible involvement of a number of amino acids in sesquiterpene synthase product specificity is discussed.
γ-Glutamyl Transpeptidase in Transgenic Tobacco Plants. Cellular Localization, Processing, and Biochemical Properties
Authors: Storozhenko Sergei , Belles-Boix Enric , Babiychuk Elena , Hérouart Didier , Davey Mark W. , Slooten Luit , Van Montagu Marc , Inzé Dirk , Kushnir Sergei
Published: July 2002

γ-Glutamyl transpeptidase (γ-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the γ-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for γ-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound γ-GT activity was localized outside the cell in transgenic plants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in two unequal subunits. Thus, Arabidopsis γ-GT is similar to the mammalian enzymes in enzymatic properties, post-translational processing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH.
The Vacuolar Targeting Signal of the 2S Albumin from Brazil Nut Resides at the C Terminus and Involves the C-Terminal Propeptide as an Essential Element
Authors: Saalbach G. , Rosso M. , Schumann U.
Published: July 2002

Genetic constructs in which different N- and C-terminal segments of Brazil nut (Bertholletia excelsa H.B.K.) 2S albumin were fused to secretory yeast invertase were transformed into tobacco (Nicotiana tabacum) plants to investigate the vacuolar targeting signal of the 2S albumin. None of the N-terminal segments, including the complete precursor containing all propeptides, was able to direct the invertase to the vacuoles. However, a short C-terminal segment comprising the last 20 amino acids of the precursor was sufficient for efficient targeting of yeast invertase to the vacuoles of the transformed tobacco plants. Further analyses showed that peptides of 16 and 13 amino acids of the C-terminal segment were still sufficient, although they had slightly lower efficiency. When segments of 9 amino acids or shorter were analyzed, a decrease to approximately 30% was observed. These segments included the C-terminal propeptide of four amino acids (Ile-Ala-Gly-Phe). When the 2S albumin was expressed in tobacco, it was also localized to the vacuoles of mesophyll cells. If the C-terminal propeptide was deleted from the 2S albumin precursor, all of this truncated 2S albumin was secreted from the tobacco cells. These results indicate that the C-terminal propeptide is necessary but not sufficient for vacuolar targeting. In addition, an adjacent segment of at least 12 amino acids of the mature protein is needed to form the complete signal for efficient targeting.
Characterization and Expression of Caffeoyl-Coenzyme A 3-O-Methyltransferase Proposed for the Induced Resistance Response of Vitis vinifera L
Authors: Busam G. , Junghanns K. T. , Kneusel R. E. , Kassemeyer H. H. , Matern U.
Published: July 2002

Cell-suspension cultures of Vitis vinifera L. cv Pinot Noir accumulated resveratrol upon fungal elicitation, and the activity of S-adenosyI-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyl-transferase (CCoAOMT), yielding feruloyl-CoA, increased to a transient maximum at 12 to 15 h. CCoAOMT cDNA was cloned from the elicited cells and was shown to encode a polypeptide highly homologous to CCoAOMTs from cells of Petroselinum species or Zinnia species. The expression of the cDNA in Escherichia coli revealed that grapevine CCoAOMT methylates both caffeoyl-and 5-hydroxyferuloyl-coenzyme A and is probably involved in phenolic esterification and lignification. Commercial plant activators induce the disease-resistance response of test plants and are considered to mimic the action of salicylic acid. Among these chemicals, 2,6-dichloroisonicotinic acid and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester provoke systemic acquired resistance (SAR) and were also shown to induce the expression of class III chitinase in grapevine. The SAR response is classified by an unchanged phenotype of tissues, but the mechanistic basis is unknown. Treatment of the cultured V. vinifera cells with either fungal elicitor or low concentrations of salicylic acid and 2,6-dichloroisonicotinic acid, respectively, raised the CCoAOMT or stilbene synthase transcript abundance, suggesting that grapevine is capable of the SAR response, whereas benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester was ineffective. The data imply for the first time (to our knowledge) that the expression of phenylpropanoid genes in grapevine is induced by SAR activators without phenotypic consequences and suggest a role for CCoAOMT and stilbene synthase in the disease-resistance response leading beyond the level of pathogenesis-related proteins as markers of the SAR.
The NAD(P)H Dehydrogenase in Barley Thylakoids Is Photoactivatable and Uses NADPH as well as NADH1
Authors: Bernhard Teicher Harald , Vibe Scheller Henrik
Published: July 2002

An improved light-dependent assay was used to characterize the NAD(P)H dehydrogenase (NDH) in thylakoids of barley (Hordeum vulgare L.). The enzyme was sensitive to rotenone, confirming the involvement of a complex I-type enzyme. NADPH and NADH were equally good substrates for the dehydrogenase. Maximum rates of activity were 10 to 19 μmol electrons mg−1chlorophyll h−1, corresponding to about 3% of linear electron-transport rates, or to about 40% of ferredoxin-dependent cyclic electron-transport rates. The NDH was activated by light treatment. After photoactivation, a subsequent light-independent period of about 1 h was required for maximum activation. The NDH could also be activated by incubation of the thylakoids in low-ionic-strength buffer. The kinetics, substrate specificity, and inhibitor profiles were essentially the same for both induction strategies. The possible involvement of ferredoxin:NADP+ oxidoreductase (FNR) in the NDH activity could be excluded based on the lack of preference for NADPH over NADH. Furthermore, thenoyltrifluoroacetone inhibited the diaphorase activity of FNR but not the NDH activity. These results also lead to the conclusion that direct reduction of plastoquinone by FNR is negligible.
RNA Polymerase Subunits Encoded by the Plastid rpoGenes Are Not Shared with the Nucleus-Encoded Plastid Enzyme1
Authors: Serino Germán , Maliga Pal
Published: July 2002

Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases. The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme. The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus. Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion ofrpoB (encoding the β-subunit) from the plastid genome. To determine if NEP and PEP share any essential subunits, therpoA, rpoC1, and rpoC2genes encoding the PEP α-, β′-, and β"-subunits were removed by targeted gene deletion from the plastid genome. We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters. Therefore, rpoA,rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery. Furthermore, our data indicate that no functional copy ofrpoA, rpoB, rpoC1, orrpoC2 that could complement the deleted plastidrpo genes exists outside the plastids.
Characterization of Euphorbia characias Latex Amine Oxidase1
Authors: Padiglia Alessandra , Medda Rosaria , Lorrai Anita , Murgia Barbara , Pedersen Jens Z. , Finazzi Agró Alessandro , Floris Giovanni
Published: July 2002

A copper-containing amine oxidase from the latex of Euphorbia characias was purified to homogeneity and the copper-free enzyme obtained by a ligand-exchange procedure. The interactions of highly purified apo- and holoenzyme with several substrates, carbonyl reagents, and copper ligands were investigated by optical spectroscopy under both aerobic and anaerobic conditions. The extinction coefficients at 278 and 490 nm were determined as 3.78 × 105m−1cm−1 and 6000 m−1cm−1, respectively. Active-site titration of highly purified enzyme with substrates and carbonyl reagents showed the presence of one cofactor at each enzyme subunit. In anaerobiosis the native enzyme oxidized one equivalent substrate and released one equivalent aldehyde per enzyme subunit. The apoenzyme gave exactly the same 1:1:1 stoichiometry in anaerobiosis and in aerobiosis. These findings demonstrate unequivocally that copper-free amine oxidase can oxidize substrates with a single half-catalytic cycle. The DNA-derived protein sequence shows a characteristic hexapeptide present in most 6-hydroxydopa quinone-containing amine oxidases. This hexapeptide contains the tyrosinyl residue that can be modified into the cofactor 6-hydroxydopa quinone.
Blue Light and Abscisic Acid Independently Induce Heterophyllous Switch in Marsilea quadrifolia1
Authors: Lin Bai-Ling , Yang Wen-Jen
Published: July 2002

In natural habitats Marsilea quadrifolia L. produces different types of leaves above and below the water level. In aseptic cultures growth conditions can be manipulated so that leaves of the submerged type are produced continuously. Under such conditions the application of either blue light or an optimal concentration of abscisic acid (ABA) induced the development of aerial-type leaves. When fluridone, an inhibitor of ABA biosynthesis, was added to the culture medium it did not prevent blue light induction of aerial leaf development. During blue light treatment the endogenous ABA level inM. quadrifolia leaves remained unchanged. However, after the plants were transferred to an enriched medium, the ABA level gradually increased, corresponding to a transition in development from the submerged type of leaves to aerial leaves. These results indicate that the blue light signal is not mediated by ABA. Therefore, in the regulation of heterophyllous determination, discrete pathways exist in response to environmental signals.
Purification and Characterization of Caffeine Synthase from Tea Leaves1
Authors: Kato Misako , Mizuno Kouichi , Fujimura Tatsuhito , Iwama Masanori , Irie Masachika , Crozier Alan , Ashihara Hiroshi
Published: July 2002

