bluuebunny/crossref_metadata_2025 huggingface dataset:
Influences of zinc on fertilisation and development of bovine oocytes in vitro
Authors: Stephenson J.L. , Brackett B.G.
Published: July 2002

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 μg added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 μg zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 μg added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 μg per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 μg zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 μg/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 μg ml of zinc in vitro than in vivo because a concentration of 3.0 μg/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 μg/ml of zinc; however, higher concentrations were inhibitory.
Ultrastructural changes during nuclear maturation in Bufo arenarum oocytes
Authors: Ramos Inés , Winik Beatriz C. , Cisint Susana , Crespo Claudia , Medina Marcela , Fernández Silvia
Published: July 2002

During progesterone-induced nuclear maturation the oocytes of Bufo arenarum undergo a series of nuclear and cytoplasmic changes. The breakdown of heterocellular communications between the follicular cell projections and the oocyte microvilli, and the consequent enlargement of the perivitelline space, were observed at the animal pole. The more evident cytoplasmic feature during nuclear maturation comprised the gathering of glycogen granules in clusters, some phagocytosed by empty vesicles. With respect to the location of these vesicles, some were observed in close proximity to the oolemma and others were freely suspended in the perivitelline space, extruded from the oocyte. Other visible events were the disruption of the annulate lamellae, the formation of an elaborate cortical endoplasmic reticulum and the rearrangement of the cortical granules in a monolayer immediately beneath the oolemma together with aggregates of endoplasmic reticulum cisternae. Our results show that during nuclear maturation the nuclear oocyte changes include a flattening of the spherical oocyte nucleus, its migration towards the surface of the animal pole, the disappearance of the nucleoli and the dissolution of the nuclear envelope.
How it all began: the monetary and financial architecture of Europe during the first global capital markets, 1648–1815
Authors: NEAL LARRY
Published: July 2002

Larry Neal, How it all began: the monetary and financial architecture of Europe during the first global capital markets, 1648–1815

The Treaty of Westphalia created the modern nation-state system of Europe and set the stage for the long-term success of financial capitalism. The new sovereign states experimented with competing monetary regimes during their wars over the next century and two-thirds while they extended and perfected the financial innovations in war finance developed during the Thirty Years War. The Dutch maintained fixed exchange rates, the French insisted on exercising monetary independence, while the English placed priority on free movement of international capital. In struggling with the
trilemma of choosing among the goals of maintaining fixed exchange rates, monetary independence and free movement of capital, the governments of early modern Europe learned many valuable lessons. By the time of the Napoleonic wars, the
innovations that emphasised reliance on financial markets rather than on financial institutions proved their superiority.
Using the Web in the democratic process. The Web-orchestrated ‘Stop the Overlay’ cyber-campaign
Authors: DUTTON WILLIAM H. , LIN WAN-YING
Published: July 2002

In the 1996 US Presidential elections, new information and communication technologies (ICTs), particularly the Internet and World Wide Web (Web), began to play a visible role in US campaigns and elections, and its role has expanded to shape the political process more generally. Case studies have shown how the Web, for example, can facilitate the rapid exchange of information that is essential to coordinating political activity. By virtue of reducing the costs of communication, it has become accessible to grassroots organisations without the resources to mount more traditional media campaigns. This study looks in-depth at one campaign – Stop the Overlay – which employed the Internet and Web to effect public policy and regulatory change locally, but with implications for California and the US. Our study led us to employ the framework of an ecology of games to discuss the interplay among the separate but interdependent decisions and games that shaped the campaign. The case shows how this Web-orchestrated campaign was one element that reconfigured the ecology of games in ways that influenced policy decisions. It accomplished this not only by altering the costs of communication, but by reshaping access and thereby changing the networks of communication among political actors.
Detection of hail by means of polarimetric radar data and hailpads: results from four storms
Authors: Nanni S , Mezzasalma P , Alberoni P P
Published: July 2002

Hailfall is one of the most dangerous and damaging phenomena associated with severe thunderstorms. Its remote identification is still under investigation, especially by means of polarimetric radar data. The need to use polarimetric measurements is a limit to operational implementation of the techniques developed up till now. The routine sampling of radar allows the computation of the differential hail signal HDR (Aydin et al., 1986) every 15 minutes. The assessment of the HDR is performed by comparison with the ground truth from a hailpad network. The spatial resolution of this network is quite coarse (one pad per 16 km2) for the collection of all the hail shafts, as they generally have a small horizontal extent and are short lived. Such bounds have been considered in the analysis of four large hailfalls recorded in 1997. This study reveals also the limitation on radar hail detection arising from the frequent occurrence of strong differential attenuation. Nevertheless, to identify regions where a sharp rain–hail transition occurs, polarimetric measurements work much better than algorithms based only on horizontal reflectivity. Results show that use of HDR gives a high probability of correct hail detection as well as an acceptable number of false alarms. Copyright © 2000 Royal Meteorological Society
The hurricane‐like Mediterranean cyclone of January 1995
Authors: Pytharoulis Ioannis , Craig George C , Ballard Susan P
Published: July 2002

The development of a hurricane‐like vortex over the Mediterranean Sea was studied using (mainly) the UK Met. Office Unified Model. The Mediterranean cyclone formed in the morning of 15 January 1995 over the sea between Greece and Sicily. Strong convection was observed prior to its genesis. During the longest part of the cyclone's lifetime, strong surface fluxes and, as a result, deep convection existed in its vicinity. Its track was influenced by the surface fluxes and the flow in the wider region. The forecast of the mesoscale and limited‐area models reproduced the general characteristics of the actual system as they appeared at the surface and upper‐air charts and at the satellite imagery. The investigation of the cyclone's characteristics gave strong evidence (including an ‘eye’ and a warm core) to support the initial assertion that it was similar to tropical cyclones and some polar lows. Baroclinic instability does not seem particularly important, although the cyclone formed at the edge of a baroclinic zone. A numerical experiment showed the vortex did not develop in the absence of surface heat and moisture fluxes. Another experiment showed that sensible and latent heat fluxes were equally important in its development. Copyright © 2000 Royal Meteorological Society
Knowledge representation for natural language processing in implemented
systems
Authors: ALI SYED S. , IWAŃSKA ŁUCJA
Published: July 2002

This special issue presents the state-of-the-art in implemented,
general-purpose
Natural Language Processing (NLP) systems that use nontrivial Knowledge
Representation
and Reasoning (KRR). These systems use full-scale implementations of
traditional KRR techniques as well as some newer knowledge-related processing

mechanisms that have been developed specifically to meet the needs of natural

language processing. The papers cover a wide range of natural language
inputs,
knowledge and formalisms, application domains and processing tasks, illustrating

the key role that knowledge representation plays in all types of NLP systems.
Reliable Change Formula Query: Temkin et al. reply
Authors: TEMKIN NANCY R. , HEATON ROBERT K. , GRANT IGOR , DIKMEN SUREYYA S.
Published: July 2002

Hinton-Bayre (2000) raises a point that may occur
to many readers who are familiar with the Reliable Change
Index (RCI). In our previous paper comparing four models
for detecting significant change in neuropsychological
performance (Temkin et al., 1999), we used a formula for
calculating Sdiff, the measure of
variability for the test–retest difference, that
differs from the one Hinton-Bayre has seen employed in
other studies of the RCI. In fact, there are two ways
of calculating Sdiff—a direct
method and an approximate method. As stated by Jacobson
and Truax (1991, p. 14), the direct method is to compute
“the standard error of the difference between the
two test scores” or equivalently
[begin square root](s12 +
s22 − 2s1s2rxx′)[end square root]
where si is the standard deviation at
time i and rxx′ is the
test–retest correlation or reliability coefficient.
Jacobson and Truax also provide a formula for the approximation
of Sdiff when one does not have access
to retest data on the population of interest, but does have a
test–retest reliability coefficient and an estimate
of the cross-sectional standard deviation, i.e., the standard
deviation at a single point in time. This approximation assumes
that the standard deviations at Time 1 and Time 2 are equal,
which may be close to true in many cases. Since we had the
longitudinal data to directly calculate the standard error of
the difference between scores at Time 1 and Time 2, we used the
direct method. Which method is preferable? When the needed data
are available, it is the one we used.
The effects of visual distraction following traumatic brain
injury
Authors: Whyte John , Fleming Megan , Polansky Marcia , Cavallucci Chris , Coslett and H. Branch
Published: July 2002

Clinical assessments of individuals with traumatic
brain injury (TBI) typically report attentional difficulties,
with distractibility prominent among these complaints.
However, laboratory-based measures have often failed to
find disproportionate distraction among patients with TBI,
as compared to control participants. In this experiment,
we tested 21 patients hospitalized for rehabilitation following
recent TBI and 21 demographically comparable control subjects
on a visual reaction time go–no-go task in which
the target was preceded or followed by a brightly colored
moving visual stimulus, appearing above the target location.
Early distractors actually served as warning stimuli, improving
accuracy and speed for both participant groups. Distractors
occurring at or shortly after the time of target presentation
had no significant impact on accuracy or response bias
in either group, but did produce slowing of RT that was
significantly greater for patients than for controls. The
distractor that produced maximal slowing occurred 100 ms
after the presentation of the target or foil. Repeated
testing sessions led to reduction in the impact of the
distractor and loss of the group difference in RT impact.
The degree of RT slowing induced by distraction was modestly
related to injury severity, as measured by the current
score on the Disability Rating Scale, and the time until
the patient first followed verbal commands. There was also
a trend of greater RT slowing among individuals with focal
orbitofrontal lesions, as assessed on neuroimaging studies.
These results document a greater susceptibility to extraneous
visual distraction among patients with TBI in comparison
to controls. The fact that this difference appears only
in the RT domain, and is greatest when the distractor follows
the target, suggests that the primary impact of visual
distractors is on response preparation and execution rather
than target detection. (JINS, 1998, 4,
127–136).
Acquired oral reading vocabulary following the onset of
amnesia in childhood
Authors: Benedict Ralph H. B. , Shapiro Anne , Duffner Patricia , Jaeger and Jeri J.
Published: July 2002

