bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Ethanolic Extract of Embelia Ribes on Dyslipidemia in Diabetic Rats
Authors: Bhandari Uma , Kanojia Raman , Pillai K. K.
Published: November 2003

Diabetes mellitus has been treated orally with herbal remedies
based on folk medicine since ancient times. Embelia ribes burm
(Myrsinaceae), known commonly as vidanga, was used in Ayurveda
for its anthelmintic activity. Ayurveda describes vidanga as pungent,
causes increase in digestive fire, and cures flatulence and colic.
A single study reported the antihyperglycemic activity of decoction
of E. ribes in glucose‐induced hyperglycemic albino rabbits.
In the present study, the lipid‐lowering and antioxidant potential
of ethanolic extract of E. ribes burm was investigated in streptozotocin
(40 mg/kg, IV, single injection)‐induced diabetes in rats.
Twenty days of orally feeding the extract (200 mg/kg) to diabetic
rats resulted in significant (P < 0.01) decrease in blood glucose,
serum total cholesterol, and triglycerides, and increase in HDLcholesterol
levels when compared to pathogenic diabetic rats. Further,
the extract also lowered the liver and pancreas thiobarbituric
acid–reactive substances (TBARSs) values (P < 0.01) when compared
to TBARS values of liver and pancreas of pathogenic diabetic
rats. The results of test drug were comparable to gliclazide
(25 mg/kg, orally), a standard antihyperglycemic agent. This is the
first pilot study to provide biochemical evidence of potential of E.
ribes in diabetic dyslipidemia.
Effect of Mild Hypoinsulinemia on Renal Hypertrophy: Growth Hormone/Insulin‐Like Growth Factor I System in Mild Streptozotocin Diabetes
Authors: Khamaisi Mogher , Flyvbjerg Allan , Haramati Ziv , Raz Gadi , Wexler Isaiah D. , Raz Itamar
Published: November 2003

The metabolic aberrations associated with diabetes mellitus
profoundly alter the growth hormone/insulin‐like growth factor I
(GH/IGF‐I) system. In severe experimental diabetes, serum IGF‐I
level is reduced, reflecting altered hepatic expression. On the other
hand, increased levels of kidney IGF‐I have been implicated in the
development of diabetic kidney disease. This study aimed to examine
the effect of mild experimental diabetes with hypoinsulinemia
on both the systemic and renal GH/IGF‐I systems in a low‐dose
streptozotocin (STZ)‐induced diabetic rat. Diabetic animals with
mild hypoinsulinemia developed renal hyperfiltration within 3 days
of diabetes, whereas the renal size increased significantly only between
30 and 48 days of diabetes. Plasma GHlevels were unchanged
during the entire course of the study, but a decrease in serum
IGF‐I, IGF‐binding protein 3 (IGFBP‐3), and IGF‐binding protein
4 (IGFBP‐4) occurred after 10, 30, and 48 days. Kidney IGF‐I
and IGF‐binding protein 1 (IGFBP‐1) mRNA expression increased
after 10 and 30 days of diabetes. A significant increase in kidney
IGFBP‐1/2, IGFBP‐3, and IGFBP‐4 proteins was seen after 48 days
of diabetes.Apositive correlations was found between renal growth
and insulin/glucose ratio (r = .57), kidney IGF‐I (r = .57), IGFBP‐1
mRNA(r = .43), IGFBP‐1/2 (r = .41), and IGFBP‐4 levels (r = .40).
These results demonstrate hyperfiltration within 3 days of diabetes
and a similar response in the IGF‐I system in mildly and severely hypoinsulinemic
rats; however, renomegaly develops slower in mildly diabetic rats at least partly due to delayed changes in the renal IGF
and IGF BPs.
Leukocyte, Rather than Tumor-produced SPARC, Determines Stroma and Collagen Type IV Deposition in Mammary Carcinoma
Authors: Sangaletti Sabina , Stoppacciaro Antonella , Guiducci Cristiana , Torrisi Maria Rosaria , Colombo Mario P.
Published: November 2003

Secreted protein, acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is a Ca2+-binding matricellular glycoprotein involved in development, wound healing, and neoplasia. However, the role of SPARC in tumors is ill defined mostly because it is expressed by both tumor and stromal cells, especially inflammatory cells. We analyzed the respective roles of host- and tumor-derived SPARC in wild-type and congenic SPARC knockout (SPARC−/−) mice on a BALB/c genetic background injected into the mammary fat pad with SPARC-producing mammary carcinoma cells derived from c-erB2 transgenic BALB/c mice. Reduced tumor growth but massive parenchyma infiltration, with large areas of necrosis and impaired vascularization were observed in SPARC−/− mice. Immunohistochemical analysis showed a defect in collagen type IV deposition in the stroma of lobular tumors from SPARC−/− mice. Chimeric mice expressing SPARC only in bone marrow–derived cells were able to organize peritumoral and perilobular stroma, whereas reciprocal chimeras transplanted with bone marrow from SPARC−/− mice developed tumors with less defined lobular structures, lacking assembled collagen type IV and with a parenchyma heavily infiltrated by leukocytes. Together, the data indicate that SPARC produced by host leukocytes, rather than the tumor, determines the assembly and function of tumor-associated stroma through the organization of collagen type IV.
Complementary Signaling through flt3 and Interleukin-7 Receptor α Is Indispensable for Fetal and Adult B Cell Genesis
Authors: Sitnicka Ewa , Brakebusch Cord , Martensson Inga-Lill , Svensson Marcus , Agace William W. , Sigvardsson Mikael , Buza-Vidas Natalija , Bryder David , M.Cilio Corrado , Ahlenius Henrik , Maraskovsky Eugene , Peschon Jacques J. , Jacobsen Sten Eirik W.
Published: November 2003