Caffeine synthase (CS), theS-adenosylmethionine-dependentN-methyltransferase involved in the last two steps of caffeine biosynthesis, was extracted from young tea (Camellia sinensis) leaves; the CS was purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg−1protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 kD as estimated by gel-filtration chromatography and 41 kD as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3- and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. However, the enzyme had no 7-N-methyltransferase activity toward xanthosine and xanthosine 5′-monophosphate. The Km values of CS for paraxanthine, theobromine, 7-methylxanthine, andS-adenosylmethionine were 24, 186, 344, and 21 μm, respectively. The possible role and regulation of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS showed little homology with other methyltransferases.
Leucine Aminopeptidase RNAs, Proteins, and Activities Increase in Response to Water Deficit, Salinity, and the Wound Signals Systemin, Methyl Jasmonate, and Abscisic Acid1
Authors: Chao Wun S. , Gu Yong-Qiang , Pautot Véronique , Bray Elizabeth A. , Walling Linda L.
Published: July 2002

LapARNAs, proteins, and activities increased in response to systemin, methyl jasmonate, abscisic acid (ABA), ethylene, water deficit, and salinity in tomato (Lycopersicon esculentum). Salicylic acid inhibited wound-induced increases of LapA RNAs. Experiments using the ABA-deficient flacca mutant indicated that ABA was essential for wound and systemin induction ofLapA, and ABA and systemin acted synergistically to induce LapA gene expression. In contrast,pin2 (proteinase inhibitor 2) was not dependent on exogenous ABA. Whereas both LapA and le4(L. esculentumdehydrin) were up-regulated by increases in ABA, salinity, and water deficit, only LapAwas regulated by octadecanoid pathway signals. Comparison ofLapA expression with that of thePR-1 (pathogenesis-related 1) andGluB (basic β-1,3-glucanase) genes indicated that these PR protein genes were modulated by a systemin-independent jasmonic acid-signaling pathway. These studies showed that at least four signaling pathways were utilized during tomato wound and defense responses. Analysis of the expression of aLapA1:GUS gene in transgenic plants indicated that theLapA1 promoter was active during floral and fruit development and was used during vegetative growth only in response to wounding, Pseudomonas syringae pv tomatoinfection, or wound signals. This comprehensive understanding of the regulation of LapA genes indicated that this regulatory program is distinct from the wound-induced pin2, ABA-responsive le4, and PR protein genes.
The Relationship between Ethylene Binding and Dominant Insensitivity Conferred by Mutant Forms of the ETR1 Ethylene Receptor
Authors: Hall Anne E. , Grace Chen Qianhong , Findell Jennifer L. , Eric Schaller G. , Bleecker Anthony B.
Published: July 2002

Ethylene responses in Arabidopsis are mediated by a small family of receptors, including theETR1 gene product. Specific mutations in the N-terminal ethylene-binding domain of any family member lead to dominant ethylene insensitivity. To investigate the mechanism of ethylene insensitivity, we examined the effects of mutations on the ethylene-binding activity of the ETR1 protein expressed in yeast. Theetr1-1 andetr1-4 mutations completely eliminated ethylene binding, while the etr1-3mutation severely reduced binding. Additional site-directed mutations that disrupted ethylene binding in yeast also conferred dominant ethylene insensitivity when the mutated genes were transferred into wild-type Arabidopsis plants. By contrast, theetr1-2 mutation did not disrupt ethylene binding in yeast. These results indicate that dominant ethylene insensitivity may be conferred by mutations that disrupt ethylene binding or that uncouple ethylene binding from signal output by the receptor. Increased dosage of wild-type alleles in triploid lines led to the partial recovery of ethylene sensitivity, indicating that dominant ethylene insensitivity may involve either interactions between wild-type and mutant receptors or competition between mutant and wild-type receptors for downstream effectors.
A Point Mutation in the Ethylene-Inducing Xylanase Elicitor Inhibits the β-1-4-Endoxylanase Activity But Not the Elicitation Activity
Authors: Furman-Matarasso Noa , Cohen Eitan , Du Quansheng , Chejanovsky Nor , Hanania Uri , Avni Adi
Published: July 2002

