bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of transgenic mice by transfection of pronuclear embryos using lipid-DNA complexes
Authors: Carballada Rosa , Relloso Miguel , Esponda Pedro
Published: September 2002

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of β-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.
Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles
Authors: Liu Rui-Hua , Li Yong-Hai , Jiao Li-Hong , Wang Xiao-Ning , Wang Hong , Wang Wei-Hua
Published: September 2002

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 ± 2.1 pmol/oocyte) and medium (13.69 ± 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 ± 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 ± 5.18 nM and 52.25 ± 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 ± 68.6 ng/ml) than from medium (40.0 ± 6.4 ng/ml) and small (41.2 ± 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 ± 45.9 ng/ml) and medium (267.5 ± 38.6 ng/ml) follicles were significantly higher than that (174.7 ± 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.
Prediction of precipitation associated with a western disturbance using a high‐resolution regional model: role of parameterisation of physical processes
Authors: Azadi M , Mohanty U C , Madan O P , Padmanabhamurty B
Published: September 2002

In this study a non‐hydrostatic version of the PSU/NCAR mesoscale model is used to simulate an active western disturbance (WD) that affected the Indian region in January 1997. The role of the planetary boundary layer (PBL) and convection parameterisation schemes in the development of the WD is investigated. Analysis and predictions for some fields, including sea level pressure, geopotential height, temperature, horizontal wind and precipitation are examined. Some statistical scores are also calculated and compared. It is found that the performance of the Hong‐Pan (as implemented in the NCAR MRF model) and Betts‐Miller (or Grell) schemes as PBL and convection parameterisation schemes respectively are best compared to the other schemes used in this study. Copyright © 2002 Royal Meteorological Society.
An operational method for forecasting total column ozone for Punta Arenas, Chile
Authors: McMahon R Monreal , Aguilar R , Valderrama V , Burt P J A
Published: September 2002

Owing to the decrease of stratospheric ozone during the southern spring (starting in September), the city of Punta Arenas (53.1°S, 70.9°W) and its surroundings are exposed to a high incidence of ultra‐violet‐B (UV‐B) radiation during this period. Ozone denatures DNA, and prolonged exposure may cause skin carcinoma and cataracts. Consequently, there is great concern among the inhabitants of this region regarding the possible effects of long‐term exposure to UV‐B. In this work we apply and further develop a pilot study carried out in 1995 which looks at the simple operational method of forecasting ozone levels some 42 hours in advance. The method is based on the prediction of the 100 mb‐temperature from ECMWF (European Centre for Medium‐Range Weather Forecasts), which is well correlated with total column ozone levels. It allows the inhabitants of the Magallanes region to be alerted to potentially high incidences of UV‐B radiation during the Austral spring. Copyright © 2002 Royal Meteorological Society.
Sensitivity of monsoon circulation and precipitation over India to model horizontal resolution and orographic effects
Authors: Wu Yihua , Raman S , Mohanty U C , Madala R V
Published: September 2002

A triple‐nested regional weather prediction model was used to investigate the effects of the model horizontal resolution and orography on southwest monsoon precipitation over India. Numerical experiments with different resolution topography and different horizontal resolution model domains were conducted. Simulation results indicate that both the distribution and intensity of simulated southwest monsoon precipitation over India is highly sensitive to model horizontal resolution and topography. The model with a finer resolution is able to predict mesoscale organization of rainfall over the land mass. Rainfall predicted over the coarse domain is much less than that observed owing to its unrealistic representation of orographic effects and mesoscale forcings. Simulated wind speed, surface pressure, and latent and sensible heat flux distributions are also sensitive to the model resolution. Larger values and more detailed structure of the distribution of the wind speed and latent and sensible heat fluxes are simulated in the finer domains. Copyright © 2002 Royal Meteorological Society.
A Ypt32p Exchange Factor Is a Putative Effector of Ypt1p
Authors: Wang Wei , Ferro-Novick Susan
Published: September 2002

