bluuebunny/crossref_metadata_2025 huggingface dataset:
In search of mountain sites suitable for wind farms in the Mediterranean area using a diagnostic wind model
Authors: Gazzilli G , Palmieri S , Siani A M , Casale G R
Published: July 2002

A mesoscale diagnostic wind model, based on mass conservation, is utilised to develop a wind climatology for the southern Appennines in Italy, with the aim of identifying areas suitable for wind energy production plants. The model includes the kinematic effects of the high ground, anabatic and katabatic flows along mountain slopes as well as blocking effects. The climatological analysis carried out appears quite effective in selecting favourable areas where a more detailed investigation based on boundary layer models and observational field studies may lead to the final specification of locations suitable for mountain wind farms. Copyright © 2001 Royal Meteorological Society
Use of dynamical concepts in weather forecasting
Authors: Carroll E B
Published: July 2002

A divergence‐based procedure for diagnosing development, based on a two‐layer model of the atmosphere, is discussed. It is pointed out that thinking in terms of vorticity advection and thermal advection is directly equivalent to considering divergence due to advective and isallobaric components of the ageostrophic wind. It is shown that such an approach is similar in basis to the quasi‐geostrophic omega equation, but it is argued that it is more suitable for subjective application. However, simply estimating vertical velocity or instantaneous pressure tendency from synoptic charts is of questionable accuracy and little operational use, especially since such diagnostics are much more reliably obtained from NWP models. It is necessary to think in terms of the development and movement of features in the flow of the upper as well as the lower troposphere, and their vertical interactions. Copyright © 1997 Royal Meteorological Society
On studying the structure of synoptic systems
Authors: Davies Huw C , Wernli Heini
Published: July 2002

There are several extant techniques for diagnosing the dynamics and structure of synoptic‐scale flow. The essence of three of these approaches (the Q‐field diagnosis, Lagrangian airstreams and the PV‐perspective) is outlined, and their relative characteristics illustrated by examining two synoptic cases. It is shown that the three approaches can independently reveal key features of the events, and that the views they offer are complementary. Copyright © 2004 Royal Meteorological Society
Conceptual apraxia and semantic memory deficit in Alzheimer's
disease: Two sides of the same coin?
Authors: DUMONT CATHERINE , SKA BERNADETTE , JOANETTE YVES
Published: July 2002

This study was designed to examine the patterns of
apraxic disturbances and the relationships between action
knowledge and other measures of semantic knowledge about
objects in 10 well-characterized Alzheimer's disease
(AD) patients. Five tasks were used to assess components
of action knowledge (action–tool relationships, pantomime
recognition, and sequential organization of action) and
praxis execution (actual use, pantomiming) according to
the cognitive model of praxis. Three tasks (verbal comprehension,
naming, and a visual semantic matching task) were used
to assess verbal–visual semantics. Considering patterns
of apraxia first, conceptual apraxia was found in 9 out
of the 10 AD patients, suggesting that it is a common feature
even in the early stages of AD. Second, we found partly
parallel deficits in tests of action-semantic and verbal–visual
semantic knowledge in 9 AD patients. Impaired action knowledge
was found only in patients with a semantic language deficit.
These findings provide no evidence that “action semantics”
may be separated from other semantic information. Our results
support the view of a unitary semantic system, given that
the representations of action-semantic and other semantic
knowledge of objects are often simultaneously disrupted
in AD. (JINS, 2000, 6, 693–703.)
The Health of the Nation Outcome Scales for Child & Adolescent Mental Health (HoNOSCA)
Authors: Gowers Simon , Bailey-Rogers Sarah J. , Shore Alison , Levine Warren
Published: July 2002

The child and adolescent version of the Health of the Nation Outcome
Scales (HoNOSCA) represents the first attempt at a routine outcome
measure for Child and Adolescent Mental Health Services in the U.K.
Extensive field trials suggested that the scales were both acceptable to
clinicians from the various disciplines working in this area and also valid
and reliable. A growing number of services are now using the scales in
audit and research, supported by the national HoNOSCA base that
provides training and co-ordinates further developments.
The Frequency of Emotional and Behavioural Disturbance in an EBD School
Authors: Place Maurice , Wilson Jo , Martin Elaine , Hulsmeier Jessica
Published: July 2002

The education of children with behavioural difficulties is under scrutiny
from various quarters. The provision of specialised school environments is
being questioned, at the same time as there is pressure to minimise the
exclusion of disruptive pupils. This study looked at the rate of psychiatric
disorder that was prevalent in a school for emotionally and behaviourally
disordered (EBD) pupils, and found, using DSM III-R criteria, that 24% of
the sample had depression, 11% had overanxiousness, and 70% had a
diagnosis of Attention Deficit Disorder with Hyperactivity (ADHD).
Educational attainment assessment showed that 75% had a reading age
that was at least 2 years below their chronological age. These results
suggest that such settings are dealing with a very disturbed group of
youngsters.
The Role of a Psychologist on the Paediatric Intensive Care Unit
Authors: Colville Gillian
Published: July 2002