Ethylene-inducing xylanase (EIX) elicits plant defense responses in certain tobacco (Nicotiana tabacum) and tomato cultivars in addition to its xylan degradation activity. It is not clear, however, whether elicitation occurs by cell wall fragments released by the enzymatic activity or by the xylanase protein interacting directly with the plant cells. We cloned the gene encoding EIX protein and overexpressed it in insect cells. To determine the relationship between the two activities, substitution of amino acids in the xylanase active site was performed. Substitution at glutamic acid-86 or -177 with glutamine (Gln), aspartic acid (Asp), or glycine (Gly) inhibited the β-1-4-endoxylanase activity. Mutants having Asp-86 or Gln-177 also lost the ability to induce the hypersensitive response and ethylene biosynthesis. However, mutants having Gln-86, Gly-86, Asp-177, or Gly-177 retained ability to induce ethylene biosynthesis and the hypersensitive response. Our data show that the xylanase activity of EIX elicitor can be separated from the elicitation process, as some of the mutants lack the former but retain the latter.
Cloning and Molecular Analyses of a Gibberellin 20-Oxidase Gene Expressed Specifically in Developing Seeds of Watermelon
Authors: Kang Hong-Gyu , Jun Sung-Hoon , Kim Junyul , Kawaide Hiroshi , Kamiya Yuji , An Gynheung
Published: July 2002

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolatedCv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed inEscherichia coli as a fusion protein, which oxidized GA12 at C-20 to the C19 compound GA9, a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter ofCv20ox was isolated and fused to the β-glucuronidase (GUS) gene. In a transient expression system, β-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.
The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm
Authors: Kubo Akiko , Fujita Naoko , Harada Kyuya , Matsuda Toshiaki , Satoh Hikaru , Nakamura Yasunori
Published: July 2002

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of thesugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in somesugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that theSugary-1 gene encodes the isoamylase gene of the rice genome.
Cloning, Expression, and Molecular Characterization of a Small Pea Gene Family Regulated by Low Levels of Ultraviolet B Radiation and Other Stresses
Authors: Brosché Mikael , Strid Åke
Published: July 2002

A pea (Pisum sativum) DNA fragment (termed MB3) was isolated by differential display of cDNAs obtained from total leaf RNA of ultraviolet B (UV-B) radiation-treated plants. Longer cDNAs were cloned by rapid amplification of cDNA ends in the 3′ to 5′ direction. Three different, but very similar, cDNAs were cloned, sadA, sadB, andsadC, the major difference between them being a 36-bp deletion in the coding region of sadB. Southern blotting confirmed the occurrence of at least three genes in the pea genome. Database comparisons of the SAD protein sequences revealed high identity (46%) and similarity (77%) with a putative tomato (Lycopersicon esculentum) short-chain alcohol dehydrogenase. Very low levels of UV-B radiation (the biologically effective radiation normalized to 300 nm = 0.08 W m−2) was shown to up-regulate expression, a dose considerably lower than that needed to induce expression of the well-known UV-B defensive chalcone synthase and phenylalanine ammonia lyase genes. RNase protection assay revealed that primarilysadA and sadC mRNA accumulation was enhanced by UV-B. In addition to UV-B irradiation, ozone fumigation, wounding, aluminum stress, and salt stress induced increased transcript levels of the sad genes in pea.
Increased Respiratory Restriction during Phosphate-Limited Growth in Transgenic Tobacco Cells Lacking Alternative Oxidase
Authors: Parsons Hannah L. , Yip Justine Y.H. , Vanlerberghe Greg C.
Published: July 2002

