Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.     

Cloning the spoT gene of Escherichia coli: identification of the spoT gene product.

We have isolated five specialized transducing lambda bacteriophages (lambda dpyrE spoT) carrying the pyrE and spoT genes of Escherichia coli. A fragment from one of these phages was used as the source of DNA to clone the spoT and pyrE genes on a multicopy plasmid, pBR322. Insertions and deletions in this plasmid were obtained. These plasmids were used to transform a minicell-producing strain, and the gene products synthesized were determined. Our experiments demonstrate that the spoT and pyrE genes are separated by about 4 magadaltons and suggest that the spoT gene product is a protein whose molecular weight is 80,000. The strain in which the spoT+ allele is carried on a plasmid produced nine times more spoT gene activity than a normal spoT+ strain when assayed in crude extracts. This strain was used to prepare partially purified gene product, guanosine 5'-diphosphate, 3'-diphosphate pyrophosphatase. The enzyme has the following characteristics. (i) It hydrolyzes pyrophosphate from the 5'-pyrophosphate of guanosine 5'-diphosphate, 3'-diphosphate, yielding GDP and pyrophosphate. (ii) Its activity is strongly stimulated by Mn2+ and slightly stimulated by salt. (iii) Its activity is inhibited by uncharged tRNA. There are also two additional activities in the cell extract which degrade guanosine in 5'-diphosphate, 3'-diphosphate in vitro but which are not specified by the spoT gene.     

Genetic control of the serum concentration of an H-2-antigen-like polypeptide chain

Mouse serum contains a protein complex consisting of at least three polypeptide chains. One of the chains with an approximate molecular weight of 40 000 is similar to the heavy chain of normal H-2 antigens. The serum concentration of this 40 000 dalton chain is under genetic control. Formal genetic analyses in B10.M and B10.S mice, their F₁ progeny and in backcrosses show that there are two codominantly expressed alleles at a single locus regulating the serum concentration. Measurements of the 40 000 dalton chain in recombinant mice suggest that the controlling locus is situated to the right of theS region.     

Calcium in isolated mitochondria from the left ventricular wall

Summary The content of calcium per mg mitochondrial protein has been measured by conventional biochemical methods in myocardial tissue of some mammalian species. In addition, a method is presented for (1) the analysis of mitochondrial volumes in the same tissues and (2) calculating the amount of calcium in units of 10⁶ mitochondria.It appears that a highly significant correlation exists between the calcium content and the number of mitochondria, with a positive correlation coefficient of 0.92. The mean mitochondrial volume in fractions of the rabbit myocardium was found to be 1.3386 m³. Electron microscopic studies demonstrate pure mitochondrial fractions and only moderate structural alterations. The method described may represent a useful supplement for the estimation of calcium fluxes in mitochondria and of alterations in their volume, number and structure under conditions of myocardial ischemia.This work was supported by grants from the Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

pyrR identical to pyrH in Salmonella typhimurium: control of expression of the pyr genes.

Mutants of Salmonella typhimurium showing constitutive synthesis of the pyrimidine biosynthetic enzymes coded for by the pyrA-F genes (G. A. O'Donavan and J. C. Gerhart, 1972) have been reinvestigated. The high rate of expression of the pyrB-F genes in these mutants as well as their pyrimidine excretion is shown to be due to mutations in the gene pyrH encoding uridine 5'-monophosphate kinase. Thus, the term pyrR used for these mutants should be replaced by the designation pyrH.     

Growth, contents of K+ and kinetics of K+(86Rb) uptake in barley cultured at different low supply rates of potassium

Plants of barley ( Hordeum vulgare L. cv. Salve) were grown with 6.5–35% relative increase of K⁺ supply per day (RKR) using a special computer-controlled culture unit. After a few days on the culture solution the plants adapted their relative growth rate (RGR) to the rate of nutrient supply. The roots of the plants remained in a low salt status irrespective of the rate of nutrient supply, whereas the concentration of K⁺ in shoots increased with RKR. Both V_max and K_m for K⁺(⁸⁶Rb) influx increased with RKR. It is concluded that with a continuous and stable K⁺ stress, the K⁺ uptake system is adjusted to provide an effective K⁺ uptake at each given RKR. Allosteric regulation of K⁺ influx does not occur and efflux of K⁺ is very small.     

Expression of the heat-shock protein HSP70 in Drosophila buzzatii lines selected for thermal resistance

The level of HSP70 expression induced by a non-lethal high temperature was examined in lines selected for increased thermal resistance and in corresponding control lines of Drosophila buzzatii, in order to test if selection for high temperature resistance leads to an increased level of HSP70 expression. The lines used were selected for up to 64 generations either as adults or through all larval stages. In adult selection lines, hard selection was implemented every second generation after mild heat hardening. In larval selection lines, larvae were exposed each generation to laboratory "natural" selection. Generally lines selected as adults showed a higher HSP70 expression than did controls, both in third instar larvae and in adults. A strong negative response to selection of HSP70 expression was found in all lines that were selected at cycling temperatures during larval development. The results suggests that a trade off between heat resistance in form of HSP70 expression and fecundity/fertility are responsible for the level of HSP70 expression. The effect of the different methods of selection on HSP70 expression suggests that heat resistance constitutes more than one trait.     