Extensive studies of mice deficient in one or several cytokine receptors have failed to support an indispensable role of cytokines in development of multiple blood cell lineages. Whereas B1 B cells and Igs are sustained at normal levels throughout life of mice deficient in IL-7, IL-7Rα, common cytokine receptor gamma chain, or flt3 ligand (FL), we report here that adult mice double deficient in IL-7Rα and FL completely lack visible LNs, conventional IgM+ B cells, IgA+ plasma cells, and B1 cells, and consequently produce no Igs. All stages of committed B cell progenitors are undetectable in FL−/− × IL-7Rα−/− BM that also lacks expression of the B cell commitment factor Pax5 and its direct target genes. Furthermore, in contrast to IL-7Rα−/− mice, FL−/− × IL-7Rα−/− mice also lack mature B cells and detectable committed B cell progenitors during fetal development. Thus, signaling through the cytokine tyrosine kinase receptor flt3 and IL-7Rα are indispensable for fetal and adult B cell development.
pH Changes in the Invaginating Synaptic Cleft Mediate Feedback from Horizontal Cells to Cone Photoreceptors by Modulating Ca2+ Channels
Authors: Hirasawa Hajime , Kaneko Akimichi
Published: November 2003

Feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the center-surround–receptive field organization of retinal neurons. Recordings from cone photoreceptors in newt retinal slices were obtained by the whole-cell patch-clamp technique, using a superfusate containing a GABA antagonist (100 μM picrotoxin). Surround illumination of the receptive field increased the voltage-dependent calcium current (ICa) in the cones, and shifted the activation voltage of ICa to negative voltages. External alkalinization also increased cone ICa and shifted its activation voltage toward negative voltages. Enrichment of the pH buffering capacity of the extracellular solution increased cone ICa, and blocked any additional increase in cone ICa by surround illumination. Hyperpolarization of the HCs by a glutamate receptor antagonist-augmented cone ICa, whereas depolarization of the HCs by kainate suppressed cone ICa. From these results, we propose the hypothesis that pH changes in the synaptic clefts, which are intimately related to the membrane voltage of the HCs, mediate the feedback from the HCs to cone photoreceptors. The feedback mediated by pH changes in the synaptic cleft may serve as an additional mechanism for the center-surround organization of the receptive field in the outer retina.
Position of eukaryotic initiation factor eIF1 on the 40S ribosomal subunit determined by directed hydroxyl radical probing
Authors: Lomakin Ivan B. , Kolupaeva Victoria G. , Marintchev Assen , Wagner Gerhard , Pestova Tatyana V.
Published: November 2003

Eukaryotic initiation factor (eIF) eIF1 maintains the fidelity of initiation codon selection by enabling 43S complexes to reject codon–anticodon mismatches, to recognize the initiation codon context, and to discriminate against establishing a codon–anticodon interaction with AUGs located <8 nt from the 5′-end of mRNA. To understand how eIF1 plays its discriminatory role, we determined its position on the 40S ribosomal subunit using directed hydroxyl radical cleavage. The cleavage of 18S rRNA in helices 23b, 24a, and 44 by hydroxyl radicals generated from Fe(II) tethered to seven positions on the surface of eIF1 places eIF1 on the interface surface of the platform of the 40S subunit in the proximity of the ribosomal P-site. The position of eIF1 on the 40S subunit suggests that although eIF1 is unable to inspect the region of initiation codon directly, its position close to the P-site is very favorable for an indirect mechanism of eIF1's action by influencing the conformation of the platform of the 40S subunit and the positions of mRNA and initiator tRNA in initiation complexes. Unexpectedly, the position of eIF1 on the 40S subunit was strikingly similar to the position on the 30S ribosomal subunit of the sequence and structurally unrelated C-terminal domain of prokaryotic initiation factor IF3, which also participates in initiation codon selection in prokaryotes.
Epigenetic Switch from Posttranscriptional to Transcriptional Silencing Is Correlated with Promoter Hypermethylation
Authors: Fojtova Miloslava , Van Houdt Helena , Depicker Anna , Kovarik Ales
Published: November 2003

