Structural studies of the capsular polysaccharides from Klebsiella types 8 and 82, a reinvestigation

The structures of the capsular polysaccharides elaborated by Klebsiella types 8 (K8) and 82 (K82) have been reinvestigated. N.m.r. spectroscopy of the original and chemically modified polysaccharides was the principal method used. It is concluded that the polysaccharides are composed of repeating units having the following structures. (Formula: see text). The presence of L-glutamic acid, linked as an amide to the carboxyl group of a uronic acid, has not been observed hitherto in bacterial polysaccharides.     

Double Triton X-114 Phase Partitioning for the Purification of Plant Cytochromes P450 and Removal of Green Pigments

A double Triton X-114 phase partitioning procedure that separates plant cytochromes P450 from green pigments and provides an extract highly enriched in total cytochromes P450 has been developed. Upon phase partitioning in Triton X-114, plant cytochromes P450 have previously been found to partition to the pigmented detergent rich phase. These partitionings were carried out using phosphate buffer. We found that the partitioning of the cytochromes P450 could be shifted to a pigment-free Triton X-114 poor phase by changing the buffer component to borate. The protein extract containing the cytochromes P450 but devoid of green pigment was subjected to a second phase partitioning step before which the buffer was changed from borate to phosphate. This second phase partitioning step produced a Triton X-114-rich phase highly enriched in cytochromes P450 proteins compared to the microsomal starting material as monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, cytochrome P450 reconstitution assays, and Western blotting. The yield of the double phase partitioning purification procedure is about 26% which is high compared to the yields obtained at similar stages of purification using column chromatography. The double phase partitioning procedure takes 3–4 h to complete. This is very fast compared to traditional purification schemes for cytochromes P450 which involve multiple of column chromatographic steps. Plant cytochromes P450 are labile, low abundant proteins that are difficult to isolate. The double Triton X-114 phase partitioning here reported thus constitutes a versatile, efficient purification procedure circumventing many of the problems previously encountered.     

Photoinhibition of Photosystem I damages both reaction centre proteins PSI-A and PSI-B and acceptor-side located small Photosystem I polypeptides

Photoinhibition of Photosystem I at chilling temperatures was investigated. Illumination of barley and cucumber leaves at 4°C induced a lowered Photosystem I activity. In barley, the reaction centre proteins PSI-A and PSI-B were both partially degraded as was the nuclear-encoded PSI-D polypeptide. Barley leaves infiltrated with KCN to increase oxidative stress, showed increased photoinhibition of Photosystem I, including reduced photochemical activity and marked degradation of several Photosystem I polypeptides. The most rapid and pronounced degradation was found in the PSI-D and PSI-E polypeptides exposed at the Photosystem I acceptor side. The PSI-A, -B, -C, -G, -H, -K and -L polypeptides were less extensively damaged. No damage of the lumenally oriented PSI-F and -N polypeptides was detected. The elevated photoinhibition of Photosystem I seen in KCN treated barley is most likely induced by a combination of increased active oxygen due to inhibited scavenging and increased accumulation of reducing power due to inhibition of the Calvin cycle. In barley, photo-inactivation of Photosystem I closely followed the degradation of PSI-A and PSI-B. Illumination of cucumber resulted in a pronounced loss of activity and appearance of specific PSI-A and PSI-B degradation products whereas the total PSI-A/B degradation was small. The PSI-A/B degradation identified in barley is interpreted to reflect a physiologically relevant process being part of a repair cycle, whereas the much smaller PSI-A/B degradation observed in cucumber is interpreted to represent an irreversible damage induced far below the temperature tolerance for cucumber.     

