bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of Protein Interactions In Vivo with Fluorescence Resonance Energy Transfer (FRET)
Authors: Damelin Marc , Silver Pamela 
Published: October 2006

INTRODUCTIONThe protocol presented here focuses on a standard microscope-based fluorescence resonance energy transfer (FRET) assay for budding yeast, but many aspects are applicable to other systems and assays. It provides information on experimental design and data analysis procedures that can be extended to FRET experiments in any system, including mammalian cell lines and other model organisms. The steps describing strain construction can be applied to studies in yeast using fluorimetry instead of microscopy. Although the data acquisition and analysis described in this protocol can be a daunting task, careful quantitative analysis is absolutely necessary because of a high degree of cross-talk and many unknown parameters that vary from cell to cell.
Preparation of Vertical SDS Slab Gels: Simultaneous Casting of Multiple-Gradient Gels
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy 
Published: October 2006

INTRODUCTIONGradient SDS-PAGE gels provide the best resolution over a wide range of molecular weights, resulting in sharper protein spots, because diffusion is minimized by the decreasing pore size in the gel. However, gradient gels are more difficult to produce reproducibly; thus, they are commonly cast with multiple-gel casters, which allows for an identical set of gels to be produced for an experiment. Presented here is a method for casting gradient gels using a multiple-gel casting system.
Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IPG Strip Equilibration
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy 
Published: October 2006

INTRODUCTIONThe equilibration step serves to saturate the IPG strip with the SDS buffer system required for the second-dimension separation. The equilibration solution consists of buffer, urea, glycerol, reductant, SDS, and dye. The buffer (50 mM Tris-HCl, pH 8.8) maintains the appropriate pH range for electrophoresis. Urea and glycerol are added to reduce the effects of electroendosmosis, thus helping improve protein transfer from the IPG strip to the second dimension. The reductant (dithiothreitol) ensures that disulfide bridges are broken. SDS ensures that the proteins are denatured and also provides a net negative charge to all proteins. Iodoacetamide, introduced during a second equilibration step, alkylates thiol groups on the proteins, preventing their reoxidation during electrophoresis, and thus reducing streaking and other artifacts in the second-dimension separation. Iodoacetamide also alkylates residual dithiothreitol, preventing point streaking and other silver staining artifacts. Finally, a tracing dye (bromophenol blue) is added to allow the electrophoresis to be monitored during the run.
EMS Mutagenesis of Arabidopsis Seed
Authors: Weigel Detlef , Glazebrook Jane 
Published: October 2006

INTRODUCTIONEMS-mutagenized Arabidopsis seed collections can be purchased from companies or acquired from other researchers. But in some cases (e.g., mutagenesis of a specific genotype), it is necessary for an investigator to generate EMS-mutagenized seed. In this protocol, EMS is used at concentrations that induce multiple point mutations in each plant, such that mutant alleles of a specific locus are found at a rate of ~1 in 2000-5000 M2 plants. This high rate of mutagenesis makes possible the screening of relatively few plants to find those with the phenotype of interest, a particular advantage if the screen is laborious or if only a small number of genes mutate to the required phenotype.
The effect of endogenous nitric oxide on mechanical ciliostimulation of human nasal mucosa
Authors: Alberty J. , Stoll W. , Rudack C. 
Published: October 2006

SummaryBackground
Endogenous nitric oxide (NO) production by the inducible NO‐synthase is enhanced in the nasal respiratory epithelium of patients with allergic rhinitis. Recent experimental data suggest endogenous NO to be strongly involved in the regulation of ciliary activity, the driving force of the mucociliary transport system.Objective
In this study, we investigated the effect of endogenous NO on mechanical stimulation of ciliary activity in a nasal mucosa explant model.Methods
Cultures of nasal mucosa explants were incubated with TNF‐α and bacterial lipopolysaccharides (LPS) to enhance endogenous NO production. Direct in vitro NO imaging was performed by the fluorescent NO‐indicator DAF‐2 DA and laser scanning confocal microscopy. Ciliary beat frequency (CBF) was determined using a photoelectric technique. Mechanical stimulation was performed by two consecutive flow increments in a closed perfusion chamber. Endogenous NO‐synthesis was blocked by l‐NAME before the second flow stimulation.Results
Under control conditions the mean rise of CBF relative to baseline was 30.2% during the first flow increment and 30.7% during the second flow increment. Blocking of the endogenous NO synthesis in TNF‐α/LPS‐stimulated cultures reduced baseline CBF by 10.6±2.1% (P<0.05) but the effect of mechanical ciliostimulation on CBF remained unchanged (36.0% vs. 38.2%).Conclusion
In conclusion, endogenous NO‐ and Ca2+‐dependent mechanical stimulation of ciliary activity probably use independent intracellular signalling pathways. The combination of both effects on ciliary activity is likely to improve the local defence against inhaled allergens in patients with nasal allergies.
Role of intercellular adhesion molecule‐1 in a murine model of toluene diisocyanate‐induced asthma
Authors: Furusho S. , Myou S. , Fujimura M. , Kita T. , Yasui M. , Kasahara K. , Nakao S. , Takehara K. , Sato S. 
Published: October 2006

SummaryBackground
Adhesion molecules such as intercellular adhesion molecule‐1 (ICAM‐1) are thought to contribute to the airway inflammation and airway hyper‐responsiveness (AHR) of allergic asthma. Some differences from allergic asthma have been noted, including airway neutrophilia, and the involvement of ICAM‐1 in toluene diisocyanate (TDI) asthma is currently unclear.Objective
We utilized mice lacking ICAM‐1 expression (ICAM‐1−/−) to investigate the role of ICAM‐1 in airway inflammation and AHR in TDI‐induced asthma.Methods
Male C57BL/6J mice (ICAM‐1+/+) and ICAM‐1−/− mice were intranasally sensitized to TDI solution or solvent alone. Airway inflammation, AHR and cytokine secretion were assessed 24 h after challenge by TDI or solvent. The production of antigen‐specific IgG and IgE by TDI sensitized and non‐sensitized mice was determined.Results
TDI challenge to ICAM‐1+/+ mice induced an increase in airway inflammatory cell numbers, AHR and cytokine secretion of TNF‐α, macrophage inflammatory protein‐2 (MIP‐2), IL‐4, IL‐5 and IFN‐γ into the bronchoalveolar lavage fluid. All these pathophysiological changes were reduced in ICAM‐1−/− mice. Serum levels of TDI‐specific IgG and IgE of ICAM‐1−/− and ICAM‐1+/+ mice were comparable.Conclusion
These results suggest that ICAM‐1 plays an essential role in airway inflammation and AHR in TDI‐induced asthma.
Changes in Plasma Adrenocorticotropin, Corticosterone, Corticosteroid‐Binding Globulin, and Hippocampal Glucocorticoid Receptor Occupancy/Translocation in Rat Pups in Response to Stress
Authors: Viau Victor , Sharma Shakti , Meaney Michael J. 
Published: October 2006

AbstractPituitary‐adrenal responses to stress in the neonatal rat have been reported to be substantially reduced compared to older animals (i.e. a stress hyporesponsive period). This supposed period of endocrine quiescence is characterized by reduced stress‐induced increases in both plasma ACTH and corticosterone. At the same time a number of authors have noted the decreased plasma corticosteroid‐binding globulin (CBG) levels of the neonate, and there is evidence for an increased percentage of free corticosterone as well as age‐related changes in the volume of distribution for corticosterone. These findings suggest that the reduced CBG levels might enhance the biological significance of existing glucocorticoid levels, beyond that assumed on the basis of plasma total corticosterone levels. We examined this question by estimating hippocampal glucocorticoid receptor occupancy and ‘translocation’ in Day 6, Day 15, and adult animals under basal and stressful conditions. The results showed that: 1) plasma ACTH levels were elevated in Day 6 animals in response to acute exposure to ether, maternal separation, and maternal separation + ether, however, ACTH responses were substantially lower than in Day 15 or adult animals; 2) Plasma total corticosterone levels followed a similar pattern; most noteworthy was the potent glucocorticoid response in Day 15 animals to the combination of maternal separation + ether; 3) Plasma CBG levels in Day 6 animals were extremely low (<3% adult values); by Day 15 CBG levels were about 25% of adult levels. Interestingly, maternal separation was associated with a substantial decrease in plasma CBG levels; 4) Hippocampal glucocorticoid receptor occupancy/translocation was similar at all ages under both basal and stress conditions. The only notable exception occurred during maternal separation in Day 15 animals, where the percentage of hippocampal glucocorticoid receptor occupancy/translocation was higher than that observed at any time in either Day 6 or adult animals. This finding is likely related to the decrease in plasma CBG that occurs following separation of Day 15 pups from the dam.Thus, despite the higher corticosterone level in the adult, the increase in glucocorticoid receptor occupancy/translocation was generally comparable across all ages either under basal conditions, or following stress. These receptor data underscore the importance of developmental changes in plasma CBG levels.
Diuretic and Natriuretic Actions of Melanin Concentrating Hormone in Conscious Sheep
Authors: Parkes David G. 
Published: October 2006

AbstractMelanin concentrating hormone (MCH) is a 19 amino‐acid peptide expressed in high concentrations within the dorso‐lateral hypothalamus of rats, sheep and man. MCH regulates skin colour and ACTH release in teleost fish, however, its physiological relevance in mammals is unclear. The present study examined the cardiovascular and metabolic actions of intracerebroventricular (i.e.v.) infusion of MCH, and the pro‐MCH derived peptide Neuropeptide‐E‐I (NEI), in conscious, chronically instrumented sheep. Human MCH (1–19) or NEI (1–13) was infused i.e.v. for 24 h into 6 sheep, and measurements were made every 10 min of arterial pressure, heart rate, cardiac output, stroke volume and peripheral blood flow/conductance. Recordings of water intake (H2Oin), urine volume (Uv), urinary Na (UNaV) and K excretion (UKV) were made, as well as hematocrit, plasma Na, K, osmolality, protein, glucose, ACTH, vasopressin, renin, endothelin, ANF, Cortisol and aldosterone concentrations. After 24 h of infusion at 10 μg/h, MCH produced a significant increase in Uv from 0.8 ± 0.2 to 1.4 ± 0.3 l/day, together with an increase in UNaV from 56 ± 8 to 107 ± 14 mmol/day, and in UKV from 202 ± 18 to 369 ± 38 mmol/day. H2Oin was unchanged. Similar renal changes were observed during i.e.v. infusion of NEI. There was no change in any cardiovascular parameter, although hematocrit showed a large decrease with infusion of both peptides after 24 h infusion. Plasma osmolality increased from 291 ±1 to 295 ± 1 mOsm/kg during MCH infusion, whereas total protein and plasma Na and K were unchanged. MCH increased plasma glucose from 3.4 ± 0.2 to 3.8 ± 0.2 mmol/l. Plasma aldosterone exhibited a 30–40% decrease following MCH or NEI infusion, whereas all other plasma concentrations remained unchanged. This study has shown that i.c.v. infusion of MCH or NEI can produce diuretic, natriuretic and kaliuretic changes in conscious sheep, triggered by a possible increase in plasma volume as indicated by the changes in hematocrit. These results, together with anatomical data reporting the presence of MCH/NEI in fluid regulatory areas of the brain, indicate that MCH/NEI may be an important peptide involved in the central control of fluid homeostasis in mammals.
Estradiol, Progesterone, and Prolactin Regulate Maternal Nest‐Building in Rabbits
Authors: González‐Mariscal G. , Melo A. I. , Jiménez P. , Beyer C. , Rosenblatt J. S. 
Published: October 2006

AbstractMaternal nest‐building in rabbits, expressed across the last third of pregnancy, consists of: digging a burrow, collecting straw and shaping it into a nest inside the burrow, plucking body hair and lining the straw nest with it. The sequential expression of these activities is correlated with specific changes in the plasma concentration of estradiol, progesterone (P), and prolactin (PRL). To further substantiate the participation of these hormones in the control of maternal nest‐building we explored in ovariectomized (ovx) New Zealand white rabbits the capacity of several combinations of such hormones to stimulate digging, straw‐carrying, and hair‐pulling. Does given estradiol benzoate (EB; 5 μg/day from days 3 to 21) plus P (2 or 10 mg/day from days 4 to 16) dug into a substrate from the fourth day of the P treatment until withdrawal of this hormone. The intensity of this effect was greater in the group treated with the high dose of P. Straw‐carrying and hair‐pulling occurred after P withdrawal in a dose‐response way. Food intake, which declines in pregnant females shortly before parturition, decreased to the same extent in both groups of ovx EB‐treated does after P withdrawal. A significant increase in PRL plasma levels was observed on day 9 in does given EB plus 2 mg P/day and at two days following P withdrawal in does given EB plus 10 mg P/day. When such ovx EB/P‐treated does were given bromocriptine to block PRL release (1 or 3 mg/Kg/day, from days 11 to 21) the expression of digging was unmodified. By contrast, bromocriptine abolished the display of straw‐carrying and hair‐pulling, and also prevented the decline in food intake normally following P withdrawal. The addition of ovine PRL to ovx EB/P‐treated does given bromocriptine reduced the expression of digging, did not restore straw‐carrying or hair‐pulling, and provoked a sharp decline in food intake. The possible mechanisms of interaction between PRL and steroid hormones for the regulation of specific aspects of the pregnant doe's physiology and behavior are discussed.
The Relative Roles of the Hypothalamus and Cortisol in the Control of Prolactin Gene Expression in the Anterior Pituitary of the Sheep Fetus
Authors: Phillips I. D. , Fielke S. L. , Young I. R. , McMillen I. C. 
Published: October 2006