Changes in the distribution of methylcytosine residues along a transgene locus of tobacco (Nicotiana tabacum) in relation to the type of gene silencing were studied in parental plant leaves, calli, and regenerated plants derived thereof. Parental-silenced HeLo1 (hemizygous for locus 1) plants show posttranscriptional silencing of the residing nptII (neomycin phosphotransferase II) transgene and cytosine methylation restricted to the 3′ end and center part of the transcribed region. Here, we report that with an increasing number of cell cycles, DNA methylation changes gradually, and methylation is introduced into the promoter during cell culture and more slowly in vegetatively propagated plants. After 24 months of callus in vitro cultivation, an epigenetic variant, designated locus 1E, was obtained in which cytosine methylation of symmetrical (CG and CNG) sites was almost complete within the 5′ end of the nptII-transcribed region and the 35S promoter. Further, methylation of nonsymmetrical sites appeared de novo in the promoter, whereas this type of methylation was significantly reduced in the 3′ end of the transcribed region when compared with locus 1. The newly established epigenetic patterns were stably transmitted from calli into regenerated plants and their progeny. The protein and steady-state RNA levels remained low in locus 1E, whereas with nuclear run-on assays, no detectable amounts of primary transcripts were found along the nptII gene, indicating that the methylated promoter became inactivated. The results suggest that a switch between posttranscriptional and transcriptional gene silencing could be a mechanism leading to irrevocable shut down of gene expression within a finite number of generations.
Phylogenetic Analyses and Expression Studies Reveal Two Distinct Groups of Calreticulin Isoforms in Higher Plants
Authors: Persson Staffan , Rosenquist Magnus , Svensson Karin , Galvão Rafaelo , Boss Wendy F. , Sommarin Marianne
Published: November 2003

Calreticulin (CRT) is a multifunctional protein mainly localized to the endoplasmic reticulum in eukaryotic cells. Here, we present the first analysis, to our knowledge, of evolutionary diversity and expression profiling among different plant CRT isoforms. Phylogenetic studies and expression analysis show that higher plants contain two distinct groups of CRTs: a CRT1/CRT2 group and a CRT3 group. To corroborate the existence of these isoform groups, we cloned a putative CRT3 ortholog from Brassica rapa. The CRT3 gene appears to be most closely related to the ancestral CRT gene in higher plants. Distinct tissue-dependent expression patterns and stress-related regulation were observed for the isoform groups. Furthermore, analysis of posttranslational modifications revealed differences in the glycosylation status among members within the CRT1/CRT2 isoform group. Based on evolutionary relationship, a new nomenclature for plant CRTs is suggested. The presence of two distinct CRT isoform groups, with distinct expression patterns and posttranslational modifications, supports functional specificity among plant CRTs and could account for the multiple functional roles assigned to CRTs.
Characterization of Leachianone G 2″ -Dimethylallyltransferase, a Novel Prenyl Side-Chain Elongation Enzyme for the Formation of the Lavandulyl Group of Sophoraflavanone G in Sophora flavescens Ait. Cell Suspension Cultures
Authors: Zhao Ping , Inoue Kenichiro , Kouno Isao , Yamamoto Hirobumi
Published: November 2003

Leachianone G (LG) 2′′-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme, was identified in Sophora flavescens Ait. cultured cells. The enzyme transfers a dimethylallyl group to the 2′′ position of another dimethylallyl group attached at position 8 of LG to form sophoraflavanone G, a branched monoterpenoid-conjugated flavanone characteristic to this plant. This membrane-bound dimethylallyltransferase required Mg2+ (optimum concentration was 10 mm) for the reaction and had an optimum pH of 8.8. It utilized dimethylallyl diphosphate as the sole prenyl donor, and the 2′-hydroxy function in LG was indispensable to the activity. The apparent K  m values for dimethylallyl diphosphate and LG were 59 and 2.3 μm, respectively. Subcellular localization of three enzymes that participated in the formation of the lavandulyl group was also investigated by sucrose density gradient centrifugation. Two prenyltransferases, naringenin 8-dimethylallyltransferase and LG 2′′-dimethylallyltransferase, were localized in the plastids, whereas 8-dimethylallylnaringenin 2′-hydroxylase, which catalyzes the crucial step in the lavandulyl-group formation, was associated with the endoplasmic reticulum. These results suggest the close cooperation between the plastids and the endoplasmic reticulum in the formation of lavandulyl groups.
Synthesis of the Arabidopsis Bifunctional Lysine-Ketoglutarate Reductase/Saccharopine Dehydrogenase Enzyme of Lysine Catabolism Is Concertedly Regulated by Metabolic and Stress-Associated Signals
Authors: Stepansky Asya , Galili Gad
Published: November 2003