While the neuropsychological literature includes
few cases of child-onset amnesia, 2 previous case studies
suggest that these patients may be able to learn new information
of a semantic or academic nature. The previous studies
were, in large part, based on neuropsychological testing
performed during adulthood and a retrospective review of
academic achievement test scores during childhood. We present
patient A.C., who acquired severe anterograde amnesia at
age 10 years but demonstrated average levels of performance
on tests of reading, spelling and arithmetic upon examination
at age 19 years. Episodic and semantic memory test scores
were severely impaired, but near normal performances were
found on tests of implicit and procedural memory. In a
prospective study, A.C. learned to read new irregular and
pseudowords and retained this learning over a 1-month period,
similar to the performance of age-matched controls. This
demonstration of postmorbid, acquired oral reading vocabulary
supports a previous conclusion that oral reading can progress
in childhood following the onset of severe anterograde
amnesia. The data also suggest that this new learning probably
reflects nondeclarative memory processes rather than preservation
of semantic memory, as was proposed in an earlier case
study. (JINS, 1998, 4, 179–189.)
Benefit–cost analysis as a mechanism for evaluating conservation policies in developing countries
Authors: SHYAMSUNDAR PRIYA
Published: July 2002

Can benefit-cost analysis (BCA) be used in the developing world in the same way in which it is used in developed market economies? The paper by Arrow et al. makes a good case for employing BCA to evaluate environmental, health, and safety regulations in the United States. It offers a number of principles to guide the use of benefit-cost analyses, some of which can be applied to developing countries. Conservation policies in the tropics can help illustrate the relevance of BCA for evaluating regulations in less industrialized countries. I use a set of studies on the Mantadia National Park to show in practical terms how and when BCA can serve as an effective evaluative tool.
Recent and emerging work in music technology in Southeast Asia
Authors: Ang Minni K.
Published: July 2002

Music technology work in Southeast Asia faces challenges unique to this region. Economic, religious and cultural
factors hinder its progress. Despite these factors, pockets of development have occurred, most notably in Singapore
and Malaysia. Academic programmes have been introduced; strides have been made in research and development; and
commercially viable music technology products have been invented and marketed. Various centres in the region are
active in music technology-related work: the Music Department at Universiti Putra Malaysia, the Electronic Music
Lab at the National University of Singapore, and Creative Resource at the International Business Park in Singapore.
The First International Music Technology Conference in Southeast Asia held in Kuala Lumpur recently received
lukewarm response in terms of turnout and contributions. People from this region have produced a relatively small
number of music technology-related publications. Music technology work in Southeast Asia can thus be said to be in
its infancy with small but significant steps being taken to expand; however, for the region to become a major contributor
on the international level, the various hindrances identified will need to be addressed and overcome.
Some parentheses around algorithmic composition
Authors: BURT WARREN
Published: July 2002

Algorithmic composition and gambling, two activities associated (in the public mind) with the use of chance, are contrasted. Gambling is based on the concepts of winning and losing. Algorithmic composition is not, or should not be. Problems of mappings of information from one medium to another are considered, along with problems of reception for artworks made with these methods. In the end, the quality of attention given to an artwork may be more critical to its reception than any methods used to construct it. Mr. Yasser’s Piano, Tuning the Furniture of Chaos and Pi and the Square Root of Two, three recent algorithmic compositions by the author, are considered in the light of these thoughts.
The Challenge to Social Work
Authors: Hollows Anne
Published: July 2002

The Framework for the Assessment of Children in Need and their Families
signifies a major achievement in the development of a coherent approach
to promoting the welfare of children while safeguarding them from harm.
In this article, the aspirations of the Framework are considered, with
particular focus on the challenges posed by the Framework, not only to the
practice of social workers but also to the status of the social work
profession. The area of professional judgement making is discussed, along
with the threats to the effective implementation of the Framework. The
author concludes that the Framework could bring about not only a better
deal for children and families service users, but a new era of professional
respect for social workers.
Xenopus Sprouty2 inhibits FGF-mediated gastrulation movements but does not affect mesoderm induction and patterning
Authors: Nutt Stephen L. , Dingwell Kevin S. , Holt Christine E. , Amaya Enrique
Published: July 2002