Biological nitrogen fixation in coniferous forest watershed areas in Central Sweden

An investigation on biological nitrogen fixation in the forest floor was carried out in five watershed areas in Central Sweden during the summers 1978 and 1979. Five different types of bottom layers with underlying soil horizons were investigated for nitrogen fixation. An inventory on the occurrence of potential nitrogen fixing lichens and alder trees was also carried out.
Low fixation rates were determined in mesic-soil mosses, F/H,layers and mineral soil. Though two orders of magnitude higher than in mesic-soil mosses, the nitrogen fixation in the investigated Sphagnum mosses must be considered as low, probably due to absence of blue-green algal associations. The contribution to total nitrogen fixation by lichens is low, but Alnus incana may play an important role. The estimated biological nitrogen fixation calculated as a weighted mean over the five watersheds is in the order of 0.5 kg N ha^-1 yr^-1.     

Conversion of saccharopine to lysine in barley

Labelled saccharopine was synthesized and showed a low conversion to lysine in barley seedlings. The results indicate a role of saccharopine in either lysine biosynthesis or catabolism.     

NickFects,Phosphorylated Derivatives of Transportan 10 for Cellular Delivery of Oligonucleotides

Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membrane is impermeable for nucleic acid polymers and, consequently, an efficient and non-toxic transfection agent is needed for their delivery into the cell. In this study we present a novel series, NickFects, of chemically modified TP10 peptide-based delivery vectors used for the cellular delivery of single-stranded oligonucleotides. These carriers, obtained by replacement of Ile8 by threonine in stearyl-TP10 and by modifying of tyrosine and/or threonine, respectively, by phosphorylation formed 300–500 nm in size peptide:oligonucleotide nanocomplexes with negative surface charges. The highest splice-correcting effect was obtained when phosphorotiate 2′-O-methyl oligonucleotides were transduced into cells by NickFect1 (NF1) or NickFect2 (NF2). In addition, we also show how a small modification (one or two negative charges) in peptide sequence can affect its ability to deliver ONs into cells and increase their potency in the splicing redirection assay. Our studies demonstrate that NF1 and NF2 have higher transfection efficacy for oligonucleotides as compared to the most commonly used transfection agent Lipofectamine™ 2000 and lead to higher biological response in cells.     

Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units

Quantification of viral infectious units is traditionally measured by methods based on forming plaques in semisolid media (PFU) or endpoint dilution of a virus-containing solution (TCID₅₀), methods that are laborious, time-consuming and take on average 3–7 days to carry out. Quantitative real-time PCR is an established method to quantify nucleic acids at high accuracy and reproducibility, routinely used for virus detection and identification. In the present study, a procedure was developed using a two-step real-time PCR and the SYBR Green detection method to study whether there are correlations between TCID₅₀/ml, PFU/ml and Ct values generated by real-time PCR enabling rapid and efficient calculation of titer equivalents when working with viruses in the research laboratory. In addition, an external standard with known concentrations was included using in vitro transcribed viral RNA, thus allowing the calculation of the amount of RNA copies needed for various applications (i.e. per plaque or TCID₅₀).The results show that there is a correlation between the three quantification methods covering a wide range of concentration of viruses. Furthermore, a general regression line between TCID₅₀ and Ct values was obtained for all viruses included in the study, which enabled recording titer equivalents using real-time PCR. Finally, by including an external standard, the amount of RNA genomes generating one TCID₅₀ or PFU for each enterovirus serotype included was determined.     

Effects of early pregnancy on regional adipose tissue metabolism

Adipose tissue metabolism was studied in needle biopsies from femoral and abdominal subcutaneous depots, in 12 healthy young women, during early (9-11 weeks) pregnancy, and 6 weeks after a legal abortion. Both during pregnant and non-pregnant conditions, a higher lipoprotein lipase (LPL) activity was seen in the femoral compared to the abdominal region, but the LPL activity was not influenced by early pregnancy. Rates of fatty acid esterification and acylglyceride synthesis were not different between regions, nor affected by pregnancy. The stimulatory effect of norepinephrine (10(-7) M) on lipolysis was significantly greater in the abdominal than in the femoral region in both the pregnant and non-pregnant condition. This difference was apparently due to higher alpha-adrenergic activity in the femoral region. Pregnancy per se had no effect on lipolytic response to norepinephrine. These findings indicate that lipid accumulation is favoured in the femoral region in young women both during pregnant and non-pregnant conditions.