Ypt1p regulates vesicle tethering and fusion events from the ER to the Golgi and through the early Golgi. Genetic studies have suggested a functional relationship between Ypt1p and Ypt31p/Ypt32p. Ypt31p and Ypt32p are a pair of functionally redundant GTPases that act after Ypt1p to mediate intra-Golgi traffic or the budding of post-Golgi vesicles from the trans-Golgi. Here we report that a novel Ypt32p exchange factor is a putative effector of Ypt1p. These findings implicate small GTP-binding proteins of the Ypt/Rab family in a signal cascade that directs membrane traffic through the secretory pathway.
Modulation of Cellular Cholesterol Transport and Homeostasis by Rab11
Authors: Hölttä-Vuori Maarit , Tanhuanpää Kimmo , Möbius Wiebke , Somerharju Pentti , Ikonen Elina
Published: September 2002

To analyze the contribution of vesicular trafficking pathways in cellular cholesterol transport we examined the effects of selected endosomal Rab proteins on cholesterol distribution by filipin staining. Transient overexpression of Rab11 resulted in prominent accumulation of free cholesterol in Rab11-positive organelles that sequestered transferrin receptors and internalized transferrin. Sphingolipids were selectively redistributed as pyrene-sphingomyelin and sulfatide cosequestered with Rab11-positive endosomes, whereas globotriaosyl ceramide and GM2 ganglioside did not. Rab11 overexpression did not perturb the transport of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate–labeled low-density lipoprotein (LDL) to late endosomes or the Niemann-Pick type C1 (NPC1)-induced late endosomal cholesterol clearance in NPC patient cells. However, Rab11 overexpression inhibited cellular cholesterol esterification in an LDL-independent manner. This effect could be overcome by introducing cholesterol to the plasma membrane by using cyclodextrin as a carrier. These results suggest that in Rab11-overexpressing cells, deposition of cholesterol in recycling endosomes results in its impaired esterification, presumably due to defective recycling of cholesterol to the plasma membrane. The findings point to the importance of the recycling endosomes in regulating cholesterol and sphingolipid trafficking and cellular cholesterol homeostasis.
A Role forCaenorhabditis elegansImportin IMA-2 in Germ Line and Embryonic Mitosis
Authors: Geles Kenneth G. , Johnson Jeffrey J. , Jong Sena , Adam Stephen A.
Published: September 2002

The importin α family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin α proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin α proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype.ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis.
ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization
Authors: Boman Annette L. , Salo Paul D. , Hauglund Melissa J. , Strand Nicole L. , Rensink Shelly J. , Zhdankina Olga
Published: September 2002

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.
DNA Repair and Transcriptional Effects of Mutations in TFIIH inDrosophilaDevelopment
Authors: Merino Carlos , Reynaud Enrique , Vázquez Martha , Zurita Mario
Published: September 2002

Mutations in XPB and XPD TFIIH helicases have been related with three hereditary human disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. The dual role of TFIIH in DNA repair and transcription makes it difficult to discern which of the mutant TFIIH phenotypes is due to defects in any of these different processes. We used haywire (hay), theDrosophila XPB homolog, to dissect this problem. Our results show that when hay dosage is affected, the fly shows defects in structures that require high levels of transcription. We found a genetic interaction between hay andcdk7, and we propose that some of these phenotypes are due to transcriptional deficiencies. We also found more apoptotic cells in imaginal discs and in the CNS of hay mutant flies than in wild-type flies. Because this abnormal level of apoptosis was not detected in cdk7 flies, this phenotype could be related to defects in DNA repair. In addition the apoptosis induced by p53 Drosophila homolog (Dmp53) is suppressed in heterozygous hay flies.
Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring γ-Tubulin Ring Complex
Authors: Takahashi Mikiko , Yamagiwa Akiko , Nishimura Tamako , Mukai Hideyuki , Ono Yoshitaka
Published: September 2002

Microtubule assembly is initiated by the γ-tubulin ring complex (γ-TuRC). In yeast, the microtubule is nucleated from γ-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors γ-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with γ-tubulin through binding with γ-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and γ-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with γ-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring γ-TuRC.
An Essential Subfamily of Drs2p-related P-Type ATPases Is Required for Protein Trafficking between Golgi Complex and Endosomal/Vacuolar System
Authors: Hua Zhaolin , Fatheddin Parvin , Graham Todd R.
Published: September 2002

The Saccharomyces cerevisiae genome contains five genes encoding P-type ATPases that are potential aminophospholipid translocases (APTs): DRS2, NEO1, and three uncharacterized open reading frames that we have namedDNF1, DNF2, and DNF3 forDRS2/NEO1 family. NEO1 is the only essential gene in APT family and seems to be functionally distinct from the DRS2/DNF genes. The drs2Δdnf1Δ dnf2Δ dnf3Δ quadruple mutant is inviable, although any one member of this group can maintain viability, indicating that there is a substantial functional overlap between the encoded proteins. We have previously implicated Drs2p in clathrin function at the trans-Golgi network. In this study, we constructed strains carrying all possible viable combinations of null alleles from this group and analyzed them for defects in protein transport. The drs2Δdnf1Δ mutant grows slowly, massively accumulates intracellular membranes, and exhibits a substantial defect in the transport of alkaline phosphatase to the vacuole. Transport of carboxypeptidase Y to the vacuole is also perturbed, but to a lesser extent. In addition, the dnf1Δ dnf2Δdnf3Δ mutant exhibits a defect in recycling of GFP-Snc1p in the early endocytic-late secretory pathways. Drs2p and Dnf3p colocalize with the trans-Golgi network marker Kex2p, whereas Dnf1p and Dnf2p seem to localize to the plasma membrane and late exocytic or early endocytic membranes. We propose that eukaryotes express multiple APT subfamily members to facilitate protein transport in multiple pathways.
Distinct Chromosome Segregation Roles for Spindle Checkpoint Proteins
Authors: Warren Cheryl D. , Brady D. Michelle , Johnston Raymond C. , Hanna Joseph S. , Hardwick Kevin G. , Spencer Forrest A.
Published: September 2002

The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage.
Agonist-induced PIP2Hydrolysis Inhibits Cortical Actin Dynamics: Regulation at a Global but not at a Micrometer Scale
Authors: van Rheenen Jacco , Jalink Kees
Published: September 2002

Phosphatidylinositol 4, 5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane has been proposed to locally regulate the actin cytoskeleton. Indeed, recent studies that use GFP-tagged pleckstrin homology domains (GFP-PH) as fluorescent PIP2sensors suggest that this lipid is enriched in membrane microdomains. Here we report that this concept needs revision. Using three distinct fluorescent GFP-tagged pleckstrin homology domains, we show that highly mobile GFP-PH patches colocalize perfectly with various lipophilic membrane dyes and, hence, represent increased lipid content rather than PIP2-enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized at ∼15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP2breakdown, and it resumes as soon as PIP2levels are back to normal. Thus, our data support a role for PIP2in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP2regulation of the cytoskeleton exist at a micrometer scale.
Isolation and Characterization of a Novel Ribosome-Inactivating Protein from Root Cultures of Pokeweed and Its Mechanism of Secretion from Roots
Authors: Park Sang-Wook , Lawrence Christopher B. , Linden James C. , Vivanco Jorge M.
Published: September 2002

Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, β-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.
VFL, the GrapevineFLORICAULA/LEAFYOrtholog, Is Expressed in Meristematic Regions Independently of Their Fate
Authors: Carmona Marı́a José , Cubas Pilar , Martı́nez-Zapater José M.
Published: September 2002

The flowering process in grapevine (Vitis vinifera) takes place in buds and extends for two consecutive growing seasons. To understand the genetic and molecular mechanisms underlying this process, we have characterized grapevine bud development, cloned the grapevineFLORICAULA/LEAFY (FLO/LFY) ortholog, VFL, and analyzed its expression patterns during vegetative and reproductive development. Flowering induction takes place during the first season. Upon induction, the shoot apical meristem begins to produce lateral meristems that will give rise to either inflorescences or tendrils. During the second season, after a winter dormancy period, buds reactivate and inflorescence meristems give rise to flower meristems. VFL is expressed in lateral meristems that give rise to inflorescence and flower meristems, consistent with a role in reproductive development. Furthermore,VFL is also detected in other meristematic regions such as the vegetative shoot apical meristem and the lateral meristems that will give rise to tendrils. VFL is also expressed in leaf primordia and in growing leaf margins until later stages of development. Accumulation of VFL transcripts in cell-proliferating regions suggests a role for VFL not only in flower meristem specification, but also in the maintenance of indeterminacy before the differentiation of derivatives of the apical meristem: flowers, leaves, or tendrils.
Biochemical Characterization of the Arabidopsis Protein Kinase SOS2 That Functions in Salt Tolerance
Authors: Gong Deming , Guo Yan , Jagendorf Andre T. , Zhu Jian-Kang
Published: September 2002