This paper highlights the many complex psychological needs of paediatric patients in intensive care. The literature on the experience of children in intensive care and their relatives is reviewed and related to the work of a paediatric psychologist. Case examples are given of direct work with children and suggestions are made of ways in which the psychologist can work in a consultation/liaison capacity. The personal impact of this work is addressed, as is the need to disseminate information to other health professionals. Further research is called for, both on the child's experience and on the development of appropriate psychological interventions.
China's First Direct Election of the Township Head: A Case Study of Buyun
Authors: He Baogang , Lang Youxing
Published: July 2002

This paper is a detailed case study of China's first direct election of the township heads, examining the driving forces for and obstacles to direct township election, and Chinese utilitarian approach toward local democracy. The paper discusses the prospect of direct township election in China and highlights the Chinese paternalist model of democracy being implemented in practice.
The eyeless homeodomain is dispensable for eye development in Drosophila
Authors: Punzo Claudio , Kurata Shoichiro , Gehring Walter J.
Published: July 2002

Pax-6 genes, known to be essential for eye development, encode an evolutionarily conserved transcription factor with two DNA-binding domains. To corroborate the contribution of each DNA-binding domain to eye formation, we generated truncated forms of the Drosophila Pax-6 gene eyeless and tested their capacity to rescue the ey2 mutant. Surprisingly, EY deleted of the homeodomain rescued the ey2 mutant and triggered ectopic eyes morphogenesis. In contrast, EY lacking the paired domain failed to rescue the ey2 mutant, led to truncation of appendages, and repressed Distal-less when misexpressed. This result suggests distinct functions mediated differentially by the two DNA-binding domains of eyeless.
Cdc2–cyclin B kinase activity links Crb2 and Rqh1–topoisomerase III
Authors: Caspari Thomas , Murray Johanne M. , Carr Antony M.
Published: July 2002

The availability of a sister chromatid, and thus the cell cycle phase in which DNA double-strand breaks (DSBs) occur, influences the choice between homologous recombination (HR) or nonhomologous end joining (NHEJ). The sequential activation and destruction of CDK–cyclin activities controls progression through the cell cycle. Here we provide evidence that the major Schizosaccharomyces pombe CDK, Cdc2–cyclin B, influences recombinational repair of radiation-induced DSBs during the G2 phase at two distinct stages. At an early stage in HR, a defect in Cdc2 kinase activity, which is caused by a single amino acid change in cyclin B, affects the formation of Rhp51 (Rad51sp) foci in response to ionizing radiation in a process that is redundant with the function of Rad50. At a late stage in HR, low Cdc2–cyclin B activity prevents the proper regulation of topoisomerase III (Top3) function, disrupting a recombination step that occurs after the assembly of Rhp51 foci. This effect of Cdc2–cyclin B kinase on Top3 function is mediated by the BRCT-domain-containing checkpoint protein Crb2, thus linking checkpoint proteins directly with recombinational repair in G2. Our data suggest a model in which CDK activity links processing of recombination intermediates to cell cycle progression via checkpoint proteins.
Yak1p, a DYRK family kinase, translocates to the nucleus and phosphorylates yeast Pop2p in response to a glucose signal
Authors: Moriya Hisao , Shimizu-Yoshida Yuki , Omori Akira , Iwashita Shintaro , Katoh Mariko , Sakai Akira
Published: July 2002

POP2 protein of Saccharomyces cerevisiae is a component of a protein complex that regulates the transcription of many genes. We found that the 97th threonine residue (Thr 97) of Pop2p was phosphorylated upon glucose limitation. The Thr 97 phosphorylation occurred within 2 min after removing glucose and was reversed within 1 min after the readdition of glucose. The effects of hexokinase mutations and glucose analogs indicate that this phosphorylation is dependent on glucose phosphorylating activity. We purified a protein kinase that phosphorylates a peptide containing Thr 97 of Pop2p and identified it as Yak1p, a DYRK family kinase. Phosphorylation of Pop2p was barely detectable in a yak1Δ strain. We found that Yak1p interacted with Bmh1p and Bmh2p only in the presence of glucose. A GFP-Yak1p fusion protein shuttled rapidly between the nucleus and the cytoplasm in response to glucose. A strain with alanine substituted for Thr 97 in Pop2p showed overgrowth in the postdiauxic transition and failed to stop the cell cycle at G1 phase in response to glucose deprivation. Thus, Yak1p and Pop2p are part of a novel glucose-sensing system in yeast that is involved in growth control in response to glucose availability.
Suppression of cap-dependent translation in mitosis
Authors: Pyronnet Stéphane , Dostie Josée , Sonenberg Nahum
Published: July 2002

Cap-dependent translation is mediated by eIF4F, a protein complex composed of three subunits as follows: eIF4E, which recognizes the mRNA 5′ cap structure; eIF4A, an RNA-helicase; and eIF4G, a scaffolding protein that binds eIF4E, eIF4A, and the eIF4E-kinase Mnk1 simultaneously. eIF4E is hypophosphorylated and cap-dependent translation is reduced at mitosis. Here, we show that 4E-BP1, a suppressor of eIF4E function, is also hypophosphorylated in mitosis, resulting in disruption of the eIF4F complex. Consequently, eIF4E is sequestered from the eIF4G/Mnk1 complex. These results explain the specific inhibition of cap-dependent translation in mitosis and also explain how eIF4E is rendered hypophosphorylated during mitosis. Furthermore, eIF4E interaction with eIF4GII is strongly decreased coincident with hyperphosphorylation of eIF4GII. Thus, inhibition of cap-dependent translation in mitosis results from a combination of phosphorylation modifications leading to eIF4F complex disruption.
A molecular link between gene-specific and chromosome-wide transcriptional repression
Authors: Chu Diana S. , Dawes Heather E. , Lieb Jason D. , Chan Raymond C. , Kuo Annie F. , Meyer Barbara J.
Published: July 2002