AbstractThe neuroendocrine control of prolactin synthesis and secretion before birth is not well understood. We have measured the changes in the level of prolactin mRNA in the anterior pituitary of the fetal sheep throughout the last 15 days of pregnancy (term = 147 ± 3 days gestation). We have also investigated the effects of surgical disconnection of the fetal hypothalamus and pituitary (HPD) with or without long term Cortisol infusion on pituitary prolactin mRNA levels and plasma prolactin concentrations in the late gestation sheep fetus. Prolactin mRNA levels were measured in anterior pituitaries collected from a series of fetal sheep (130–134 days, n = 6; 135–140 days, n = 6; 141–145 days, n = 6) in late gestation. HPD was carried out in ten fetal sheep at 105–115 days gestation and five intact fetal sheep were used as controls. In the HPD group, either saline (HPD + saline group, n = 5) or Cortisol was infused (3.5mg/24 h) for 5 days from 134–136 days gestation (HPD + Cortisol group, n = 5). There was an increase in the ratio of prolactin mRNA:18S rRNA in the fetal pituitary between 130–134 days (0.46 + 0.08, n = 6) and 135–140 days (1.27 ± 0.17 n = 6) which was maintained after 141 days gestation, (1.27 ± 0.11, n = 6). The mean prolactin mRNA: 18 S rRNA ratio was significantly higher (P<0.05) in intact fetal sheep (1.41 ± 0.16, n = 4) than in the HPD fetal sheep after either saline (0.54 ± 0.14, n = 4) or Cortisol (0.74 ± 0.24, n = 5) administration. The mean plasma concentration of prolactin was also higher in the intact group (28.3 ± 3.9 ng/ml) when compared with the HPD + saline group (8.0±3.3 ng/ml) or the HPD+cortisol group (5.6 ± 1.9 ng/ml). We have demonstrated that there is a strong hypothalamic drive to prolactin synthesis and secretion in the fetus and that Cortisol does not act directly at the fetal pituitary to stimulate prolactin synthesis and secretion in late gestation.
Acetylcholine Stimulates Growth Hormone Secretion in the Neonatal Rat Pituitary
Authors: Pintér I. , Makara G. B. , Ács Zs. 
Published: October 2006

AbstractThe direct effect of acetylcholine on pituitary growth hormone secretion during the postnatal period of the rat was studied using a superfusion system. Acetylcholine elicited a dose related stimulatory effect on growth hormone (GH) secretion in the pituitaries from 2‐day old rats. M, muscarinic agonist McN A343 mimicked the GH releasing effect of acetylcholine, nicotine was ineffective. The GH release elicited by acetylcholine diminished with postnatal development, it was small by the end of the third postnatal week and was not demonstrable in the adult pituitaries. The effect of acetylcholine was potently antagonized by pirenzepine (M1 antagonist) and 4‐DAMP (M3 and M1 antagonist) but not by methoctramine (M2 antagonist). It is concluded that unlike in the adult, in the newborn rat the cholinergic regulation of pituitary GH secretion plays a prominent role directly at pituitary level most likely via M1 receptors.
The annual cycle of Synechococcus (cyanobacteria) in the northern Levantine Basin shelf waters (Eastern Mediterranean)
Authors: Uysal Zahit , Köksalan İrem 
Published: October 2006

AbstractAbundance of picoplanktonic chroococcoid marine cyanobacteria Synechococcus was monitored weekly over the year 1998 in shallow coastal waters of the northern Levantine Basin. The ambient physical, chemical and biological variables (temperature, salinity, Secchi disk depth, total suspended sediment, nitrate, phosphate, Chl a and phytoplankton) were also measured. Synechococcus was found to be more abundant during summer and early autumn and less during winter and early spring. At the surface and 15 m depth, cell concentrations were in the range 6.4 × 103–1.5 × 105 and 3.2 × 103–1.6 × 105 cells·ml−1, respectively. Based on the Pearson product–moment correlation analysis, a highly significant correlation between Synechococcus abundance and ambient temperature was observed (n = 40, r = 0.558, P < 0.01). As Synechococcus forms blooms that usually do not last more than a week, the short time‐scale survey achieved in this study was appropriate to reveal its abundance dynamics. Several factors such as rapid changes in nutrient concentration (especially nitrate), phytoplankton, light availability, temperature, salinity, freshwater input and vertical mixing played a relevant role on the abundance of Synechococcus over the year in the highly dynamic shallow coastal waters of the Levantine Basin.
Identification and characterization of PEBP as a calpain substrate
Authors: Chen Qinghua , Wang Susan , Thompson Stephanie N. , Hall Edward D. , Guttmann Rodney P. 
Published: October 2006