We found that mitochondrial alternative oxidase (AOX) protein and the capacity for CN-resistant respiration are dramatically increased in wild-type tobacco (Nicotiana tabacum) suspension-cultured cells in response to growth under P limitation, and antisense (AS8) tobacco cells unable to induce AOX under these conditions have altered growth and metabolism. Specifically, we found that the respiration of AS8 cells was restricted during P-limited growth, when the potential for severe adenylate control of respiration (at the level of C supply to the mitochondrion and/or at the level of oxidative phosphorylation) is high due to the low cellular levels of ADP and/or inorganic P. As a result of this respiratory restriction, AS8 cells had altered growth, morphology, cellular composition, and patterns of respiratory C flow to amino acid synthesis compared with wild-type cells with abundant AOX protein. Also, AS8 cells under P limitation displayed high in vivo rates of generation of active oxygen species compared with wild-type cells. This difference could be abolished by an uncoupler of mitochondrial oxidative phosphorylation. Our results suggest that induction of non-phosphorylating AOX respiration (like induction of adenylate and inorganic P-independent pathways in glycolysis) is an important plant metabolic adaptation to P limitation. By preventing severe respiratory restriction, AOX acts to prevent both redirections in C metabolism and the excessive generation of harmful active oxygen species in the mitochondrion.
The Activity of the Maize Opaque2 Transcriptional Activator Is Regulated Diurnally
Authors: Ciceri Pietro , Locatelli Franca , Genga Annamaria , Viotti Angelo , Schmidt Robert J.
Published: July 2002

The maize (Zea maysL.) Opaque2 (O2) protein is an endosperm-specific transcriptional activator whose DNA-binding activity is regulated diurnally by a phosphorylation/dephosphorylation mechanism. We show that theO2 transcript undergoes pronounced oscillations during the day-night cycle. The highest level of the O2 message is present at midday and the lowest level at midnight. The level ofO2 transcript follows a diurnal rhythm that appears controlled by the circadian clock. Two different endosperm-expressed DNA-binding proteins, PBF (prolamin box-binding factor) and OHP1 (O2-heterodimerizing protein 1), were also analyzed. While thePBF message levels oscillate diurnally, the steady-state levels of OHP1 transcript were constant through the day and night. We present data showing that the seed is not directly involved in the perception of the light signal, but presumably responds to diurnal fluxes of nutrients into the endosperm. Moreover, we show that the O2 protein is not involved in the regulation of its own transcript levels. These data indicate that O2 activity is down-regulated at night by both a reduction in O2transcript and by hyperphosphorylation of residual O2 protein, and suggest that regulatory gene activity during endosperm development may be acutely sensitive to a diurnal signal(s) emanating from the plant and passing into the developing seeds.
A Putative Role for the Tomato Genes DUMPY and CURL-3 in Brassinosteroid Biosynthesis and Response
Authors: Koka Chala V. , Cerny R. Eric , Gardner Randy G. , Noguchi Takahiro , Fujioka Shozo , Takatsuto Suguru , Yoshida Shigeo , Clouse Steven D.
Published: July 2002

Thedumpy (dpy) mutant of tomato (Lycopersicon esculentum Mill.) exhibits short stature, reduced axillary branching, and altered leaf morphology. Application of brassinolide and castasterone rescued the dpyphenotype, as did C-23-hydroxylated, 6-deoxo intermediates of brassinolide biosynthesis. The brassinolide precursors campesterol, campestanol, and 6-deoxocathasterone failed to rescue, suggesting thatdpy may be affected in the conversion of 6-deoxocathasterone to 6-deoxoteasterone, similar to the Arabidopsisconstitutive photomorphogenesis and dwarfism(cpd) mutant. Measurements of endogenous brassinosteroid levels by gas chromatography-mass spectrometry were consistent with this hypothesis. To examine brassinosteroid-regulated gene expression in dpy, we performed cDNA subtractive hybridization and isolated a novel xyloglucan endotransglycosylase that is regulated by brassinosteroid treatment. The curl-3(cu-3) mutant (Lycopersicon pimpinellifolium [Jusl.] Mill.) shows extreme dwarfism, altered leaf morphology, de-etiolation, and reduced fertility, all strikingly similar to the Arabidopsis mutantbrassinosteroid insensitive 1 (bri1). Primary root elongation of wild-type L. pimpinellifoliumseedlings was strongly inhibited by brassinosteroid application, whilecu-3 mutant roots were able to elongate at the same brassinosteroid concentration. Moreover, cu-3 mutants retained sensitivity to indole-3-acetic acid, cytokinins, gibberellin, and abscisic acid while showing hypersensitivity to 2,4-dichlorophenoxyacetic acid in the root elongation assay. Thecu-3 root response to hormones, coupled with itsbri1-like phenotype, suggests that cu-3may also be brassinosteroid insensitive.
Enhancement of Na+ Uptake Currents, Time-Dependent Inward-Rectifying K+ Channel Currents, and K+Channel Transcripts by K+ Starvation in Wheat Root Cells
Authors: Buschmann Peter H. , Vaidyanathan Rama , Gassmann Walter , Schroeder Julian I.
Published: July 2002