Gene-specific and chromosome-wide mechanisms of transcriptional regulation control development in multicellular organisms. SDC-2, the determinant of hermaphrodite fate in Caenorhabditis elegans, is a paradigm for both modes of regulation. SDC-2 represses transcription of X chromosomes to achieve dosage compensation, and it also represses the male sex-determination gene her-1 to elicit hermaphrodite differentiation. We show here that SDC-2 recruits the entire dosage compensation complex to her-1, directing thisX-chromosome repression machinery to silence an individual, autosomal gene. Functional dissection of her-1 in vivo revealed DNA recognition elements required for SDC-2 binding, recruitment of the dosage compensation complex, and transcriptional repression. Elements within her-1 differed in location, sequence, and strength of repression, implying that the dosage compensation complex may regulate transcription along the X chromosome using diverse recognition elements that play distinct roles in repression.
The Axin-like protein PRY-1 is a negative regulator of a canonical Wnt pathway in C. elegans
Authors: Korswagen Hendrik C. , Coudreuse Damien Y.M. , Betist Marco C. , van de Water Sandra , Zivkovic Danica , Clevers Hans C.
Published: July 2002

Axin, APC, and the kinase GSK3β are part of a destruction complex that regulates the stability of the Wnt pathway effector β-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3β, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. We have shown previously that the EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/β-catenin and POP-1/Tcf. Here, we have investigated how BAR-1 is regulated by the EGL-20 pathway. First, we have characterized a negative regulator of the EGL-20 pathway,pry-1. We show that pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. Our results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. We show that PRY-1 interacts with BAR-1, SGG-1, and APR-1 and that overexpression of PRY-1 inhibitsmab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that it can functionally interact with vertebrate destruction complex components. Finally, we show that SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. We conclude that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/β-catenin signaling in C. elegans.
Ecologic Genomics of DNA: Upstream Bending in Prokaryotic Promoters
Authors: Bolshoy Alexander , Nevo Eviatar
Published: July 2002

After our analysis of the distribution of predicted intrinsic curvature along all available complete prokaryotic genomes, the genomes were divided into two groups. Curvature distribution in all prokaryotes of the first group indicated a substantial fraction of promoters characterized by intrinsic DNA curvature located within or upstream of the promoter region. We did not find this peculiar DNA curvature distribution in prokaryotes in the second group. Remarkably, all bacteria of the first group were mesophilic, whereas many prokaryotes of the second group were hyperthermophilic. We hypothesize that DNA curvature plays a biologic role in gene regulation in mesophilic as opposed to hyperthermophilic prokaryotes, i.e., DNA curvature presumably has a functional adaptive significance determined by temperature selection.
Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing
Authors: Alderborn Anders , Kristofferson Anna , Hammerling Ulf
Published: July 2002

The characterization of naturally occurring variations in the human genome has evoked an immense interest during recent years. Variations known as biallelic Single-Nucleotide Polymorphisms (SNPs) have become increasingly popular markers in molecular genetics because of their wide application both in evolutionary relationship studies and in the identification of susceptibility to common diseases. We have addressed the issue of SNP genotype determination by investigating variations within the Renin–Angiotensin–Aldosterone System (RAAS) using pyrosequencing, a real-time pyrophosphate detection technology. The method is based on indirect luminometric quantification of the pyrophosphate that is released as a result of nucleotide incorporation onto an amplified template. The technical platform employed comprises a highly automated sequencing instrument that allows the analysis of 96 samples within 10 to 20 minutes. In addition to each studied polymorphic position, 5–10 downstream bases were sequenced for acquisition of reference signals. Evaluation of pyrogram data was accomplished by comparison of peak heights, which are proportional to the number of incorporated nucleotides. Analysis of the pyrograms that resulted from alternate allelic configurations for each addressed SNP revealed a highly discriminating pattern. Homozygous samples produced clear-cut single base peaks in the expected position, whereas heterozygous counterparts were characterized by distinct half-height peaks representing both allelic positions. Whenever any of the allelic bases of an SNP formed a homopolymer with adjacent bases, the nonallelic signal was added to those of the SNP. This feature did not, however, influence SNP readability. Furthermore, the multibase reading capacity of the described system provides extensive flexibility in regard to the positioning of sequencing primers and allows the determination of several closely located SNPs in a single run.
Automation for Genomics, Part One: Preparation for Sequencing
Authors: Meldrum Deirdre
Published: July 2002

In the past four years, automation for genomics has enabled a 43-fold increase in the total finished human genomic sequence in the world. This two-part noncomprehensive review will provide an overview of different types of automation equipment used in genome sequencing. Part One focuses on equipment involved in DNA preparation, DNA sequencing reactions, and other automated procedures for preparing DNA for running on sequencers or subsequent analysis; it also includes information on the development of these machines at various genome centers. Part Two, to be published in the next issue, will cover sequencing machinery and array technology, and conclude with a look at the future technologies that will revolutionize molecular biology. “Alternate” sequencing technologies (including mass spectrometry, biochips, and single-molecule analysis) will also be examined.
Gene Structure Prediction and Alternative Splicing Analysis Using Genomically Aligned ESTs
Authors: Kan Zhengyan , Rouchka Eric C. , Gish Warren R. , States David J.
Published: July 2002

With the availability of a nearly complete sequence of the human genome, aligning expressed sequence tags (EST) to the genomic sequence has become a practical and powerful strategy for gene prediction. Elucidating gene structure is a complex problem requiring the identification of splice junctions, gene boundaries, and alternative splicing variants. We have developed a software tool, Transcript Assembly Program (TAP), to delineate gene structures using genomically aligned EST sequences. TAP assembles the joint gene structure of the entire genomic region from individual splice junction pairs, using a novel algorithm that uses the EST-encoded connectivity and redundancy information to sort out the complex alternative splicing patterns. A method called polyadenylation site scan (PASS) has been developed to detect poly-A sites in the genome.TAP uses these predictions to identify gene boundaries by segmenting the joint gene structure at polyadenylated terminal exons. Reconstructing 1007 known transcripts, TAP scored a sensitivity (Sn) of 60% and a specificity (Sp) of 92% at the exon level. The gene boundary identification process was found to be accurate 78% of the time. TAP also reports alternative splicing patterns in EST alignments. An analysis of alternative splicing in 1124 genic regions suggested that more than half of human genes undergo alternative splicing. Surprisingly, we saw an absolute majority of the detected alternative splicing events affect the coding region. Furthermore, the evolutionary conservation of alternative splicing between human and mouse was analyzed using an EST-based approach. (See http://stl.wustl.edu/∼zkan/TAP/)
Genome-Wide Epistatic Interaction Analysis Reveals Complex Genetic Determinants of Circadian Behavior in Mice
Authors: Shimomura Kazuhiro , Low-Zeddies Sharon S. , King David P. , Steeves Thomas D.L. , Whiteley Andrew , Kushla Jani , Zemenides Peter D. , Lin Andrew , Vitaterna Martha Hotz , Churchill Gary A. , Takahashi Joseph S.
Published: July 2002

Genetic heterogeneity underlies many phenotypic variations observed in circadian rhythmicity. Continuous distributions in measures of circadian behavior observed among multiple inbred strains of mice suggest that the inherent contributions to variability are polygenic in nature. To identify genetic loci that underlie this complex behavior, we have carried out a genome-wide complex trait analysis in 196 (C57BL/6J X BALB/cJ)F2 hybrid mice. We have characterized variation in this panel of F2 mice among five circadian phenotypes: free-running circadian period, phase angle of entrainment, amplitude of the circadian rhythm, circadian activity level, and dissociation of rhythmicity. Our genetic analyses of these phenotypes have led to the identification of 14 loci having significant effects on this behavior, including significant main effect loci that contribute to three of these phenotypic measures: period, phase, and amplitude. We describe an additional locus detection method, genome-wide genetic interaction analysis, developed to identify locus pairs that may interact epistatically to significantly affect phenotype. Using this analysis, we identified two additional pairs of loci that have significant effects on dissociation and activity level; we also detected interaction effects in loci contributing to differences of period, phase, and amplitude. Although single gene mutations can affect circadian rhythms, the analysis of interstrain variants demonstrates that significant genetic complexity underlies this behavior. Importantly, most of the loci that we have detected by these methods map to locations that differ from the nine known clock genes, indicating the presence of additional clock-relevant genes in the mammalian circadian system. These data demonstrate the analytical value of both genome-wide complex trait and epistatic interaction analyses in further understanding complex phenotypes, and point to promising approaches for genetic analysis of such phenotypes in other mammals, including humans.
A Fine Physical Map of the Rice Chromosome 4
Authors: Zhao Qiang , Zhang Yu , Cheng Zhukuan , Chen Mingsheng , Wang Shengyue , Feng Qi , Huang Yucheng , Li Ying , Tang Yesheng , Zhou Bo , Chen Zhehua , Yu Shuliang , Zhu Jingjie , Hu Xin , Mu Jie , Ying Kai , Hao Pei , Zhang Lei , Lu Yiqi , Zhang Lei S. , Liu Yilei , Yu Zhen , Fan Danlin , Weng Qijun , Chen Ling , Lu Tingting , Liu Xiaohui , Jia Peixin , Sun Tongguo , Wu Yongrui , Zhang Yujun , Lu Ying , Li Can , Wang Rong , Lei Haiyan , Li Tao , Hu Hao , Wu Mei , Zhang Runquan , Guan Jianping , Zhu Jia , Fu Gang , Gu Minghong , Hong Guofan , Xue Yongbiao , Wing Rod , Jiang Jiming , Han Bin
Published: July 2002

As part of an international effort to completely sequence the rice genome, we have produced a fine bacterial artificial chromosome (BAC)-based physical map of the Oryza sativa japonicaNipponbare chromosome 4 through an integration of 114 sequenced BAC clones from a taxonomically related subspecies O. sativa indica Guangluai 4 and 182 RFLP and 407 expressed sequence tag (EST) markers with the fingerprinted data of the Nipponbare genome. The map consists of 11 contigs with a total length of 34.5 Mb covering 94% of the estimated chromosome size (36.8 Mb). BAC clones corresponding to telomeres, as well as to the centromere position, were determined by BAC-pachytene chromosome fluorescence in situ hybridization (FISH). This gave rise to an estimated length ratio of 5.13 for the long arm and 2.9 for the short arm (on the basis of the physical map), which indicates that the short arm is a highly condensed one. The FISH analysis and physical mapping also showed that the short arm and the pericentromeric region of the long arm are rich in heterochromatin, which occupied 45% of the chromosome, indicating that this chromosome is likely very difficult to sequence. To our knowledge, this map provides the first example of a rapid and reliable physical mapping on the basis of the integration of the data from two taxonomically related subspecies.

[The following individuals and institutions kindly provided reagents, samples, or unpublished information as indicated in the paper: S. McCouch, T. Sasaki, and Monsanto.]
Regulation of Distinct Attractive and Aversive Mechanisms Mediating Benzaldehyde Chemotaxis inCaenorhabditis elegans
Authors: Nuttley William M. , Harbinder Singh , van der Kooy Derek
Published: July 2002

Olfactory-mediated chemotaxis in nematodes provides a relatively simple system to study biological mechanisms of information processing. Analysis of the kinetics of chemotaxis in response to 100% benzaldehyde revealed an initial attractive response that is followed by a strong aversion to the odorant. We show that this behavior is mediated by two genetically separable attraction- and aversion-mediating response pathways. The attraction initially dominates behavior but with prolonged exposure habituation leads to a behavioral change, such that the odorant becomes repulsive. This olfactory habituation is susceptible to dishabituation, thereby re-establishing the attractive response to the odorant. Re-examination of the putative olfactory adaptation mutantadp-1(ky20)revealed that the phenotype observed in this line is due to a supersensitivity to a dishabituating stimulus, rather than a defect in the adaptation to odorants per se. A modified benzaldehyde chemotaxis assay was developed and used for the isolation of a mutant with a specific defect in habituation kinetics, expressed as a persistence of the attractive response.
Overexpression of and RNA Interference with the CCAAT Enhancer-Binding Protein on Long-Term Facilitation ofAplysiaSensory to Motor Synapses
Authors: Lee Jin-A , Kim Hyong-Kyu , Kim Kyung-Hee , Han Jin-Hee , Lee Yong-Seok , Lim Chae-Seok , Chang Deok-Jin , Kubo Tai , Kaang Bong-Kiun
Published: July 2002

In the marine molluskAplysia, the CCAAT/enhancer-binding protein, ApC/EBP, serves as an immediate early gene in the consolidation of long-term facilitation in the synaptic connection between the sensory and motor neurons of the gill-withdrawal reflex. To further examine the role of ApC/EBP as a molecular switch of a stable form of long-term memory, we cloned the full-length coding regions of two alternatively spliced forms, the short and long form of ApC/EBP. Overexpression of each isoform by DNA microinjection resulted in a l6-fold increase in the expression of the coinjected luciferase reporter gene driven by an ERE promoter. In addition, when we overexpressed ApC/EBP inAplysiasensory neurons, we found that the application of a single pulse of 5-HT that normally induced only short-term facilitation now induced long-term facilitation. Conversely, when we attempted to block the synthesis of native ApC/EBP by microinjecting double-strand RNA or antisense RNA, we blocked long-term facilitation in a sequence-specific manner. These data support the idea that ApC/EBP is both necessary and sufficient to consolidate short-term memory into long-term memory. Furthermore, our results suggest that this double-strand RNA interference provides a powerful tool in the study of the genes functioning in learning and memory inAplysiaby specifically inhibiting both the constitutive and induced expression of the genes.
A Necessity for MAP Kinase Activation in Mammalian Spatial Learning
Authors: Selcher Joel C. , Atkins Coleen M. , Trzaskos James M. , Paylor Richard , Sweatt J. David
Published: July 2002

Although the biochemical mechanisms underlying learning and memory have not yet been fully elucidated, mounting evidence suggests that activation of protein kinases and phosphorylation of their downstream effectors plays a major role. Recent findings in our laboratory have shown a requirement for the mitogen-activated protein kinase (MAPK) cascade in hippocampal synaptic plasticity. Therefore, we used an inhibitor of MAPK activation, SL327, to test the role of the MAPK cascade in hippocampus-dependent learning in mice. SL327, which crosses the blood-brain barrier, was administered intraperitoneally at several concentrations to animals prior to cue and contextual fear conditioning. Administration of SL327 completely blocked contextual fear conditioning and significantly attenuated cue learning when measured 24 hr after training. To determine whether MAPK activation is required for spatial learning, we administered SL327 to mice prior to training in the Morris water maze. Animals treated with SL327 exhibited significant attenuation of water maze learning; they took significantly longer to find a hidden platform compared with vehicle-treated controls and also failed to use a selective search strategy during subsequent probe trials in which the platform was removed. These impairments cannot be attributed to nonspecific effects of the drug during the training phase; no deficit was seen in the visible platform task, and injection of SL327 following training produced no effect on the performance of these mice in the hidden platform task. These findings indicate that the MAPK cascade is required for spatial and contextual learning in mice.
Genetic Manipulation of the Metabolism of Polyamines in Poplar Cells. The Regulation of Putrescine Catabolism
Authors: Bhatnagar Pratiksha , Minocha Rakesh , Minocha Subhash C.
Published: July 2002