In plants, excess cellular lysine (Lys) is catabolized into glutamic acid and acetyl-coenzyme A; yet, it is still not clear whether this pathway has other functions in addition to balancing Lys levels. To address this issue, we examined the effects of stress-related hormones, abscisic acid (ABA), and jasmonate, as well as various metabolic signals on the production of the mRNA and polypeptide of the bifunctional Lys-ketoglutarate reductase (LKR)/saccharopine dehydrogenase (SDH) enzyme, which contains the first two linked enzymes of Lys catabolism. The level of LKR/SDH was strongly enhanced by ABA, jasmonate, and sugar starvation, whereas excess sugars and nitrogen starvation reduced its level; thus this pathway appears to fulfill multiple functions in stress-related and carbon/nitrogen metabolism. Treatments with combination of hormones and/or metabolites, as well as use of ABA mutants in conjunction with the tester sugars mannose and 3-O-methyl-glucose further supported the idea that the hormonal and metabolic signals apparently operate through different signal transduction cascades. The stimulation of LKR/SDH protein expression by ABA is regulated by a signal transduction cascade that contains the ABI1-1 and ABI2-1 protein phosphatases. By contrast, the stimulation of LKR/SDH protein expression by sugar starvation is regulated by the hexokinase-signaling cascade in a similar manner to the repression of many photosynthetic genes by sugars. These findings suggest a metabolic and mechanistic link between Lys catabolism and photosynthesis-related metabolism in the regulation of carbon/nitrogen partitioning.
Translation Start Sequences Affect the Efficiency of Silencing ofAgrobacterium tumefaciensT-DNA Oncogenes
Authors: Lee Hyewon , Humann Jodi L. , Pitrak Jennifer S. , Cuperus Josh T. , Parks T. Dawn , Whistler Cheryl A. , Mok Machteld C. , Ream L. Walt
Published: November 2003

Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin. Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase). A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP). Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis. Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt. Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences. Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS. Sequences required for oncogene silencing included a translation start site. A transgene encoding a translatable sense-strand RNA from the 5′ end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM. Silencing A. tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease.
Ozone-Induced Cell Death in Tobacco Cultivar Bel W3 Plants. The Role of Programmed Cell Death in Lesion Formation
Authors: Pasqualini Stefania , Piccioni Claudia , Reale Lara , Ederli Luisa , Della Torre Guido , Ferranti Francesco
Published: November 2003

Treatment of the ozone-sensitive tobacco (Nicotiana tabacum L. cv Bel W3) with an ozone pulse (150 nL L–1 for 5 h) induced visible injury, which manifested 48 to 72 h from onset of ozone fumigation. The “classical” ozone symptoms in tobacco cv Bel W3 plants occur as sharply defined, dot-like lesions on the adaxial side of the leaf and result from the death of groups of palisade cells. We investigated whether this reaction had the features of a hypersensitive response like that which results from the incompatible plant-pathogen interaction. We detected an oxidative burst, the result of H2O2 accumulation at 12 h from the starting of fumigation. Ozone treatment induced deposition of autofluorescent compounds and callose 24 h from the start of treatment. Total phenolic content was also strongly stimulated at the 10th and 72nd h from starting fumigation, concomitant with an enhancement in phenylalanine ammonia-lyase a and phenylalanine ammonia-lyase b expression, as evaluated by reverse transcriptase-polymerase chain reaction. There was also a marked, but transient, increase in the mRNA level of pathogenesis-related-1a, a typical hypersensitive response marker. Overall, these results are evidence that ozone triggers a hypersensitive response in tobacco cv Bel W3 plants. We adopted four criteria for detecting programmed cell death in ozonated tobacco cv Bel W3 leaves: (a) early release of cytochrome c from mitochondria; (b) activation of protease; (c) DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling of DNA 3′-OH groups; and (d) ultrastructural changes characteristic of programmed cell death, including chromatin condensation and blebbing of plasma membrane. We, therefore, provide evidence that ozone-induced oxidative stress triggers a cell death program in tobacco cv Bel W3.
Interactive Effects of Jasmonic Acid, Salicylic Acid, and Gibberellin on Induction of Trichomes in Arabidopsis
Authors: Traw M. Brian , Bergelson Joy
Published: November 2003

Leaf trichomes protect plants from attack by insect herbivores and are often induced following damage. Hormonal regulation of this plant induction response has not been previously studied. In a series of experiments, we addressed the effects of artificial damage, jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Artificial damage and jasmonic acid caused significant increases in trichome production of leaves. The jar1-1 mutant exhibited normal trichome induction following treatment with jasmonic acid, suggesting that adenylation of jasmonic acid is not necessary. Salicylic acid had a negative effect on trichome production and consistently reduced the effect of jasmonic acid, suggesting negative cross-talk between the jasmonate and salicylate-dependent defense pathways. Interestingly, the effect of salicylic acid persisted in the nim1-1 mutant, suggesting that the Npr1/Nim1 gene is not downstream of salicylic acid in the negative regulation of trichome production. Last, we found that gibberellin and jasmonic acid had a synergistic effect on the induction of trichomes, suggesting important interactions between these two compounds.
Changes in Surface Area of Intact Guard Cells Are Correlated with Membrane Internalization
Authors: Shope Joseph C. , DeWald Daryll B. , Mott Keith A.
Published: November 2003