Excessive low-affinity Na+ uptake is toxic to the growth of glycophytic plants. Recently, several reports have suggested that the interaction between K+ and Na+ uptake might represent a key factor in determining the Na+ tolerance of plants. We investigated the effects of K+ starvation on Na+ and K+ uptake mechanisms in the plasma membrane of wheat (Triticum aestivum L.) root cortex cells using the patch-clamp technique. Unexpectedly, K+starvation of wheat seedlings was found to enhance the magnitude and frequency of occurrence of time-dependent inward-rectifying K+ channel currents (IK  +  in). We examined whether the transcription of a wheat root K+  in channel gene is induced by K+ starvation. A cDNA coding for a wheat root K+ channel homolog, TaAKT1 (accession no.AF207745), was isolated. TaAKT1 mRNA levels were up-regulated in roots in response to withdrawal of K+ from the growth medium. Furthermore, K+ starvation caused an enhancement of instantaneous Na+ currents (INa  +). Electrophysiological analyses suggested that IK  +  in and INa  + are not mediated by the same transport protein based on: (a) different activation curves, (b) different time dependencies, (c) different sensitivities to external Ca2+, and (d) different cation selectivities. These data implicate a role for INa  + in Na+ uptake and stress during K+ starvation, and indicate that K+  in channels may contribute to K+-starvation-induced K+ uptake in wheat roots.
AVP2, a Sequence-Divergent, K+-Insensitive H+-Translocating Inorganic Pyrophosphatase from Arabidopsis
Authors: Drozdowicz Yolanda M. , Kissinger Jessica C. , Rea Philip A.
Published: July 2002

Plant vacuolar H+-translocating inorganic pyrophosphatases (V-PPases; EC 3.6.1.1) have been considered to constitute a family of functionally and structurally monotonous intrinsic membrane proteins. Typified by AVP1 (V. Sarafian, Y. Kim, R.J. Poole, P.A. Rea [1992] Proc Natl Acad Sci USA 89: 1775–1779) from Arabidopsis, all characterized plant V-PPases share greater than 84% sequence identity and catalyze K+-stimulated H+ translocation. Here we describe the molecular and biochemical characterization of AVP2 (accession no. AF182813), a sequence-divergent (36% identical) K+-insensitive, Ca2+-hypersensitive V-PPase active in both inorganic pyrophosphate hydrolysis and H+ translocation. The differences between AVP2 and AVP1 provide the first indication that plant V-PPases from the same organism fall into two distinct categories. Phylogenetic analyses of these and other V-PPase sequences extend this principle by showing that AVP2, rather than being an isoform of AVP1, is but one representative of a novel category of AVP2-like (type II) V-PPases that coexist with AVP1-like (type I) V-PPases not only in plants, but also in apicomplexan protists such as the malarial parasite Plasmodium falciparum.
Cucumber Mosaic Virus Infection Affects Sugar Transport in Melon Plants
Authors: Shalitin Dror , Wolf Shmuel
Published: July 2002

Viral infection often affects carbon assimilation and metabolism in host plants. To better understand the effect of cucumber mosaic virus (CMV) infection on sugar transport, carbohydrate levels and the amounts of the various sugars in the phloem sap were determined in infected melon (Cucumis melo L.) plants. Source leaves infected with CMV were characterized by high concentrations of reducing sugars and relatively low starch levels. The altered level of carbohydrates was accompanied by increased respiration and decreased net photosynthetic rates in the infected leaves. Although stachyose was the predominant sugar in phloem sap collected from petioles of control leaves, sucrose (Suc) was a major sugar in the phloem sap of infected leaves. Moreover, analyses of the newly fixed14CO2 revealed a high proportion of radioactive Suc in the phloem sap of infected leaves 60 min post-labeling. The alteration in phloem sap sugar composition was found in source, but not old, leaves. Moreover, elevations in Suc concentration were also evident in source leaves that did not exhibit symptoms or contain detectable amounts of virus particles. The mode by which CMV infection may cause alterations in sugar transport is discussed in terms of the mechanism by which sugars are loaded into the phloem of cucurbit plants.