We investigated the catabolism of putrescine (Put) in a non-transgenic (NT) and a transgenic cell line of poplar (Populus nigra × maximowiczii) expressing a mouse (Mus musculus) ornithine (Orn) decarboxylase (odc) cDNA. The transgenic cells produce 3- to 4-fold higher amounts of Put than the NT cells. The rate of loss of Put from the cells and the initial half-life of cellular Put were determined by feeding the cells with [U-14C]Orn and [1,4-14C]Put as precursors and following the loss of [14C]Put in the cells at various times after transfer to label-free medium. The amount of Put converted into spermidine as well as the loss of Put per gram fresh weight were significantly higher in the transgenic cells than the NT cells. The initial half-life of exogenously supplied [14C]Put was not significantly different in the two cell lines. The activity of diamine oxidase, the major enzyme involved in Put catabolism, was comparable in the two cell lines even though the Put content of the transgenic cells was severalfold higher than the NT cells. It is concluded that in poplar cells: (a) exogenously supplied Orn enters the cells and is rapidly converted into Put, (b) the rate of Put catabolism is proportional to the rate of its biosynthesis, and (c) the increased Put degradation occurs without significant changes in the activity of diamine oxidase.
Subcellular Localization of Spinach Cysteine Synthase Isoforms and Regulation of Their Gene Expression by Nitrogen and Sulfur
Authors: Takahashi H. , Saito K.
Published: July 2002

Subcellular localization and regulation of the spinach (Spinacia oleracea) cysteine synthase (O-acetyl-L-serine[thiol]-lyase, EC 4.2.99.8) isoforms (CysA, CysB, and CysC) were determined in transgenic tobacco (Nicotiana tabacum) and in spinach cell cultures. The 5[prime] regions of CysB and CysC encoding the chloroplastic (CysB-TP) and the putative mitochondrial (CysC-TP) transit peptide (TP) sequences were fused to a bacterial β-glucuronidase gene (gus) and expressed in tobacco under the control of the cauliflower mosaic virus 35S promoter. Subcellular fractionation of transgenic tobacco showed transportation of β-glucuronidase proteins to chloroplasts by CysB-TP and to mitochondria by CysC-TP, respectively, indicating that both presequences were sufficient to act specifically as chloroplastic and mitochondrial TPs in vivo. The mRNA expression patterns of CysA (cytoplasmic form), CysB, and CysC genes under nitrogen- and sulfur-starved conditions were characterized in spinach cells cultures. In sulfur-starved cells, only slight differences (approximately 1.2- to 1.5-fold) in the mRNA levels of CysA and CysB were observed during the short-term (0–24 h) cultivation periods compared with cells growth in Murashige-Skoog medium. However, under nitrogen and nitrogen/sulfur double-deficient stress conditions, mRNA levels of CysC increased up to 500% of the original level within 72 h.
Mechanism of Proton Permeation through Chloroplast Lipid Membranes
Authors: Fuks B. , Homble F.
Published: July 2002

Electrical measurements were carried out to investigate the contribution of chloroplast lipids to the passive proton permeability of both the thylakoid and inner-envelope membranes. Permeability coefficient and conductance to protons were measured for solvent-free bilayers made from monogalactosyldiglyceride:digalactosyldiglycerid:sulfoquinovosyldiglyceride :phosphatidylglycer ol (2:1:0.5:0.5, w/w) in the presence of a pH gradient of 7.4/8.1. The permeability coefficient for protons in glycolipids was 5.5 [plus or minus] 1.1 x 10–4 cm s-1 (n = 14). To determine whether this high H+ permeability could be explained by the presence of lipid contaminants such as weak acids, we investigated the effects of (a) bovine serum albumin, which can remove some amphiphilic molecules such as free fatty acids, (b) 6-ketocholestanol, which increases the membrane dipole potential, (c) oleic acid, and (d) chlorodecane, which increases the dielectric constant of the lipid bilayer. Our results show that free fatty acids are inefficient protonophores, as compared with carbonylcyanide-m-chlorphenylhydrazone, and that the hypothesis of a weak acid mechanism is not valid with glycolipid bilayers. In the presence of deuterium oxide the H+ conductance was reduced significantly, indicating that proton transport through the glycolipid matrix could occur directly by a hydrogen bond process. The passive transport of H+ through the glycolipid matrix is discussed with regard to the activity of the thylakoid ATP synthase and the inner-envelope H+-ATPase.
Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Plants Overproducing Fe-Superoxide Dismutase in Chloroplasts
Authors: Van Camp W. , Capiau K. , Van Montagu M. , Inze D. , Slooten L.
Published: July 2002