The Arabidopsis Salt Overly Sensitive 2(SOS2) gene encodes a serine/threonine (Thr) protein kinase that has been shown to be a critical component of the salt stress signaling pathway. SOS2 contains a sucrose-non-fermenting protein kinase 1/AMP-activated protein kinase-like N-terminal catalytic domain with an activation loop and a unique C-terminal regulatory domain with an FISL motif that binds to the calcium sensorSalt Overly Sensitive 3. In this study, we examined some of the biochemical properties of the SOS2 in vitro. To determine its biochemical properties, we expressed and isolated a number of active and inactive SOS2 mutants as glutathione S-transferase fusion proteins in Escherichia coli. Three constitutively active mutants, SOS2T168D, SOS2T168DΔF, and SOS2T168DΔ308, were obtained previously, which contain either the Thr-168 to aspartic acid (Asp) mutation in the activation loop or combine the activation loop mutation with removal of the FISL motif or the entire regulatory domain. These active mutants exhibited a preference for Mn2+ relative to Mg2+ and could not use GTP as phosphate donor for either substrate phosphorylation or autophosphorylation. The three enzymes had similar peptide substrate specificity and catalytic efficiency. Salt overly sensitive 3 had little effect on the activity of the activation loop mutant SOS2T168D, either in the presence or absence of calcium. The active mutant SOS2T168DΔ308 could not transphosphorylate an inactive protein (SOS2K40N), which indicates an intramolecular reaction mechanism of SOS2 autophosphorylation. Interestingly, SOS2 could be activated not only by the Thr-168 to Asp mutation but also by a serine-156 or tyrosine-175 to Asp mutation within the activation loop. Our results provide insights into the regulation and biochemical properties of SOS2 and the SOS2 subfamily of protein kinases.
Arabidopsis UVR8 Regulates Ultraviolet-B Signal Transduction and Tolerance and Contains Sequence Similarity to Human Regulator of Chromatin Condensation 1
Authors: Kliebenstein Daniel J. , Lim Jackie E. , Landry Laurie G. , Last Robert L.
Published: September 2002

To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis.
Regulation of Transcript Levels of the Arabidopsis Cytochrome P450 Genes Involved in Brassinosteroid Biosynthesis
Authors: Bancosİ Simona , Nomura Takahito , Sato Tatsuro , Molnár Gergely , Bishop Gerard J. , Koncz Csaba , Yokota Takao , Nagy Ferenc , Szekeres Miklós
Published: September 2002

Cytochrome P450 enzymes of the closely related CYP90 and CYP85 families catalyze essential oxidative reactions in the biosynthesis of brassinosteroid (BR) hormones. Arabidopsis CYP90B1/DWF4 and CYP90A1/CPD are responsible for respective C-22 and C-23 hydroxylation of the steroid side chain and CYP85A1 catalyzes C-6 oxidation of 6-deoxo intermediates, whereas the functions of CYP90C1/ROT3, CYP90D1, and CYP85A2 are still unknown. Semiquantitative reverse transcriptase-polymerase chain reaction analyses show that transcript levels of CYP85 and CYP90genes are down-regulated by brassinolide, the end product of the BR biosynthesis pathway. Feedback control of the CYP90C1,CYP90D1, and CYP85A2 genes by brassinolide suggests that the corresponding enzymes might also participate in BR synthesis. CYP85 andCYP90 mRNAs show strong and transient accumulation during the 1st week of seedling development, as well as characteristic organ-specific distribution. Transcripts of CYP90A1 andCYP85A2 are preferentially represented in shoots andCYP90C1, CYP90D1, andCYP85A1 mRNAs are more abundant in roots, whereasCYP90B1 is ubiquitously expressed. Remarkably, the spatial pattern of CYP90A1 expression is maintained in the BR-insensitive cbb2 mutant, indicating the independence of organ-specific and BR-dependent regulation. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in shoots and roots of Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum) reveal similar partitioning patterns of BR intermediates in these species. Inverse correlation between CYP90A1/CPDtranscript levels and the amounts of the CYP90A1 substrate 6-deoxocathasterone in shoots and roots suggests that transcriptional regulation plays an important role in controlling BR biosynthesis.
The Altered Pattern of Amylose Accumulation in the Endosperm of Low-Amylose Barley Cultivars Is Attributable to a Single Mutant Allele of Granule-Bound Starch Synthase I with a Deletion in the 5′-Non-Coding Region
Authors: Patron Nicola J. , Smith Alison M. , Fahy Brendan F. , Hylton Christopher M. , Naldrett Mike J. , Rossnagel Brian G. , Denyer Kay
Published: September 2002

Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5′-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxyancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity.
elongated mesocotyl1, a Phytochrome-Deficient Mutant of Maize
Authors: Sawers Ruairidh J.H. , Linley Philip J. , Farmer Phyllis R. , Hanley Nicole P. , Costich Denise E. , Terry Matthew J. , Brutnell Thomas P.
Published: September 2002

To begin the functional dissection of light signal transduction pathways of maize (Zea mays), we have identified and characterized the light-sensing mutant elm1(elongated mesocotyl1). Seedlings homozygous forelm1 are pale green, show pronounced elongation of the mesocotyl, and fail to de-etiolate under red or far-red light. Etiolated elm1 mutants contain no spectrally active phytochrome and do not deplete levels of phytochrome A after red-light treatment. High-performance liquid chromatography analyses show that elm1 mutants are unable to convert biliverdin IXα to 3Z-phytochromobilin, preventing synthesis of the phytochrome chromophore. Despite the impairment of the phytochrome photoreceptors, elm1 mutants can be grown to maturity in the field. Mature plants retain aspects of the seedling phenotype and flower earlier than wild-type plants under long days. Thus, theelm1 mutant of maize provides the first direct evidence for phytochrome-mediated modulation of flowering time in this agronomically important species.
Cloning and Functional Characterization of a β-Pinene Synthase from Artemisia annua That Shows a Circadian Pattern of Expression
Authors: Lu Shan , Xu Ran , Jia Jun-Wei , Pang Jihai , Matsuda Seiichi P.T. , Chen Xiao-Ya
Published: September 2002

Artemisia annua plants produce a broad range of volatile compounds, including monoterpenes, which contribute to the characteristic fragrance of this medicinal species. A cDNA clone, QH6, contained an open reading frame encoding a 582-amino acid protein that showed high sequence identity to plant monoterpene synthases. The prokaryotically expressed QH6 fusion protein converted geranyl diphosphate to (−)-β-pinene and (−)-α-pinene in a 94:6 ratio. QH6 was predominantly expressed in juvenile leaves 2 weeks postsprouting. QH6 transcript levels were transiently reduced following mechanical wounding or fungal elicitor treatment, suggesting that this gene is not directly involved in defense reaction induced by either of these treatments. Under a photoperiod of 12 h/12 h (light/dark), the abundance of QH6 transcripts fluctuated in a diurnal pattern that ebbed around 3 h before daybreak (9th h in the dark phase) and peaked after 9 h in light (9th h in the light phase). The contents of (−)-β-pinene in juvenile leaves and in emitted volatiles also varied in a diurnal rhythm, correlating strongly with mRNA accumulation. WhenA. annua was entrained by constant light or constant dark conditions, QH6 transcript accumulation continued to fluctuate with circadian rhythms. Under constant light, advanced cycles of fluctuation of QH6 transcript levels were observed, and under constant dark, the cycle was delayed. However, the original diurnal pattern could be regained when the plants were returned to the normal light/dark (12 h/12 h) photoperiod. This is the first report that monoterpene biosynthesis is transcriptionally regulated in a circadian pattern.
The importance of assessment
Authors: Rockman Ilene F.
Published: September 2002

States that assessment is a process for quality improvement and libraries, as administrative and academic units, have an important role to play in the continuous improvement of their parent organizations. Looks at how student performance can be assessed and concludes that this can help us to understand better why students fail or master certain courses, which will help in developing change and improvement in the area of student learning.
Instant messaging reference: users’ evaluation of library chat
Authors: Ruppel Margie , Condit Fagan Jody
Published: September 2002

This article analyzes survey results of university students who used Morris Messenger, the instant messaging (IM) reference service at Southern Illinois University Carbondale’s Morris Library. It focuses on the complete results of two surveys, including a comparison of IM reference and traditional reference desk experiences. An overview of the IM reference system and usage data are also discussed. Survey respondents indicated overall enthusiasm for the IM reference service and provided useful suggestions for improvements, which are also listed.
Revolution or evolution: reference planning in ARL libraries
Authors: Jackson Rebecca
Published: September 2002

Reference statistics over the past ten years at the Association of Research Libraries (ARL) reference desks have been gradually declining. In this survey, the author examined whether libraries are changing their services in response to these declines. A survey was conducted of ARL heads of reference services. Results show that reference services are responding to changes in statistics and resources by reorganizing, changing staffing levels, adding new services, and eliminating some services that are no longer useful. Librarians are performing more than reference activities, including collection development, and instruction. In addition, services for distant users are becoming more prevalent. Finally, libraries are conducting needs assessments to discover whether their users are satisfied with the current services and collections in their libraries. Areas for further research are explored.
Tools for evaluation of electrical performance of chip‐on‐board devices
Authors: Cordery A. , Kilbey N. , Suthiwongsunthorn N.
Published: September 2002

The present paper discusses the development of a test methodology for evaluation of the electrical performance of flip‐chip devices. A dedicated test chip was designed for this experiment. The test structure contains passive and active semiconductor devices manufactured using CMOS technology. Bond pads were designed to facilitate bumping. A Printed Circuit Board (PCB) housing the flipped devices was also designed for easy access to the individual devices. A test set‐up for measuring the structures was developed and key device parameters to monitor the electrical performance of the structures were identified.The results show that the proposed test structure is a suitable tool for determining the electrical parameters of flip‐chip devices. The experimental set‐up is universal and can be adapted to suit different custom‐designed flip‐chip test structures. In addition, the developed test set‐up is computer controlled and allows easy adaptation to different measurement techniques and devices.
Conductance and Viscosity Studies of Ion Solvation in Mixed DMSO-MeOH Solvent
Authors: Chauhan Mohinder S. , Sharma Kishore C. , Kumar Girish , Chauhan Suvarcha
Published: September 2002

Solvation behavior of Li+, Na+, K+, Ag+, Cl-, Br-, I- and ClO4- ions is reported at 25 °C from conductance and viscosity measurements of LiClO4, NaClO4, KClO4, AgClO4, LiCl, Bu4NClO4, Bu4NBr, Bu4NI, Ph4PBPh4, Bu4NBPh4, NaBPh4, Ph4PBr and NaBr in mixed DMSO-MeOH solvent. Anomalous behavior of Ag+ is interpreted as resulting from specific ion-solvent interactions. However, Li+, Na+ and K+ interact preferentially with DMSO electrostatically through oxygen atom of SO group. Initial decrease in solvation of alkali metal ions on addition of MeOH to pure DMSO is interpreted as an indication of strong intermolecular interactions between solvent components preferably through the hydrogen bond formation. The region of relatively strong intermolecular interactions is found between 10 and 40 mole % MeOH. The viscosity studies imply the existence of interactions between Br- or I- and DMSO. The studies however, indicate poor solvation of anions in this solvent system.
Covalent Analogues of DNA Base-Pairs and Triplets IV. Synthesis of Trisubstituted Benzenes Bearing Purine and/or Pyrimidine Rings by Cyclotrimerization of 6-Ethynylpurines and/or 5-Ethynyl-1,3-dimethyluracil
Authors: Hocek Michal , Stará Irena G. , Starý Ivo , Dvořáková Hana
Published: September 2002