Guard cells must maintain the integrity of the plasma membrane as they undergo large, rapid changes in volume. It has been assumed that changes in volume are accompanied by changes in surface area, but mechanisms for regulating plasma membrane surface area have not been identified in intact guard cells, and the extent to which surface area of the guard cells changes with volume has never been determined. The alternative hypothesis—that surface area remains approximately constant because of changes in shape—has not been investigated. To address these questions, we determined surface area for intact guard cells of Vicia faba as they underwent changes in volume in response to changes in the external osmotic potential. We also estimated membrane internalization for these cells. Epidermal peels were subjected to external solutions of varying osmotic potential to shrink and swell the guard cells. A membrane-specific fluorescent dye was used to identify the plasma membrane, and confocal microscopy was used to acquire a series of optical paradermal sections of the guard cell pair at each osmotic potential. Solid digital objects representing the guard cells were created from the membrane outlines identified in these paradermal sections, and surface area, volume, and various linear dimensions were determined for these solid objects. Surface area decreased by as much as 40% when external osmotic potential was increased from 0 to 1.5 MPa, and surface area varied linearly with volume. Membrane internalization was approximated by determining the amount of the fluorescence in the cell's interior. This value was shown to increase approximately linearly with decreases in the cell's surface area. The changes in surface area, volume, and membrane internalization were reversible when the guard cells were returned to a buffer solution with an osmotic potential of approximately zero. The data show that intact guard cells undergo changes in surface area that are too large to be accommodated by plasma membrane stretching and shrinkage and suggest that membrane is reversibly internalized to maintain cell integrity.
Vascularization, High-Volume Solution Flow, and Localized Roles for Enzymes of Sucrose Metabolism during Tumorigenesis byAgrobacterium tumefaciens
Authors: Wächter Rebecca , Langhans Markus , Aloni Roni , Götz Simone , Weilmünster Anke , Koops Ariane , Temguia Leopoldine , Mistrik Igor , Pavlovkin Jan , Rascher Uwe , Schwalm Katja , Koch Karen E. , Ullrich Cornelia I.
Published: November 2003

Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens. Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host. Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment. CO2 release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks. However, tumor water loss contributed little to water flow to host shoots. Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance. Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors. Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization. Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase. Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport. Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment.
Analysis of the Alternative Oxidase Promoters from Soybean
Authors: Thirkettle-Watts David , McCabe Tulene C. , Clifton Rachel , Moore Carolyn , Finnegan Patrick M. , Day David A. , Whelan James
Published: November 2003

Alternative oxidase (Aox) is a nuclear-encoded mitochondrial protein. In soybean (Glycine max), the three members of the gene family have been shown to be differentially expressed during normal plant development and in response to stresses. To examine the function of the Aox promoters, genomic fragments were obtained for all three soybean genes: Aox1, Aox2a, and Aox2b. The regions of these fragments immediately upstream of the coding regions were used to drive β-glucuronidase (GUS) expression during transient transformation of soybean suspension culture cells and stable transformation of Arabidopsis. The expression patterns of the GUS reporter genes in soybean cells were in agreement with the presence or absence of the various endogenous Aox proteins, determined by immunoblotting. Deletion of different portions of the upstream regions identified sequences responsible for both positive and negative regulation of Aox gene expression in soybean cells. Reporter gene analysis in Arabidopsis plants showed differential tissue expression patterns driven by the three upstream regions, similar to those reported for the endogenous proteins in soybean. The expression profiles of all five members of the Arabidopsis Aox gene family were examined also, to compare with GUS expression driven by the soybean upstream fragments. Even though the promoter activity of the upstream fragments from soybean Aox2a and Aox2b displayed the same tissue specificity in Arabidopsis as they do in soybean, the most prominently expressed endogenous genes in all tissues of Arabidopsis were of the Aox1 type. Thus although regulation of Aox expression generally appears to involve the same signals in different species, different orthologs of Aox may respond variously to these signals. A comparison of upstream sequences between soybean Aox genes and similarly expressed Arabidopsis Aox genes identified common motifs.
Microspore Separation in thequartet 3Mutants of Arabidopsis Is Impaired by a Defect in a Developmentally Regulated Polygalacturonase Required for Pollen Mother Cell Wall Degradation
Authors: Rhee Seung Y. , Osborne Erin , Poindexter Patricia D. , Somerville Chris R.
Published: November 2003

Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development. Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging. QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica). The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity. In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings. Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage. Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development.
Sudden neonatal death in PACAP‐deficient mice is associated with reduced respiratory chemoresponse and susceptibility to apnoea
Authors: Cummings Kevin J. , Pendlebury Jonathan D. , Sherwood Nancy M. , Wilson Richard J. A.
Published: November 2003