AbstractCalpains are calcium‐ and thiol‐dependent proteases whose dysregulation has been implicated in a number of diseases and conditions such as cardiovascular dysfunction, ischemic stroke, and Alzheimer's disease (AD). While the effects of calpain activity are evident, the precise mechanism(s) by which dysregulated calpain activity results in cellular degeneration are less clear. In order to determine the impact of calpain activity, there is a need to identify the range of specific calpain substrates. Using an in vitro proteomics approach we confirmed that phosphatidylethanolamine‐binding protein (PEBP) as a novel in vitro and in situ calpain substrate. We also observed PEBP proteolysis in a model of brain injury in which calpain is clearly activated. In addition, with evidence of calpain dysregulation in AD, we quantitated protein levels of PEBP in postmortem brain samples from the hippocampus of AD and age‐matched controls and found that PEBP levels were approximately 20% greater in AD. Finally, with previous evidence that PEBP may act as a serine protease inhibitor, we tested PEBP as an inhibitor of the proteasome and found that PEBP inhibited the chymostrypsin‐like activity of the proteasome by ∼30%. Together these data identify PEBP as a potential in vivo calpain substrate and indicate that increased PEBP levels may contribute to impaired proteasome function.
Protective effects of an anti‐inflammatory cytokine, interleukin‐4, on motoneuron toxicity induced by activated microglia
Authors: Zhao Weihua , Xie Wenjie , Xiao Qin , Beers David R. , Appel Stanley H. 
Published: October 2006

AbstractMicroglia‐mediated cytotoxicity has been implicated in models of neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease, but few studies have documented how neuroprotective signals might mitigate such cytotoxicity. To explore the neuroprotective mechanism of anti‐inflammatory cytokines, we applied interleukin‐4 (IL‐4) to primary microglial cultures activated by lipopolysaccharide as well as to activated microglia cocultured with primary motoneurons. lipopolysaccharide increased nitric oxide and superoxide (O2·−) and decreased insulin‐like growth factor‐1 (IGF‐1) release from microglial cultures, and induced motoneuron injury in microglia‐motoneuron cocultures. However, lipopolysaccharide had minimal effects on isolated motoneuron cultures. IL−4 interaction with microglial IL‐4 receptors suppressed and nitric oxide release, and lessened lipopolysaccharide‐induced microglia‐mediated motoneuron injury. The extent of nitric oxide suppression correlated directly with the extent of motoneuron survival. Although IL‐4 enhanced release of free IGF‐1 from microglia in the absence of lipopolysaccharide, it did not enhance free IGF‐1 release in the presence of lipopolysaccharide. These data suggest that IL‐4 may provide a significant immunomodulatory signal which can protect against microglia‐mediated neurotoxicity by suppressing the production and release of free radicals.
Antisense‐Mediated Depletion of Tomato Chloroplast Omega‐3 Fatty Acid Desaturase Enhances Thermal Tolerance
Authors: Liu Xun‐Yan , Yang Jing‐Hua , Li Bin , Yang Xiu‐Mei , Meng Qing‐Wei 
Published: October 2006

AbstractA chloroplast‐localized tomato (Lycopersicon esculentum Mill.) ω‐3 fatty acid desaturase gene (LeFAD7) was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine‐rich regions, of which three regions were highly conserved throughout the whole ω‐3 fatty acid desaturase gene family. Southern blotting analysis showed that LeFAD7 was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7 was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7 was induced by chilling stress (4 °C), but was inhibited by high temperature (45 °C), in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7 DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18:3 content could maintain a higher maximal photochemical efficiency (Fv/Fm) and O2 evolution rate than wild‐type plants. These results suggested that silence of the LeFAD7 gene alleviated high‐temperature stress. There was also a correlation between the low content of 18:3 resulting from silence of the LeFAD7 gene and tolerance to high‐temperature stress.(Managing editor: Li‐Hui Zhao)