Ni-Catalyzed cyclotrimerizations of 6-ethynylpurines 3 or 5-ethynyl-1,3-dimethyluracil (4) afforded the 1,2,4-tris(purin-6-yl)benzenes 7 or 1,2,4-tris(1,3-dimetyhyluracil-5-yl)benzene (9), respectively. The symmetrical 1,3,5-tris(purin-6-yl)benzenes 8 were also formed as minor products in very low yields. Co-cyclotrimerization of 9-benzyl-6-ethynylpurine (3a) with 4 afforded the tris(purinyl)benzene 7a as a major product along with 1,2-bis(9-benzylpurin-6-yl)-4-(1,3-dimethyluracil-5-yl)benzene (10) and a complex mixture of other derivatives and isomers. Compounds 7-10 are analogues of Hoogsteen base-triplets.
Trends in BB/pellet gun injuries in children and teenagers in the United States, 1985–99
Authors: Nguyen M H , Annest J L , Mercy J A , Ryan G W , Fingerhut L A
Published: September 2002

Objective:To characterize national trends in non-fatal BB/pellet gun related injury rates for persons aged 19 years or younger in relation to trends in non-fatal and fatal firearm related injury rates and discuss these trends in light of injury prevention and violence prevention efforts.

Setting:The National Electronic Injury Surveillance System (NEISS) includes approximately 100 hospitals with at least six beds that provide emergency services. These hospitals comprise a stratified probability sample of all US hospitals with emergency departments. The National Vital Statistics System (NVSS) is a complete census of all death certificates filed by states and is compiled annually.

Methods:National data on BB/pellet gun related injuries and injury rates were examined along with fatal and non-fatal firearm related injuries and injury rates. Non-fatal injury data for all BB/pellet gun related injury cases from 1985 through 1999, and firearm related injury cases from 1993 through 1999 were obtained from hospital emergency department records using the NEISS. Firearm related deaths from 1985 through 1999 were obtained from the NVSS.

Results:BB/pellet gun related injury rates increased from age 3 years to a peak at age 13 years and declined thereafter. In contrast, firearm related injury and death rates increased gradually until age 13 and then increased sharply until age 18 years. For persons aged 19 years and younger, BB/pellet gun related injury rates increased from the late 1980s until the early 1990s and then declined until 1999; these injury rates per 100 000 population were 24.0 in 1988, 32.8 in 1992, and 18.3 in 1999. This trend was similar to those for fatal and non-fatal firearm related injury rates per 100 000 which were 4.5 in 1985, 7.8 in 1993, and 4.3 in 1999 (fatal) and 38.6 in 1993 and 16.3 in 1999 (non-fatal). In 1999, an estimated 14 313 (95% confidence interval (CI) 12 025 to 16 601) cases with non-fatal BB/pellet gun injuries and an estimated 12 748 (95% CI 7881–17 615) cases with non-fatal firearm related injuries among persons aged 19 years and younger were treated in US hospital emergency departments.

Conclusions:BB/pellet gun related and firearm related injury rates show similar declines since the early 1990s. These declines coincide with a growing number of prevention efforts aimed at reducing injuries to children from unsupervised access to guns and from youth violence. Evaluations at the state and local level are needed to determine true associations.
Pediatric motor vehicle related injuries in the Navajo Nation: the impact of the 1988 child occupant restraint laws
Authors: Phelan K J , Khoury J , Grossman D C , Hu D , Wallace L J D , Bill N , Kalkwarf H
Published: September 2002

Background:Navajo motor vehicle mortality is the highest among the 12 Indian Health Service (IHS) administrative areas. In July 1988, the Navajo Nation enacted a primary enforcement safety belt use and a child restraint law.

Objective:Assess the impact of the laws on the rate and severity of pediatric (0–19 years) motor vehicle injury resulting in hospitalizations in the Navajo Nation.

Methods:Hospitalizations associated with motor vehicle related injury discharges were identified byInternational Classification of Diseases, 9th revision, CM E codes, 810–825 (.0,.1) from the Navajo IHS hospital discharge database. Age specific rates for the period before the law, 1983–88, were compared with those after enactment and enforcement, 1991–95. Severity of injury, measured by the abbreviated injury scale (AIS) score and new injury severity score (NISS), was determined with ICDMAP-90 software. Wilcoxon rank sum and χ2tests were used for analysis.