Pituitary adenylate cyclase‐activating polypeptide (PACAP)‐deficient mice are more prone to sudden death during postnatal weeks 1–3 than wild‐type littermates. Given that PACAP is localized in brainstem regions associated with respiratory chemosensitivity, we examined whether PACAP‐null neonates have reduced respiratory responses to hypoxia and hypercapnia. Using unrestrained, whole‐body, flow‐through plethysmography we found that, by postnatal day 4, the PACAP‐null neonates had significantly reduced ventilation during baseline breathing, and blunted responses to both hypoxia (10% O2–90% N2) and hypercapnia (8% CO2–92% air). To determine whether the respiratory phenotype of the PACAP‐null mice may contribute to their greater neonatal mortality, we used ECG to examine respiration and cardiovascular function of littermates. We demonstrate that, under conditions that exacerbate mortality of knockout but not wild‐type animals, PACAP‐deficient mice experience prolonged apnoeas that precede atrio‐ventricular block. Both apnoeas and atrio‐ventricular block were absent in wild‐type littermates. These data suggest that PACAP‐deficiency results in higher neonatal mortality primarily as a result of respiratory control defects and raise the possibility that mutations in genes encoding components of the PACAP signalling pathways may contribute to neonatal breathing disorders in humans.
Localised calcium release events in cells from the muscle of guinea‐pig gastric fundus
Authors: Parsons and S. P. , Bolton T. B.
Published: November 2003

After enzymatic dispersion of the muscle of the guinea‐pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non‐propagating) intracellular calcium ([Ca2+]i) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio‐temporal characteristics (full width and full duration at half‐maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell‐wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium‐free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2‐APB (100 μm), ryanodine (10 or 50 μm) or U‐73122 (1 μm). 2‐APB (100 μm), xestospongin C (XeC, 10 μm) or U‐73122 (1 μm) blocked both spontaneous localized calcium release and localized release stimulated by 10 μm carbachol. Ryanodine (50 μm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.
Mexiletine block of wild‐type and inactivation‐deficient human skeletal muscle hNav1.4 Na+ channels
Authors: Wang Ging Kuo , Russell Corinna , Wang Sho‐Ya
Published: November 2003

Mexiletine is a class 1b antiarrhythmic drug used for ventricular arrhythmias but is also found to be effective for paramyotonia congenita, potassium‐aggravated myotonia, long QT–3 syndrome, and neuropathic pain. This drug elicits tonic block of Na+ channels when cells are stimulated infrequently and produces additional use‐dependent block during repetitive pulses. We examined the state‐dependent block by mexiletine in human skeletal muscle hNav1.4 wild‐type and inactivation‐deficient mutant Na+ channels (hNav1.4‐L443C/A444W) expressed in HEK293t cells with a β1 subunit. The 50% inhibitory concentrations (IC50) for the inactivated‐state block and the resting‐state block of wild‐type Na+ channels by mexiletine were measured as 67.8 ± 7.0 μm and 431.2 ± 9.4 μm, respectively (n= 5). In contrast, the IC50 for the block of open inactivation‐deficient mutant channels at +30 mV by mexiletine was 3.3 ± 0.1 μm (n= 5), which was within the therapeutic plasma concentration range (2.8–11 μm). Estimated on‐ and off‐rates for the open‐state block by mexiletine at +30 mV were 10.4 μm−1 s−1 and 54.4 s−1, respectively. Use‐dependent block by mexiletine was greater in inactivation‐deficient mutant channels than in wild‐type channels during repetitive pulses. Furthermore, the IC50 values for the block of persistent late hNav1.4 currents in chloramine‐T‐pretreated cells by mexiletine was 7.5 ± 0.8 μm (n= 5) at +30 mV. Our results together support the hypothesis that the in vivo efficacy of mexiletine is primarily due to the open‐channel block of persistent late Na+ currents, which may arise during various pathological conditions.
Hypoxia preconditioning attenuates bladder overdistension‐induced oxidative injury by up‐regulation of Bcl‐2 in the rat
Authors: Yu Hong‐Jeng , Chien Chiang‐Ting , Lai Yu‐Jen , Lai Ming‐Kuen , Chen Chau‐Fong , Levin Robert M. , Hsu Su‐Ming
Published: November 2003

We explored whether hypoxic preconditioning minimizes oxidative injury induced by overdistension/emptying in the rat bladder. For hypoxic preconditioning, female Wistar rats were placed in a hypobaric chamber (380 Torr) 15 h day−1 for 28 days. Overdistension was induced by infusion of two times the threshold volume of saline into the bladder and was maintained for 1 or 2 h, followed by drainage/emptying. During overdistension (ischaemia) and emptying (reperfusion) periods, a bursting increase of reactive oxygen species (ROS) from the bladder was originated from the large numbers of infiltrating leucocytes and scattered resident cells, including urothelial, submucosal, and smooth muscle cells. ROS impaired the voiding function by a reduction of bladder afferent and efferent nerve activity and bethanecol‐ or ATP‐induced detrusor contraction. ROS enhanced pro‐apoptotic mechanisms, including increases in the Bax/Bcl‐2 ratio, CPP32 expression, and poly(ADP‐ribose) polymerase (PARP) fragments with subsequent apoptotic cell formation in the insulted bladders. Hypoxia preconditioning up‐regulated Bcl‐2 expression in the bladder and significantly reduced the levels of ROS and apoptosis detected in the overdistension/emptying bladders and preserved partial voiding function. Bcl‐2 up‐regulation by hypoxia preconditioning contributes protection against overdistension/emptying‐induced oxidative stress and injury in the bladder.
Future of primary healthcare education: current problems and potential solutions
Authors: Lord J
Published: November 2003

This review examines the origins of primary care and the pressures currently faced in terms of patient expectation, regulation, accountability, and work force shortages. It recognises the appropriateness of adding to the burden in primary care further by the shift both of more services and more medical education from secondary care. Some conclusions are drawn concerning potential solutions including skill mix changes, centralisation of services, a change in attitudes to professional mistakes, increased protected development time, evidence based education, and academic, leadership, and feedback skills for general practitioners. Six recommendations are offered as a prescription for organisational and educational change.
Neuropsychiatric non-motor aspects of Parkinson’s disease
Authors: Thanvi B R , Munshi S K , Vijaykumar N , Lo T C N
Published: November 2003

Parkinson’s disease is often recognised as a motor disease characterised by rest tremor, rigidity, bradykinesia, and postural disturbances. However, there are several non-motor aspects of the disease that are of at least equal importance in the management of patients with Parkinson’s disease. They include depression, cognitive impairment, anxiety, and psychosis among others. It is important to recognise them, as they are common and they contribute significantly to patients’ morbidity, quality of life, and institutionalisation to long term care homes. In addition to the disease duration and severity, other factors including drugs may contribute to their occurrence. Pathogenesis of these aspects is not fully understood, though there has been a significant increase in the knowledge in recent years. Management of these aspects involves a multidisciplinary approach.
SUPPORTING DECISION-MAKING IN SOFTWARE ENGINEERING WITH PROCESS SIMULATION AND EMPIRICAL STUDIES
Authors: RUS IOANA , HALLING MICHAEL , BIFFL STEFAN
Published: November 2003

Decision-making is a complex and important task in software engineering. The current state-of-the-practice is rather non-systematic as it typically relies upon personal judgment and experience without using explicit models. Empirical studies can help but they are costly to conduct and, to some extent, context dependent. Typically it is not efficient or even possible to conduct empirical studies for a large number of context parameter variations. Process simulation offers decision support as well, but currently suffers from a lack of empirical knowledge on the determinants of underlying system dynamics. In this paper we present an assessment of empirical knowledge and simulation techniques for the area of quality assurance planning. There is a strong interdependency between process simulation and empirical models for decision-making in this area: (a) profound empirical knowledge enables process simulation to support decision-making, and (b) the analysis of simulation results can point out situations and factors for which conducting empirical studies would be most worthwhile. This paper discusses critically some of the most important challenges for decision-making in the area of quality assurance planning.
A QUALITY-DRIVEN DECISION-SUPPORT METHOD FOR IDENTIFYING SOFTWARE ARCHITECTURE CANDIDATES
Authors: SVAHNBERG MIKAEL , WOHLIN CLAES , LUNDBERG LARS , MATTSSON MICHAEL
Published: November 2003

To sustain the qualities of a software system during evolution, and to adapt the quality attributes as the requirements evolve, it is necessary to have a clear software architecture that is understood by all developers and to which all changes to the system adheres. This software architecture can be created beforehand, but must also be updated as the domain of the software, and hence the requirements on the software system evolve. Creating a software architecture for a system or part of a system so that the architecture fulfils the desired quality requirements is often hard. In this paper we propose a decision-support method to aid in the understanding of different architecture candidates for a software system. We propose a method that is adaptable with respect to both the set of potential architecture candidates and quality attributes relevant for the system's domain to help in this task. The method creates a support framework, using a multi-criteria decision method, supporting comparison of different software architecture candidates for a specific software quality attribute and vice versa, and then uses this support framework to reach a consensus on the benefits and liabilities of the different software architecture candidates and to increase the confidence in the resulting architecture decision.
SUPPORTING SOFTWARE PROCESS DECISIONS USING BI-DIRECTIONAL SIMULATION
Authors: RAFFO DAVID M. , SETAMANIT SIRI-ON
Published: November 2003