Signal transduction through the FGF receptor is essential for the specification of the vertebrate body plan. Blocking the FGF pathway in early Xenopus embryos inhibits mesoderm induction and results in truncation of the anterior–posterior axis. The Drosophilagene sprouty encodes an antagonist of FGF signaling, which is transcriptionally induced by the pathway, but whose molecular functions are poorly characterized. We have cloned Xenopus sprouty2 and show that it is expressed in a similar pattern to known FGFs and is dependent on the FGF/Ras/MAPK pathway for its expression. Overexpression of Xsprouty2 in both embryos and explant assays results in the inhibition of the cell movements of convergent extension. Although blocking FGF/Ras/MAPK signaling leads to an inhibition of mesodermal gene expression, these markers are unaffected by Xsprouty2, indicating that mesoderm induction and patterning occurs normally in these embryos. Finally, using Xenopus oocytes we show that Xsprouty2 is an intracellular antagonist of FGF-dependent calcium signaling. These results provide evidence for at least two distinct FGF-dependent signal transduction pathways: a Sprouty-insensitive Ras/MAPK pathway required for the transcription of most mesodermal genes, and a Sprouty-sensitive pathway required for coordination of cellular morphogenesis.
Dual role for Drosophila epidermal growth factor receptor signaling in early wing disc development
Authors: Wang Shu-Huei , Simcox Amanda , Campbell Gerard
Published: July 2002

Cell fate decisions in the early Drosophila wing disc assign cells to compartments (anterior or posterior and dorsal or ventral) and distinguish the future wing from the body wall (notum). Here we show that EGF-receptor (EGFR) signaling stimulated by its ligand, Vein, has a fundamental role in regulating two of these cell fate choices: (1) Vn/EGFR signaling directs cells to become notum by antagonizing wing development and by activating notum-specifying genes; (2) Vn/EGFR signaling directs cells to become part of the dorsal compartment by induction of apterous, the dorsal selector gene, and consequently also controls wing development, which depends on an interaction between dorsal and ventral cells.
Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase
Authors: Tomonaga Takeshi , Nagao Koji , Kawasaki Yosuke , Furuya Kanji , Murakami Akiko , Morishita Jun , Yuasa Tatsuro , Sutani Takashi , Kearsey Stephen E. , Uhlmann Frank , Nasmyth Kim , Yanagida Mitsuhiro
Published: July 2002

Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase. The universal role of cohesin during anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromere-associated Rad21 did not change from metaphase to anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.
Maternal Environment and Genotype Interact to Establish Diabesity in Mice
Authors: Reifsnyder Peter C. , Churchill Gary , Leiter Edward H.
Published: July 2002

Obesity, a major risk factor for type II diabetes, is becoming more prevalent in Western populations consuming high calorie diets while expending less energy both at the workplace and at home. Most human obesity, and probably most type II diabetes as well, reflects polygenic rather than monogenic inheritance. We have genetically dissected a polygenic mouse model of obesity-driven type II diabetes by outcrossing the obese, diabetes-prone, NZO (New Zealand Obese)/HlLt strain to the relatively lean NON (Nonobese Nondiabetic)/Lt strain, and then reciprocally backcrossing obese F1 mice to the lean NON/Lt parental strain. A continuous distribution of body weights was observed in a population of 203 first backcross males. The 22% of first backcross males developing overt diabetes showed highest peripubertal weight gains and earliest development of hyperinsulinemia. We report a complex diabetes-predisposing (“diabesity”) QTL (Quantitative Trait Loci) on chromosome 1 contributing significant main effects to increases in body weight, plasma insulin, and plasma glucose. NZO contributed QTL with significant main effects on adiposity parameters on chromosomes 12 and 5. A NON QTL on chromosome 14 interacted epistatically with the NZO obesity QTL on chromosome 12 to increase adiposity. Although the main effect of the diabetogenic QTL on chromosome 1 was on rapid growth rather than adiposity, it interacted epistatically with the obesity QTL on chromosome 12 to increase plasma glucose levels. Additional complex epistatic interactions eliciting significant increases in body weight and/or plasma glucose were found between the NZO-contributed QTL on chromosome 1 and other NZO-contributed QTL on chromosomes 15 and 17, as well as with an NON-contributed QTL on chromosome 2. We further show that certain of these intergenic interactions are predicated on, or enhanced by, the maternal postparturitional environment. We show by cross-fostering experiments that the maternal environmental influence in part is because of the presence of early obesity-inducing factors in the milk of obese F1 dams. We also discuss a strategy for using recombinant congenic strains to separate and reassemble interacting QTL for future study.
The Large-Scale Organization of the Centromeric Region inBetaSpecies
Authors: Gindullis Frank , Desel Christine , Galasso Incoronata , Schmidt Thomas
Published: July 2002