A chimeric gene consisting of the coding sequence for chloroplastic Fe superoxide dismutase (FeSOD) from Arabidopsis thaliana, coupled to the chloroplast targeting sequence from the pea ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, was expressed in Nicotiana tabacum cv Petit Havana SR1. Expression of the transgenic FeSOD protected both the plasmalemma and photosystem II against superoxide generated during illumination of leaf discs impregnated with methyl viologen. By contrast, overproduction of a mitochondrial MnSOD from Nicotiana plumbaginifolia in the chloroplasts of cv SR1 protected only the plasmalemma, but not photosystem II, against methyl viologen (L. Slooten, K. Capiau, W. Van Camp, M. Van Montagu, C. Sybesma, D. Inze [1995] Plant Physiol 107: 737–750). The difference in effectiveness correlates with different membrane affinities of the transgenic FeSOD and MnSOD. Overproduction of FeSOD does not confer tolerance to H2O2, singlet oxygen, chilling-induced photoinhibition in leaf disc assays, or to salt stress at the whole plant level. In nontransgenic plants, salt stress led to a 2- to 3-fold increase in activity, on a protein basis, of FeSOD, cytosolic and chloroplastic Cu/ZnSOD, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. In FeSOD-overproducing plants under salt stress, the induction of cytosolic and chloroplastic Cu/ZnSOD was suppressed, whereas induction of a water-soluble chloroplastic ascorbate peroxidase isozyme was promoted.
Changes in the Activity and mRNA of Cinnamyl Alcohol Dehydrogenase during Tracheary Element Differentiation in Zinnia
Authors: Sato Y. , Watanabe T. , Komamine A. , Hibino T. , Shibata D. , Sugiyama M. , Fukuda H.
Published: July 2002

Changes in the enzymatic activity of cinnamyl alcohol dehydrogenase (CAD) and in the expression of a gene for CAD during tracheary element (TE) differentiation were investigated in cultures of single cells isolated from the mesophyll of zinnia (Zinnia elegans). In cultures in which TE differentiation was induced (TE-inductive cultures), CAD activity increased from h 36 after the start of culture (12 h before the start of thickening of the secondary cell wall) and peaked at h 72, when lignin was actively being deposited. In control cultures in which TE differentiation was not induced, CAD activity remained at a very low level for 5 d. Some isoforms of CAD were detected only in the TE-inductive cultures by native gel electrophoresis and subsequent staining for CAD activity. A cDNA clone for CAD, ZCAD1, was isolated from Z. elegans using a cDNA clone for CAD from Aralia cordata as the probe. RNA gel-blot analysis revealed that in the TE-inductive cultures the level of ZCAD1 mRNA increased from h 36 and peaked at h 48 to 60. No such increases were observed in control cultures. These results indicated that both the gene expression and the activity of CAD are strictly regulated, in association with lignification, during TE differentiation in Z. elegans.
Soybean Root Nodule Acid Phosphatase
Authors: Penheiter A. R. , Duff SMG. , Sarath G.
Published: July 2002

Acid phosphatases are ubiquitous enzymes that exhibit activity against a variety of substrates in vitro, although little is known about their intracellular function. In this study we report the isolation, characterization, and partial sequence of the major acid phosphatase from soybean (Glycine max L.) root nodules. The phosphatase was purified predominantly as a heterodimer with subunits of 28 and 31 kD; homodimers of both subunits were also observed and exhibited phosphatase activity. In addition to the general phosphatase substrate, p-nitrophenyl phosphate, the heterodimeric form of the enzyme readily hydrolyzed 5[prime]-nucleotides, flavin mononucleotide, and O-phospho-L-Tyr. Low or negligible activity was observed with ATP or polyphosphate. Purified nodule acid phosphatase was stimulated by magnesium, inhibited by calcium and EDTA, and competitively inhibited by cGMP and cAMP with apparent Ki values of 7 and 12 [mu]M, respectively. Partial N-terminal and internal sequencing of the nodule acid phosphatase revealed homology to the soybean vegetative storage proteins. There was a 17-fold increase in enzyme activity and a noticeable increase in protein levels detected by immunoblotting methods during nodule development. Both of these parameters were low in young nodules and reached a peak in mature, functional nodules, suggesting that this enzyme is important for efficient nodule metabolism.
Spatial and Temporal Analyses of Expansion and Cell Cycle in Sunflower Leaves1
Authors: Granier Christine , Tardieu François
Published: July 2002

We have investigated the spatial distributions of expansion and cell cycle in sunflower (Helianthus annuus L.) leaves located at two positions on the stem, from leaf initiation to the end of expansion. Relative expansion rate (RER) was analyzed by following the deformation of a grid drawn on the lamina; relative division rate (RDR) and flow-cytometry data were obtained in four zones perpendicular to the midrib. Calculations for determining in situ durations of the cell cycle and of S-G2-M in the epidermis are proposed. Area and cell number of a given leaf zone increased exponentially during the first two-thirds of the development duration.RER and RDR were constant and similar in all zones of a leaf and in all studied leaves during this period. Reduction in RER occurred afterward with a tip-to-base gradient and lagged behind that of RDR by 4 to 5 d in all zones. After a long period of constancy, cell-cycle duration increased rapidly and simultaneously within a leaf zone, with cells blocked in the G0-G1 phase of the cycle. Cells that began their cycle after the end of the period with exponential increase in cell number could not finish it, suggesting that they abruptly lost their competence to cross a critical step of the cycle. Differences in area and in cell number among zones of a leaf and among leaves of a plant essentially depended on the timing of two events, cessation of exponential expansion and of exponential division.
Cyanogenesis in Cassava1
Authors: White Wanda L.B. , Arias-Garzon Diana I. , McMahon Jennifer M. , Sayre Richard T.
Published: July 2002

In the cyanogenic crop cassava (Manihot esculenta, Crantz), the final step in cyanide production is the conversion of acetone cyanohydrin, the deglycosylation product of linamarin, to cyanide plus acetone. This process occurs spontaneously at pH greater than 5.0 or enzymatically and is catalyzed by hydroxynitrile lyase (HNL). Recently, it has been demonstrated that acetone cyanohydrin is present in poorly processed cassava root food products. Since it has generally been assumed that HNL is present in all cassava tissues, we reinvestigated the enzymatic properties and tissue-specific distribution of HNL in cassava. We report the development of a rapid two-step purification protocol for cassava HNL, which yields an enzyme that is catalytically more efficient than previously reported (Hughes, J., Carvalho, F., and Hughes, M. [1994] Arch Biochem Biophys 311: 496–502). Analyses of the distribution of HNL activity and protein indicate that the accumulation of acetone cyanohydrin in roots is due to the absence of HNL, not to inhibition of the enzyme. Furthermore, the absence of HNL in roots and stems is associated with very low steady-state HNL transcript levels. It is proposed that the lack of HNL in cassava roots accounts for the high acetone cyanohydrin levels in poorly processed cassava food products.
Cloning and Characterization of AtRGP11
Authors: Delgado Ivan J. , Wang Zhaohong , de Rocher Amy , Keegstra Kenneth , Raikhel Natasha V.
Published: July 2002

A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsisthaliana,Reversibly GlycosylatedPolypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.
Partial Purification and Characterization of the Maize Mitochondrial Pyruvate Dehydrogenase Complex1
Authors: Thelen Jay J. , Miernyk Jan A. , Randall Douglas D.
Published: July 2002

The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1β-, E1α-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg2+. Michaelis constants for the substrates were 47, 3, 77, and 1 μm for pyruvate, coenzyme A (CoA), NAD+, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD+ and CoA, and the inhibition constants were 15 and 47 μm, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg2+.
Second Messengers Mediate Increases in Cytosolic Calcium in Tobacco Protoplasts
Authors: Volotovski Igor D. , Sokolovsky Sergei G. , Molchan Olga V. , Knight Marc R.
Published: July 2002

Addition of membrane-permeable cyclic GMP (cGMP) and cyclic AMP (cAMP) were shown to cause elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) in tobacco (Nicotiana plumbaginofolia) protoplasts. Under the same conditions these cyclic nucleotides were shown to provoke a physiological swelling response in the protoplasts. Nonmembrane-permeable cAMP and cGMP were unable to trigger a detectable [Ca2+]cytresponse. Cyclic-nucleotide-mediated elevations in [Ca2+]cyt involved both internal and external Ca2+ stores. Both cAMP- and cGMP-mediated [Ca2+]cyt elevations could be inhibited by the Ca2+-channel blocker verapamil. Addition of inhibitors of phosphodiesterases (isobutylmethylxanthine and zaprinast) and the adenylate cyclase agonist forskolin to the protoplasts (predicted to elevate in vivo cyclic-nucleotide concentrations) caused elevations in [Ca2+]cyt. Addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine before forskolin significantly inhibited the forskolin-induced [Ca2+]cytelevation. Taken together, these data suggest that a potential communication point for cross-talk between signal transduction pathways using cyclic nucleotides in plants is at the level of Ca2+signaling.
Chilling Delays Circadian Pattern of Sucrose Phosphate Synthase and Nitrate Reductase Activity in Tomato1
Authors: Jones Tamara L. , Tucker Dawn E. , Ort Donald R.
Published: July 2002

Overnight low-temperature exposure inhibits photosynthesis in chilling-sensitive species such as tomato (Lycopersicon esculentum) and cucumber by as much as 60%. In an earlier study we showed that one intriguing effect of low temperature on chilling-sensitive plants is to stall the endogenous rhythm controlling transcription of certain nuclear-encoded genes, causing the synthesis of the corresponding transcripts and proteins to be mistimed when the plant is rewarmed. Here we show that the circadian rhythm controlling the activity of sucrose phosphate synthase (SPS) and nitrate reductase (NR), key control points of carbon and nitrogen metabolism in plant cells, is delayed in tomato by chilling treatments. Using specific protein kinase and phosphatase inhibitors, we further demonstrate that the chilling-induced delay in the circadian control of SPS and NR activity is associated with the activity of critical protein phosphatases. The sensitivity of the pattern of SPS activity to specific inhibitors of transcription and translation indicates that there is a chilling-induced delay in SPS phosphorylation status that is caused by an effect of low temperature on the expression of a gene coding for a phosphoprotein phosphatase, perhaps the SPS phosphatase. In contrast, the chilling-induced delay in NR activity does not appear to arise from effects on NR phosphorylation status, but rather from direct effects on NR expression. It is likely that the mistiming in the regulation of SPS and NR, and perhaps other key metabolic enzymes under circadian regulation, underlies the chilling sensitivity of photosynthesis in these plant species.