Trans-splicing of a mutated glycosylasparaginase mRNA sequence by a group I ribozyme deficient in hydrolysis.

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using the Tetrahymena ribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3' splice site (side reaction) without affecting self-splicing [Haugen, P., Andreassen, M., Birgisdottir, A.B. & Johansen, S.D. (2004) Eur. J. Biochem. 271, 1015-1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3' splice site, for application in group I ribozyme-mediated trans-splicing of RNA. The improved ribozyme catalyses both cis-splicing and trans-splicing in vitro of a human glycosylasparaginase mRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis.     

TOC fluctuations in a humic lake as related to catchment acidification, season and climate

Studies of fluctuations in total organic carbon (TOC) were performedin both the reference basin and the acidified basin of experimental LakeSkjervatjern, in order to separate effects of various catchment and in-lakeprocesses. Nearly five years of catchment acidification did not inducesignificant changes in TOC. TOC concentrations was not related clearly toprecipitation or runoff. In both basins, there was a regular, seasonal patternwith a gradual increase in TOC concentrations from spring to late autumn.Minima in concentrations occurred during periods with frozen ground inwinter, irrespective of discharge patterns. The decrease from 10 mg C l^-1 in autumn to 1--2 mg C l^-1 in latewinter, was only seen in surface layers. Runoff was the major loss routefor surface TOC in the lake. Photo-oxidation, bacterial oxidation, andsedimentation combined yielded maximum loss rates of 3%of surface TOC d^-1. Below a depth of 1 m 0.5%d^-1 was lost to these same processes. The surface microlayerhad 5--10 times more TOC than the bulk water on average, and could haveeffects on gas exchange and sub-surface light. Despite the oxidation ofTOC, the short residence time of the lake and rapid replacement of TOCfrom the catchment was the major determinant of lake water TOC.     

Effect of CO2 enhancement in an outdoor algal production system usingTetraselmis

One of the objectives of microalgal culture is to provide reliable production technology for important live aquaculture feed organisms. Presented here are the results of experiments designed to provide a better understanding of the relationship between inorganic carbon availability and algal production.Our results suggest that through additions of CO₂ gas we were able to maintain sufficient dissolved carbon to stabilize outdoor algal cultures. Increases in the rate of addition of CO₂ increased levels of dissolved CO₂, total dissolved inorganic carbon (CO₂), and decreased pH in the growth medium. This translated into improved buffering capacity of the culture medium and higher growth rate. A minimum of 2.4 mM CO₂ was found necessary to maintain a maximal growth rate of 0.7 doublings/day. We also found that the increased productivity more than offsets the cost of adding the CO₂.     

Spastic Paraplegia Type 7 Is Associated with Multiple Mitochondrial DNA Deletions

Spastic paraplegia 7 is an autosomal recessive disorder caused by mutations in the gene encoding paraplegin, a protein located at the inner mitochondrial membrane and involved in the processing of other mitochondrial proteins. The mechanism whereby paraplegin mutations cause disease is unknown. We studied two female and two male adult patients from two Norwegian families with a combination of progressive external ophthalmoplegia and spastic paraplegia. Sequencing of SPG7 revealed a novel missense mutation, c.2102A>C, p.H 701P, which was homozygous in one family and compound heterozygous in trans with a known pathogenic mutation c.1454_1462del in the other. Muscle was examined from an additional, unrelated adult female patient with a similar phenotype caused by a homozygous c.1047insC mutation in SPG7. Immunohistochemical studies in skeletal muscle showed mosaic deficiency predominantly affecting respiratory complex I, but also complexes III and IV. Molecular studies in single, microdissected fibres showed multiple mitochondrial DNA deletions segregating at high levels (38–97%) in respiratory deficient fibres. Our findings demonstrate for the first time that paraplegin mutations cause accumulation of mitochondrial DNA damage and multiple respiratory chain deficiencies. While paraplegin is not known to be directly associated with the mitochondrial nucleoid, it is known to process other mitochondrial proteins and it is possible therefore that paraplegin mutations lead to mitochondrial DNA deletions by impairing proteins involved in the homeostasis of the mitochondrial genome. These studies increase our understanding of the molecular pathogenesis of SPG7 mutations and suggest that SPG7 testing should be included in the diagnostic workup of autosomal recessive, progressive external ophthalmoplegia, especially if spasticity is present.     