In higher eukaryotes, the DNA composition of centromeres displays a high degree of variation, even between chromosomes of a single species. However, the long-range organization of centromeric DNA apparently follows similar structural rules. In our study, a comparative analysis of the DNA at centromeric regions ofBetaspecies, including cultivated and wild beets, was performed using a set of repetitive DNA sequences. Our results show that these regions inBetagenomes have a complex structure and consist of variable repetitive sequences, including satellite DNA, Ty3-gypsy-like retrotransposons, and microsatellites. Based on their molecular characterization and chromosomal distribution determined by fluorescent in situ hybridization (FISH), centromeric repeated DNA sequences were grouped into three classes. By high-resolution multicolor-FISH on pachytene chromosomes and extended DNA fibers we analyzed the long-range organization of centromeric DNA sequences, leading to a structural model of a centromeric region of the wild beet speciesBeta procumbens. The chromosomal mutants PRO1 and PAT2 contain a single wild beet minichromosome with centromere activity and provide, together with cloned centromeric DNA sequences, an experimental system toward the molecular isolation of individual plant centromeres. In particular, FISH to extended DNA fibers of the PRO1 minichromosome and pulsed-field gel electrophoresis of large restriction fragments enabled estimations of the array size, interspersion patterns, and higher order organization of these centromere-associated satellite families. Regarding the overall structure,Betacentromeric regions show similarities to their counterparts in the few animal and plant species in which centromeres have been analyzed in detail.

[The sequence data described in this paper have been submitted to the EMBL data library under accession nos.AJ278751,AJ278752, andAJ243337.
Isoproterenol Increases CREB Phosphorylation and Olfactory Nerve–Evoked Potentials in Normal and 5-HT-Depleted Olfactory Bulbs in Rat Pups Only at Doses That Produce Odor Preference Learning
Authors: Yuan Qi , Harley Carolyn W. , Bruce Jamie C. , Darby-King Andrea , McLean John H.
Published: July 2002

Norepinephrine (NE) and serotonin (5-HT) are important modulators of early odor preference learning. NE can act as an unconditioned stimulus (UCS), whereas 5-HT facilitates noradrenergic actions. In this study, we examined the phosphorylation of an important transcription factor, cAMP response element binding protein (CREB), which has been implicated in long-term-memory formation (McLean et al. 1999) during NE-induced odor preference learning in normal and olfactory bulb 5-HT-depleted rat pups. We also examined NE modulation of olfactory nerve–evoked field potentials (ON-EFPs) in anesthetized normal and bulbar 5-HT depleted pups. Systemic injection of 2 mg/kg isoproterenol (β-adrenoceptor agonist) induced odor preference learning, enhanced pCREB expression in the olfactory bulbs at 10 min after odor pairing, and increased ON-EFPs in normal rat pups but not in bulbar 5-HT-depleted rat pups. A dose of 6 mg/kg isoproterenol, which was ineffective in modulating these measures in normal rat pups, induced odor preference learning, enhanced phosphorylated CREB (pCREB) expression, and increased ON-EFPs in bulbar 5-HT-depleted pups. These outcomes suggest that NE and 5-HT promote specific biochemical and electrophysiological changes that may critically underlie odor preference learning.
Lesion of the Ventral Periaqueductal Gray Reduces Conditioned Fear but Does Not Change Freezing Induced by Stimulation of the Dorsal Periaqueductal Gray
Authors: Vianna Daniel M.L. , Graeff Frederico G. , Landeira-Fernandez Jesus , Brandão Marcus L.
Published: July 2002

Previously-reported evidence showed that freezing to a context previously associated with footshock is impaired by lesion of the ventral periaqueductal gray (vPAG). It has also been shown that stepwise increase in the intensity of the electrical stimulation of the dorsal periaqueductal gray (dPAG) produces alertness, then freezing, and finally escape. These aversive responses are mimicked by microinjections of GABA receptor antagonists, such as bicuculline, or blockers of the glutamic acid decarboxylase (GAD), such as semicarbazide, into the dPAG. In this work, we examined whether the expression of these defensive responses could be the result of activation of ventral portion of the periaqueductal gray. Sham- or vPAG electrolytic–lesioned rats were implanted with an electrode in the dPAG for the determination of the thresholds of freezing and escape responses. The vPAG electrolytic lesions were behaviorally verified through a context-conditioned fear paradigm. Results indicated that lesion of the vPAG disrupted conditioned freezing response to contextual cues associated with footshocks but did not change the dPAG electrical stimulation for freezing and escape responses. In a second experiment, lesion of the vPAG also did not change the amount of freezing and escape behavior produced by microinjections of semicarbazide into the dPAG. These findings indicate that freezing and escape defensive responses induced by dPAG stimulation do not depend on the integrity of the vPAG. A discussion on different neural circuitries that might underlie different inhibitory and active defensive behavioral patterns that animals display during threatening situations is presented.
Pharmacological Analysis of Nod Factor-Induced Calcium Spiking inMedicago truncatula. Evidence for the Requirement of Type IIA Calcium Pumps and Phosphoinositide Signaling
Authors: Engstrom Eric M. , Ehrhardt David W. , Mitra Raka M. , Long Sharon R.
Published: July 2002

