Direct monitoring of the calcium concentration in the sarcoplasmic and endoplasmic reticulum of skeletal muscle myotubes

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100-1000 microM range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 microM, whereas that measured with erAEQ is significantly lower, i.e. around 200 microM. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed.     

Ultrasound‐activated Knoevenagel condensation of malononitrile with carbonylic compounds catalysed by alkaline‐doped saponites

α,β‐Unsaturated nitriles have been synthesized by Knoevenagel condensation of a carbonylic compound with malononitrile, assisted by sonochemical irradiation. Two alkaline‐promoted clays (Li⁺‐ and Cs⁺‐exchanged saponites) have been employed as catalysts. The influence of the carbonylic compound (benzaldehyde or cyclohexanone) and the use of a solvent on the catalytic activity have been studied. Remarkable increase in the conversion values has been found when the reaction is activated by ultrasound, as compared with the thermal activation. In this green, solvent‐free procedure, α,β‐unsaturated nitriles have been produced in very high yields (97%) when the Cs⁺‐saponite is used as catalyst. Copyright © 2004 Society of Chemical Industry     

A subunit interaction in chloroplast ATP synthase determined by genetic complementation between chloroplast and bacterial ATP synthase genes

F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.     

Micromanipulation and cloning studies on buffalo oocytes and embryos using nucleus transfer

1. Nine male volunteers were exposed to the pyrethroid insecticide cyfluthrin. The study was performed in an exposure room, where an aerosol containing cyfluthrin was sprayed to obtain atmospheres with mean cyfluthrin concentrations of 160 and 40 micrograms/m3. Four volunteers were exposed for 10, 30 and 60 min at 160 micrograms/m3 and another five volunteers were exposed for 60 min at 40 micrograms/m3. For 160 micrograms/m3 exposure urine samples were collected before and immediately after exposure as well as for the periods 1-2, 2-3, 3-4, 4-5, 5-6, 6-12 and 12-24 h after exposure. For 40 micrograms/m3 exposure urine samples were collected before and 2 h after exposure. 2. The main urinary cyfluthrin metabolites, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylycyclopropane carboxylic acid (DCCA) and 4-fluoro-3-phenoxybenzoic acid (FPBA), were determined. The limit of detection (LOD) for all metabolites was 0.0025 microgram in an urine sample of 5 ml (0.5 microgram/l). After inhalative exposure of 40 micrograms cyfluthrin/m3 air for 60 min, the amount of metabolites in urine collected in the first 2 h after exposure was less than the LOD, namely 0.14 microgram for cis-DCCA, 0.15-0.28 microgram for trans-DCCA and 0.12-0.23 microgram for FPBA. 3. Of the metabolites, 93% was excreted within the first 24 h (peak excretion rates between 0.5 and 3 h) after inhalative exposure of 160 micrograms/m3. The mean half-lives were 6.9 h for cis-DCCA, 6.2 h for trans-DCCA and 5.3 h for FPBA. 4. The mean trans-:cis-DCCA ratio was 1.9 for the time course as well as for each subject. 5. The amount of metabolites in urine depends on the applied dose, on the exposure time and shows interindividual differences.     

Prognostic significance of color Doppler findings in head and neck tumors

This summary of the practice parameters describes the assessment, differential diagnosis, and treatment of children, adolescents, and adults who present with symptoms of attention-deficit/hyperactivity disorder. The rationales for specific recommendations are based on a review of the scientific literature and clinical consensus which is contained in the complete document. Assessment includes clinical interviews with the child and parents and standardized rating scales from parent and teachers. Testing of intelligence and academic achievement is usually required. Comorbidity is common. The cornerstones of treatment are support and education of parents, appropriate school placement, and psychopharmacology. The primary medications are psychostimulants, but antidepressants and alpha-adrenergic agonists are used in special circumstances. Other treatments such as behavior modification, school consultation, family therapy, and group therapy address remaining symptoms.     

Coupling GIS with Hydrologic and Hydraulic Flood Modelling

Geographic Information Systems (GIS) have been recognised as a powerful means to integrate and analyse data from various sources in the context of comprehensive floodplain management. As part of this comprehensive approach to floodplain management, it is very important to be able to predict the consequences of different scenarios in terms of flooded areas and associated risk. Hydrologic and hydraulic modelling plays a crucial role and there is much to gain in incorporating these modelling capabilities in GIS. This is still a rather complex task and research is being done on the full integration of these models. Interfacing between these models and GIS may be a very efficient way of overcoming the difficulties and getting very good results in terms of engineering practice. This paper presents results based on the use of Intergraph GIS coupled with Idrisi GIS. Using these two systems substantially increased the flexibility of using GIS as a tool for flood studies. A lumped (XSRAIN) and a distributed (OMEGA) hydrologic models were used to simulate flood hydrographs. The well known HEC-2 Hydraulic model was used to compute flooded areas. These models were applied in the Livramento catchment with very good results. The computation of flooded areas for different flood scenarios, and its representation in GIS, can be used in the assessment of affected property and associated damages. This is a very useful GIS-based approach to floodplain management.