Heterotrophic high-cell-density fed-batch and continuous-flow cultures of <Emphasis Type="Italic">Galdieria sulphuraria</Emphasis> and production of phycocyanin

Production of biomass and phycocyanin (PC) were investigated in highly pigmented variants of the unicellular rhodophyte Galdieria sulphuraria, which maintained high specific pigment concentrations when grown heterotrophically in darkness. The parental culture, G. sulphuraria 074G was grown on solidified growth media, and intensely coloured colonies were isolated and grown in high-cell-density fed-batch and continuous-flow cultures. These cultures contained 80–110 g L⁻¹ biomass and 1.4–2.9 g L⁻¹ PC. The volumetric PC production rates were 0.5–0.9 g L⁻¹ day⁻¹. The PC production rates were 11–21 times higher than previously reported for heterotrophic G. sulphuraria 074G grown on glucose and 20–287 times higher than found in phototrophic cultures of Spirulina platensis, the organism presently used for commercial production of PC.     

FGF10 signaling controls stomach morphogenesis

Maintenance of progenitor cell properties in development is required for proper organogenesis of most organs, including those derived from the endoderm. FGF10 has been shown to play a role in both lung and pancreatic development. Here we find that FGF10 signaling controls stomach progenitor maintenance, morphogenesis and cellular differentiation. Through a characterization of the initiation of terminal differentiation of the three major gastric regions in the mouse, forestomach, corpus and antrum, we first describe the existence of a "secondary transition" event occurring in mouse stomach between E15.5 and E16.5. This includes the formation of terminally differentiated squamous cells, parietal, chief and gastric endocrine cells from a pre-patterned gastric progenitor epithelium. Expression analysis of both FGF and Notch signaling components suggested a role of these networks in such progenitors, which was tested through ectopically expressing FGF10 in the developing posterior stomach. These data provide evidence that gastric gland specification and progenitor cell maintenance is controlled by FGF10. The glandular proliferative niche was disrupted in pPDX-FGF10(FLAG) mice leading to aberrant gland formation, and endocrine and parietal cell differentiation was attenuated. These effects were paralleled by changes in Hes1, Shh and Wnt6 expression, suggesting that FGF10 acts in concert with multiple morphogenetic signaling systems during gastric development.     

Feasibility Study of Performing an Life Cycle Assessment on Crude Palm Oil Production in Malaysia (9 pp)

Background, Goal and Scope The palm oil industry is one of the leading industries in Malaysia. With a yearly production of more than 13 million tons of crude palm oil (CPO) and plantations covering 11% of the Malaysian land area it is an industry to be reckoned with, also when it comes to environmental impacts. One way to describe and present the environmental impacts is through a life cycle assessment, LCA. This assessment aims to introduce the concept of LCA and perform a screening LCA on crude palm oil production in Malaysia including the stages of plantation, transport and milling. The assessment is largely based on general data and is thus meant to function as an indication of the environmental threads posed by CPO production and as a guideline to CPO producers and local universities on how to perform an LCA on a palm oil scenario. Due to the general data background the results of this report should not be quoted directly for decision making. The Functional Unit, to which all masses and emissions in this assessment have been adjusted, is the production of 1000 kg of CPO in Malaysia. Method Initially an overview of palm oil production was obtained and the outlines and borders of the assessment were determined along with the specific goal and scope of the assessment. The data for the assessment was collected from three different sources: - 1. Earlier studies and statistics on palm oil production in Malaysia - 2. Studies on similar processes, when palm oil related processes were not available - 3. General data from the SimaPro 5 database - The European Eco-Indicator 99 method and European databases included in the LCA software SimaPro 5 have been used for the impact calculations. Results and Discussion The impact processes related to the plantation are the on-site energy use (mainly diesel) and the production of artificial fertilizer. Pesticide use contributes a minor impact due to widely used integrated biological poet management. For transportation the only impact is from combustion of diesel and at the mill the boiler is the sole significant contributor – positively through electricity production and negatively by emissions from the boiler. Impacts from POME (Palm Oil Mill Effluent) are not dealt with in the main assessment, but touched upon in alternative scenarios. The results clearly show that fertilizer production is the most polluting process in the system followed by transportation and the boiler emissions at a tie. The most significant impacts from the system are respiratory inorganics and depletion of fossil fuels, of which the boiler emission is the main responsible for the prior and fertilizer production and transportation are responsible for the latter. It is also evident from the results that crude palm oil production is a significant environmental impact generator in Malaysia due to the vast production quantities. Alternative scenarios revealed that there are significant impact savings to be made by introduction of environmental investments, both regarding the overall impacts and in particularly regarding CO2 emissions. Conclusion A screening LCA was successfully conducted on the Malaysian crude palm oil production thus promising potentials for the palm oil industry to conduct their own inventories and assessments using specific company data. Crude palm oil production in Malaysia is responsible for app. 3.5% of the total environmental impacts in the country and must thus be given attention to reduce impacts. Alternatives such as optimized use of organic fertilizer, environmentally friendlier artificial fertilizer production, rail transport, approved filters at the mill boiler stack and biogas harvest from POME digestion must thus be promoted in the industry. Recommendation . The Malaysian palm oil industry should take steps towards introducing LCA. Exhaustive inventories are likely to open the eyes of many companies towards implementing environmental investments and improve the international competitiveness. In order to retrieve results with a greater accuracy in the future, databases must be created containing life cycle data from Malaysian scenarios and normalization and weighting factors must be designed to reflect Malaysian conditions. The Malaysian authorities must create incentives through increased tariffs on electricity and diesel and/or financial support for cleaner technology investments.