Bacterial Nod factors trigger a number of cellular responses in root hairs of compatible legume hosts, which include periodic, transient increases in cytosolic calcium levels, termed calcium spiking. We screened 13 pharmaceutical modulators of eukaryotic signal transduction for effects on Nod factor-induced calcium spiking. The purpose of this screening was 2-fold: to implicate enzymes required for Nod factor-induced calcium spiking in Medicago sp., and to identify inhibitors of calcium spiking suitable for correlating calcium spiking to other Nod factor responses to begin to understand the function of calcium spiking in Nod factor signal transduction. 2-Aminoethoxydiphenylborate, caffeine, cyclopiazonic acid (CPA), 2,5-di-(t-butyl)-1,4-hydroquinone, and U-73122 inhibit Nod factor-induced calcium spiking. CPA and U-73122 are inhibitors of plant type IIA calcium pumps and phospholipase C, respectively, and implicate the requirement for these enzymes in Nod factor-induced calcium spiking. CPA and U-73122 inhibit Nod factor-induced calcium spiking robustly at concentrations with no apparent toxicity to root hairs, making CPA and U-73122 suitable for testing whether calcium spiking is causal to subsequent Nod factor responses.
Glucosylation Activity and Complex Formation of Two Classes of Reversibly Glycosylated Polypeptides
Authors: Langeveld Sandra M.J. , Vennik Marco , Kottenhagen Marijke , van Wijk Ringo , Buijk Ankie , Kijne Jan W. , de Pater Sylvia
Published: July 2002

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs. Sequence comparisons showed the existence of two classes of RGP proteins, designated RGP1 and RGP2. Glucosylation activity of RGP1 and RGP2 from wheat and rice was studied. After separate expression of Rgp1 andRgp2 in Escherichia coli or yeast (Saccharomyces cerevisiae), only RGP1 showed self-glucosylation. In Superose 12 fractions from wheat endosperm extract, a polypeptide with a molecular mass of about 40 kD is glucosylated by UDP-glucose. Transgenic tobacco (Nicotiana tabacum) plants, overexpressing either wheatRgp1 or Rgp2, were generated. Subsequent glucosylation assays revealed that in RGP1-containing tobacco extracts as well as in RGP2-containing tobacco extracts UDP-glucose is incorporated, indicating that an RGP2-containing complex is active. Gel filtration experiments with wheat endosperm extracts and extracts from transgenic tobacco plants, overexpressing either wheatRgp1 or Rgp2, showed the presence of RGP1 and RGP2 in high-molecular mass complexes. Yeast two-hybrid studies indicated that RGP1 and RGP2 form homo- and heterodimers. Screening of a cDNA library using the yeast two-hybrid system and purification of the complex by an antibody affinity column did not reveal the presence of other proteins in the RGP complexes. Taken together, these results suggest the presence of active RGP1 and RGP2 homo- and heteromultimers in wheat endosperm.
Physical Association of Starch Biosynthetic Enzymes with Starch Granules of Maize Endosperm (Granule-Associated Forms of Starch Synthase I and Starch Branching Enzyme II)
Authors: Mu-Forster C. , Huang R. , Powers J. R. , Harriman R. W. , Knight M. , Singletary G. W. , Keeling P. L. , Wasserman B. P.
Published: July 2002

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.
Purification and Characterization of Neutral and Alkaline Invertase from Carrot
Authors: Lee H. S. , Sturm A.
Published: July 2002

Neutral and alkaline invertase were identified in cells of a suspension culture of carrot (Daucus carota L.) and purified to electrophoretic homogeneity. Neutral invertase is an octamer with a molecular mass of 456 kD and subunits of 57 kD, whereas alkaline invertase is a tetramer with a molecular mass of 504 kD and subunits of 126 kD. Both enzymes had sharp pH profiles, with maximal activities at pH 6.8 for neutral invertase and pH 8.0 for alkaline invertase, and both hydrolyzed sucrose with typical hyperbolic kinetics and similar Km values of about 20 mM at pH 7.5. Neutral invertase also hydrolyzed raffinose and stachyose and, therefore, is a β-fructofuranosidase. In contrast, alkaline invertase was highly specific for sucrose. Fructose acted as a competitive inhibitor of both enzymes, with Ki values of about 15 mM. Glucose was a noncompetitive inhibitor of both neutral and alkaline invertase, with a Ki of about 30 mM. Neither enzyme was inhibited by HgCl2. Alkaline invertase was markedly inhibited by CaCl2, MgCl2, and MnCl2, and neutral invertase was not. In contrast to alkaline invertase, neutral invertase was inhibited by the nucleotides ATP, CTP, GTP, and UTP.
Purification, Characterization, and Structural Analysis of a Plant Low-Temperature-Induced Protein
Authors: Boothe J. G. , Sonnichsen F. D. , de Beus M. D. , Johnson-Flanagan A. M.
Published: July 2002

We have purified to near homogeneity a recombinant form of the protein BN28 (rBN28), expressed in response to low temperature in Brassica napus plants, and we have determined its solution structure. Antibodies raised against rBN28 were used to characterize the recombinant and native proteins. Similar to many other low-temperature-induced proteins, BN28 is extremely hydrophilic, such that it remains soluble following boiling. Immunoblot analysis of subcellular fractions indicated that BN28 was not strongly associated with cellular membranes and was localized exclusively within the soluble fraction of the cell. Contrary to predicted secondary structure that suggested significant helical content, circular dichroism analysis revealed that rBN28 existed in aqueous solution largely as a random coil. However, the helical propensity of the protein could be demonstrated in the presence of trifluoroethanol. Nuclear magnetic resonance analysis further showed that rBN28 was in fact completely unstructured (100% coil) in aqueous solution. Although it had earlier been speculated that BN28-like proteins from Arabidopsis thaliana might possess antifreeze protein activity (S. Kurkela and M. Franck [1990] Plant Mol Biol 15: 137–144), no such activity could be detected in ice recrystallization assays with rBN28.
Spaceflight Exposure Effects on Transcription, Activity, and Localization of Alcohol Dehydrogenase in the Roots of Arabidopsis thaliana
Authors: Porterfield D. M. , Matthews S. W. , Daugherty C. J. , Musgrave M. E.
Published: July 2002

Although considerable research and speculation have been directed toward understanding a plant's perception of gravity and the resulting gravitropic responses, little is known about the role of gravity-dependent physical processes in normal physiological function. These studies were conducted to determine whether the roots of plants exposed to spaceflight conditions may be experiencing hypoxia. Arabidopsis thaliana (L.) Heynh. plants were grown in agar medium during 6 or 11 d of spaceflight exposure on shuttle missions STS-54 (CHROMEX-03) and STS-68 (CHROMEX-05), respectively. The analysis included measurement of agar redox potential and root alcohol dehydrogenase (ADH) activity, localization, and expression. ADH activity increased by 89% as a result of spaceflight exposure for both CHROMEX-03 and -05 experiments, and ADH RNase protection assays revealed a 136% increase in ADH mRNA. The increase in ADH activity associated with the spaceflight roots was realized by a 28% decrease in oxygen availability in a ground-based study; however, no reduction in redox potential was observed in measurements of the spaceflight bulk agar. Spaceflight exposure appears to effect a hypoxic response in the roots of agar-grown plants that may be caused by changes in gravity-mediated fluid and/or gas behavior.
Floral Scent Production in Clarkia breweri (Onagraceae) (II. Localization and Developmental Modulation of the Enzyme S-Adenosyl-L-Methionine:(Iso)Eugenol O-Methyltransferase and Phenylpropanoid Emission)
Authors: Wang J. , Dudareva N. , Bhakta S. , Raguso R. A. , Pichersky E.
Published: July 2002

We have previously shown (R.A. Raguso, E. Pichersky [1995] Plant Syst Evol 194: 55–67) that the strong, sweet fragrance of Clarkia breweri (Onagraceae), an annual plant native to California, consists of 8 to 12 volatile compounds, including 4 phenylpropanoids. Although some C. breweri plants emit all 4 phenylpropanoids (eugenol, isoeugenol, methyleugenol, and isomethyleugenol), other C. breweri plants do not emit the latter 2 compounds. Here we report that petal tissue was responsible for the bulk of the phenylpropanoid emission. The activity of S-adenosyl-L-methionine: (iso)eugenol O-methyltransferase (IEMT), a novel enzyme that catalyzes the methylation of the para-4[prime]-hydroxyl of both eugenol and (iso)eugenol to methyleugenol and isomethyleugenol, respectively, was also highest in petal tissue. IEMT activity was absent from floral tissues of plants not emitting (iso)methyleugenol. A C. breweri cDNA clone encoding IEMT was isolated, and its sequence was shown to have 70% identity to S-adenosyl-L-methionine:caffeic acid O-methyltransferase. The protein encoded by this cDNA can use eugenol and isoeugenol as substrates, but not caffeic acid. Steady-state IEMT mRNA levels were positively correlated with levels of IEMT activity in the tissues, and no IEMT mRNA was observed in flowers that do not emit (iso)methyleugenol. Overall, the data show that the floral emission of (iso)methyleugenol is controlled at the site of emission, that a positive correlation exists between volatile emission and IEMT activity, and that control of the level of IEMT activity is exerted at a pretranslational step.
Regulation and Tissue-Specific Distribution of mRNAs for Three Extracellular Invertase Isoenzymes of Tomato Suggests an Important Function in Establishing and Maintaining Sink Metabolism
Authors: Godt D. E. , Roitsch T.
Published: July 2002