Results:Discharge rates (SE) for motor vehicle injury (per 100 000) decreased significantly in all age groups: 0–4 years (62 (7) to 28 (4)), 5–11 years (55.3 (6) to 26 (4)), and 15–19 years (139 (14) to 68 (7)); p=0.0001. In children 0–4 years, the median AIS score decreased from 1.5 (1,3) (25th, 75th centile) to 1 (1,2), p=0.06, and the median NISS decreased from 3.5 (1,9) to 2 (1,5), p=0.07. The proportion of children with NISS scores >4 decreased significantly for the 0–4 year age group (p=0.03).

Conclusions:Concurrent with enactment of the Navajo Nation occupant and child restraint laws there was a reduction in the rate of motor vehicle related hospital discharges for children. Severity of injury declined in very young Navajo children. The effect of enactment and enforcement of this Native American child occupant restraint law may serve as an example of an effective injury control effort directed at Native American children.
How old is that child? Validating the accuracy of age assignments in observational surveys of vehicle restraint use
Authors: Moeller S , Berger L , Salvador J G , Helitzer D
Published: September 2002

Objectives:Many large scale observational studies of child restraint usage require observers to estimate the ages of the vehicle occupants. The accuracy of age assignments were assessed and possible methods to improve observational accuracy in research and field studies of child restraint use were identified.

Methods:The validation study was performed at fast food restaurants in a metropolitan area. Three, two person teams observed 449 occupants of vehicles with at least one child passenger. The drivers were then interviewed to obtain the actual ages of the vehicle occupants. The primary outcome measure was the per cent of age assignments that were correct by age category (infant, toddler, school age, teen, adult). The observers had previously conducted a statewide child restraint observation study and were trained in estimating age categories.

Results:A total of 391 (87%) of the 449 occupants were assigned to their correct age categories. Incorrect assignments were more common among infants (22% incorrect), but few infants (nine) were observed. The most frequent error was classifying adults (19 years and older) as teenagers (13–18 years).

Conclusion:Trained, experienced observers approached 90% accuracy in their assignment of children to specific age categories. Additional study is required to determine whether these results are applicable to different age categories and observers.

It is recommended that the National Highway Traffic Safety Administration, the Centers for Disease Control and Prevention, state and local agencies, and other sponsors of observational surveys consider observer competence as an important variable. The validity of age assignments can be assessed by randomly interviewing a sample of drivers. More accurate age estimates will improve decisions regarding prevention programs, funding, and policies.
Evaluation of the THINK FIRST For KIDS injury prevention curriculum for primary students
Authors: Greene A , Barnett P , Crossen J , Sexton G , Ruzicka P , Neuwelt E
Published: September 2002

THINK FIRST is a national non-profit organization whose mission is to educate young people about the prevention of brain and spinal cord injuries. This paper describes a study conducted by THINK FIRST of Oregon on one of its injury prevention curricula, THINK FIRST for KIDS.
MINING WEB USAGE GRAPHS USING EXAMPLE SEARCH SPACE
Authors: MAHESWARI V. UMA , SIROMONEY A. , MEHATA K. M.
Published: September 2002

Web mining refers to the process of discovering potentially useful and previously unknown information or knowledge from web data. A graph-based framework is used for classifying Web users based on their navigation patterns. GOLEM is a learning algorithm that uses the example space to restrict the solution search space. In this paper, this algorithm is modified for the graph-based framework. GOLEM is appropriate in this application where the solution search space is very large. An experimental illustration is presented.
AN INTELLIGENT HYBRID APPROACH FOR CONTENT-BASED IMAGE RETRIEVAL
Authors: KULKARNI S. , VERMA B.
Published: September 2002

The paper presents an intelligent hybrid approach for content-based image retrieval based on texture feature. The proposed approach employs an Auto–Associative Neural Network (AANN) for feature extraction and a Multi–Layer Perceptron (MLP) with a single hidden layer for the classification. Two intelligent approaches such as AANN–MLP and statistical–MLP were investigated. The performance of the proposed approaches was evaluated on a large benchmark database of texture patterns. The results are very promising compared to other existing traditional and intelligent techniques. Some of the experimental results conducted during the investigation, comparative analysis of the results and suggestions to select the appropriate techniques for texture feature extraction and classification are presented in this paper.