In this paper, we present a "forward-looking" decision support framework that integrates timely metrics data with a simulation based defect model of the software development process in order to support software project management decisions regarding product quality. These predictions are evaluated using Outcome Based Control Limits (OBCLs) and Bi-Directional (both forward and reverse) simulation models of the software development process. The Bi-Directional models provide useful guidance to the program manager when evaluating possible corrective actions on a project that has gone outside the OBCLs. The forward model predicts the potential performance impacts of the proposed corrective actions. The reverse simulation models are new and take the desired system outcome as the starting point and represent the system as it evolves backward in time to identify the necessary starting point to achieve the desired outcome. The approach is presented using an illustrative example.
Site Specificity of Atherosclerosis
Authors: VanderLaan Paul A. , Reardon Catherine A. , Getz Godfrey S.
Published: November 2003

Atherosclerosis is a complex disease process that affects very specific sites of the vasculature. It is recognized that hemodynamic forces are largely responsible for dictating which vascular sites are either susceptible or resistant to developing atherosclerosis. In addition, a number of systemic and local factors also modulate the pathogenesis of the disease. By studying the development of atherosclerosis in mice, investigators have gained insights into the molecular mechanisms of this disease, although studies have largely focused on a single vascular site. Here, we review those recent studies in which vascular site-specific effects on atherosclerosis were reported when more than 1 site was examined. We assess the hypothesis that regional differences in the hemodynamic profile prime the endothelial phenotype to respond distinctly to such systemic risk factors as hypercholesterolemia, genetics, immune status, gender, and oxidative stress. Because a given treatment may differentially affect the development of atherosclerotic lesions throughout the vasculature, the sites chosen for study are critically important. By accounting for the complex interplay of factors that may operate at these different sites, a more complete understanding of the overriding mechanisms that control the initiation and progression of the atherosclerotic lesion may be realized.
Regulation of Endogenous Reactive Oxygen Species in Platelets Can Reverse Aggregation
Authors: Clutton Patricia , Miermont Anne , Freedman Jane E.
Published: November 2003

Objective—

While much is known about the normal activation of platelets, there have been few observations demonstrating reversibility of the aggregation process. Inhibition of phosphoinositide 3-kinase (PI3-kinase) has been shown to cause platelet disaggregation. In addition, NO is a known potent inhibitor of platelet function. In this study, the role of PI3-kinase in the regulation of endogenous platelet NO and the relevance to platelet function was determined.

Methods and Results—

Incubation of platelets with PI3-kinase inhibitors led to a dose-dependent increase in platelet NO and cGMP levels that were temporally related to the period of platelet disaggregation. Addition of ferroheme myoglobin eliminated both the augmented NO release and disaggregation. PI3-kinase inhibition decreased the functional activation of NADPH oxidase and this corresponded to decreased superoxide release. To confirm these findings, platelets from NOS III-deficient mice were studied. These platelets did not release NO, and PI3-kinase inhibition led to decreased superoxide but not platelet disaggregation.

Conclusion—

Overall, these results indicate that platelet-derived NO contributes to the process of platelet disaggregation. PI3-kinase plays a role in regulating NADPH oxidase-generated superoxide in platelets and, by altering the bioactivity of platelet NO, may be a potential method for reversing platelet aggregation and thrombus formation.
Thrombomodulin and Tissue Factor Pathway Inhibitor in Endocardium of Rapidly Paced Rat Atria
Authors: Yamashita Takeshi , Sekiguchi Akiko , Iwasaki Yu-ki , Sagara Kouichi , Hatano Seiji , Iinuma Hiroyuki , Aizawa Tadanori , Fu Long-Tai
Published: November 2003

Background—

Atrial fibrillation (AF) is well known as one of the cardiogenic causes for thromboembolism. Although decreased flow and hypercoagulable state of the blood in the fibrillating atrium have been emphasized as the underlying mechanisms, endocardial dysfunction in maintaining the local coagulation balance could also contribute to the thrombogenesis in AF.

Methods and Results—

The paroxysmal AF model was created by rapid atrial pacing in anesthetized rats. To test the hypothesis that AF induces local coagulation imbalance by disturbing the atrial endocardial function, the gene expression of intrinsic anticoagulant factors, thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI), were determined by means of ribonuclease protection assay, Western blotting, and immunohistochemistry. Rapid atrial pacing for 8 hours significantly decreased TM and TFPI mRNA levels in the left atrium but not in the ventricle, leading to the downregulation of their immunoreactive proteins. Immunohistochemical analysis revealed that TM and TFPI were expressed predominantly in the endocardial cells of the normal atrium, presumably preventing local blood coagulation, and that rapid atrial pacing induced the loss of TM and TFPI expression in the endocardium, leading to deficiency in anticoagulant barriers between the atria and the blood.

Conclusions—

Rapid atrial pacing acutely downregulated the gene expression of TM and TFPI in the atrial endocardium, thereby inducing local coagulation imbalance on the internal surface of the atrial cavity. These results would support the validity of supplement of anticoagulant molecules deficient in AF.