The aim of the present study was to gain insight into the contribution of extracellular invertases for sink metabolism in tomato (Lycopersicon esculentum L.). The present study shows that extracellular invertase isoenzymes are encoded by a gene family comprising four members: Lin5, Lin6, Lin7, and Lin8. The regulation of mRNA levels by internal and external signals and the distribution in sink and source tissues has been determined and compared with mRNA levels of the intracellular sucrose (Suc)-cleaving enzymes Suc synthase and vacuolar invertase. The specific regulation of Lin5, Lin6, and Lin7 suggests an important function of apoplastic cleavage of Suc by cell wall-bound invertase in establishing and maintaining sink metabolism. Lin6 is expressed under conditions that require a high carbohydrate supply. The corresponding mRNA shows a sink tissue-specific distribution and the concentration is elevated by stress-related stimuli, by the growth-promoting phytohormone zeatin, and in response to the induction of heterotrophic metabolism. The expression of Lin5 and Lin7 in gynoecia and stamens, respectively, suggests an important function in supplying carbohydrates to these flower organs, whereas the Lin7 mRNA was found to be present exclusively in this specific sink organ.
Expression of a Vacuolar Protein (VP24) in Anthocyanin-Producing Cells of Sweet Potato in Suspension Culture
Authors: Nozue M. , Yamada K. , Nakamura T. , Kubo H. , Kondo M. , Nishimura M.
Published: July 2002

VP24, an abundant protein of 24 kD, was found to accumulate in the anthocyanin-containing vacuoles of cells of sweet potato (lpomoea batatas) in suspension culture. Light-induced expression of VP24 was analyzed by immunoblotting in three different cell lines that produced anthocyanins at different rates. The expression of VP24 was closely correlated with the accumulation of anthocyanin in these cell lines. Immunocytochemical detection of VP24 with specific antibodies on thin sections showed that VP24 was localized in the intravacuolar pigmented globules (cyanoplasts) in the anthocyanin-containing vacuoles and not in the tonoplast. No VP24 immunogold labeling was detected in the vacuoles of the cell line that does not produce anthocyanin. We suggest that VP24 may be involved in the formation of the cyanoplast via an interaction with anthocyanin, and that it may play an important role in the trapping in vacuoles of large amounts of anthocyanins that have been transported into these vacuoles.
Alteration of Hormone Levels in Transgenic Tobacco Plants Overexpressing the Rice Homeobox Gene OSH1
Authors: Kusaba Shinnosuke , Kano-Murakami Yuriko , Matsuoka Makoto , Tamaoki Masanori , Sakamoto Tomoaki , Yamaguchi Isomaro , Fukumoto Masashi
Published: July 2002

The rice (Oryza sativaL.) homeobox gene OSH1 causes morphological alterations when ectopically expressed in transgenic rice, Arabidopsis thaliana, and tobacco (Nicotiana tabacum L.) and is therefore believed to function as a morphological regulator gene. To determine the relationship between OSH1 expression and morphological alterations, we analyzed the changes in hormone levels in transgenic tobacco plants exhibiting abnormal morphology. Levels of the plant hormones indole-3-acetic acid, abscisic acid, gibberellin (GA), and cytokinin (zeatin and trans-zeatin [Z]) were measured in leaves of OSH1-transformed and wild-type tobacco. Altered plant morphology was found to correlate with changes in hormone levels. The more severe the alteration in phenotype of transgenic tobacco, the greater were the changes in endogenous hormone levels. Overall, GA1 and GA4 levels decreased and abscisic acid levels increased compared with wild-type plants. Moreover, in the transformants, Z (active form of cytokinin) levels were higher and the ratio of Z to Z riboside (inactive form) also increased. When GA3 was supplied to the shoot apex of transformants, internode extension was restored and normal leaf morphology was also partially restored. However, such GA3-treated plants still exhibited some morphological abnormalities compared with wild-type plants. Based on these data, we propose the hypothesis that OSH1 affects plant hormone metabolism either directly or indirectly and thereby causes changes in plant development.
Induction of a Carbon-Starvation-Related Proteolysis in Whole Maize Plants Submitted to Light/Dark Cycles and to Extended Darkness1
Authors: Brouquisse Renaud , Gaudillère Jean-Pierre , Raymond Philippe
Published: July 2002

Three-week-old maize (Zea mays L.) plants were submitted to light/dark cycles and to prolonged darkness to investigate the occurrence of sugar-limitation effects in different parts of the whole plant. Soluble sugars fluctuated with light/dark cycles and dropped sharply during extended darkness. Significant decreases in protein level were observed after prolonged darkness in mature roots, root tips, and young leaves. Glutamine and asparagine (Asn) changed in opposite ways, with Asn increasing in the dark. After prolonged darkness the increase in Asn accounted for most of the nitrogen released by protein breakdown. Using polyclonal antibodies against a vacuolar root protease previously described (F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283–292) or the 20S proteasome, we showed that the increase in proteolytic activities was related to an enrichment of roots in the vacuolar protease, with no change in the amount of 20S proteasome in either roots or leaves. Our results show that no significant net proteolysis is induced in any part of the plant during normal light/dark cycles, although changes in metabolism and growth appear soon after the beginning of the dark period, and starvation-related proteolysis probably appears in prolonged darkness earlier in sink than in mature tissues.