Light,temperature and nitrogen starvation effects on the total lipid and fatty acid content and composition ofSpirulina platensis UTEX 1928

The total lipid and fatty acid content ofSpirulina platensis UTEX 1928 was 7.2 and 2.2% respectively of cellular dry weight under controlled conditions supporting high growth rates. With increases in irradiance from 170 to 870 μmol photon m^?2 s^?1, growth rate increased, total lipid decreased, and fatty acid composition was unaffected. At 1411 μmol photon m^?2 s^?1, total lipid increased slightly and percent composition of the fatty acid gamma linolenic acid increased. Growth and total lipid content ofS. platensis were affected by changes in growth temperature from 25 to 38 °C. With increased growth rate, total lipid content increased. This suggests that the storage of carbon increases at temperatures supporting high growth rates. The degree of saturation increased with temperature. Although the percent composition of gamma linolenic acid was higher at lower growth temperature, production was still primarily a function of growth rate. The effect of temperature on fatty acid content and degree of saturation was of secondary importance. Nitrogen starvation increased total lipid content but decreased fatty acid content as a percentage of dry weight; composition of the fatty acids was unaffected. N-starvation appeared to suspend synthesis of long chain fatty acids inS. platensis, suggesting that some other compound stores fixed carbon when nitrogen is limiting. It was concluded that fatty acid production inS. platensis is maximized by optimizing culture conditions for growth.     

Melatonin implantation during spring and summer does not affect the seasonal rhythm of feeding in anadromous Arctic charr (Salvelinus alpinus)

Plasma melatonin levels in the high-latitude teleost Arctic charr (Salvelinus alpinus) are constantly low during summer when feeding activity is high, and high during the dark winter when they eat little and loose weight. The question arises if melatonin is involved in the phase-setting of annual rhythms of feeding and growth and if low summer melatonin production is permissive for high summer growth in this species. The present study was therefore set out to compare the seasonal appetite and growth rhythms in Arctic charr with constantly high plasma melatonin levels from February throughout the Arctic summer (melatonin implanted, average mid-day plasma melatonin levels 1,106 ± 147 pg/ml) with those of fish with natural plasma melatonin levels (vehicle implanted and untreated fish with average mid-day plasma melatonin levels of 94 ± 13 and 58 ± 6 pg/ml, respectively). Feed intake, body mass or body length, as well as the timing of the seasonal growth rhythm, were not affected by the high summer plasma melatonin level. Further, Arctic charr fasted for 3 months had a 24 h plasma profile of melatonin which was consistently higher throughout the scotophase compared to fed charr. Although the daily melatonin production seems to be affected by the energy status of the fish, melatonin does not seem to be directly involved in regulation of the seasonal feeding and growth rhythm in the high-latitude, anadromous Arctic charr.     

Identification of novel splice variants of the human catalytic subunit Cbeta of cAMP-dependent protein kinase.

Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed Calpha, Cbeta, Cgamma and PrKX have been identified. Here we demonstrate that the human Cbeta gene encodes six splice variants, designated Cbeta1, Cbeta2, Cbeta3, Cbeta4, Cbeta4ab and Cbeta4abc. The Cbeta splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the Cbeta gene. The previously identified human Cbeta variant has been termed Cbeta1, and is similar to the Cbeta isoform identified in the mouse, ox, pig and several other mammals. Human Cbeta2, which is the homologue of bovine Cbeta2, has no homologue in the mouse. Human Cbeta3 and Cbeta4 are homologous to the murine Cbeta3 and Cbeta2 splice variants, whereas human Cbeta4ab and Cbeta4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the Cbeta splice variants reveal tissue specific expression. Cbeta1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The Cbeta3 and Cbeta4 splice variants were uniquely expressed in human brain in contrast to Cbeta2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various Cbeta splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP.