Formation and activity of covalent conjugates of poliovirus and ligands binding to cell surface structures

Disulfide-linked conjugates of poliovirus with streptavidin or concanavalin A were formed and the binding of the conjugates to mouse L cells that lack natural poliovirus receptors was studied. The conjugate with streptavidin was specifically bound to biotinylated L cells, but not to unmodified L cells. The conjugate with conA was bound to L cells in the absence of, but not in the presence of alpha-methyl mannoside. Incubation of L cells with bound conjugates did not produce virus, although the conjugates were highly infectious in HeLa cells, containing natural poliovirus receptors. This suggests that the artificially bound virus was unable to penetrate the L cells and start replication. The possibility that binding of the virus to the natural receptor is required for efficient infection is discussed.     

Regulation of threonine biosynthesis in barley seedlings (Hordeum vulgare L.)

Homoserine kinase was purified 700-fold by fractional ammonium sulfate precipitation, heat treatment, CM-Sephadex C-50 and DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-100 gel filtration. The reaction products O-phosphohomoserine and ADP were the only compounds which caused considerable inhibition of homoserine kinase activity. Product inhibition studies showed non-competitive inhibition between ATP and O-phosphohomoserine and between homoserine and O-phosphohomoserine, and competitive inhibition between ATP and ADP. ADP showed non-competitive inhibition versus homoserine at suboptimal concentrations of ATP. At saturating concentrations of ATP no effect of ADP was observed. The homoserine kinase activity was negligible in the absence of K⁺ and the K_m value for K⁺ was observed to be 4.3 mmol l^–1. A non-competitive pattern was observed with respect to the substrates homoserine and ATP. Threonine synthase in the first green leaf of 6-day-old barley seedlings was partially purified 15-fold by ammonium sulfate fractionation and Sephadex G-100 gel chromatography. Threonine synthase was shown to require pyridoxal 5-phosphate as coenzyme for optimum activity and the enzyme was strongly activated by S-adenosyl-L-methionine. The optimum pH for threonine synthase activity was 7 to 8.Abbreviations PLP Pyridoxal 5-phosphate - SAM S-adenosyl-L-methionine - HSP O-phosphohomoserine     

A sensitive and specific multiplex PCR approach for sex identification of ursine and tremarctine bears suitable for non‐invasive samples

We report a new approach for molecular sex identification of extant Ursinae and Tremarctinae bears. Two Y‐specific fragments (SMCY and 318.2) and one X‐specific fragment (ZFX) are amplified in a multiplex PCR, yielding a double test for male‐specific amplification and an internal positive control. The primers were designed and tested to be bear‐specific, thereby minimizing the risk of cross‐amplification in other species including humans. The high sensitivity and small amplicon sizes (100, 124, 160 base pairs) facilitate analysis of non‐invasively obtained DNA material. DNA from tissue and blood as well as from 30 non‐invasively collected hair and faeces yielded clear and easily interpretable results. The fragments were detected both by standard gel electrophoresis and automated capillary electrophoresis.     

Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide

A fragment of the amyloid beta protein, βA(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that βA(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, βA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by βA(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to βA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 • - ) - and hydroxyl ( •OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of •OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of •OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to βA(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that βA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 • - can lead to HOCl and further formation of •OH, which both have a cytotoxic potential.     

Accumulation of ammonium in Norway spruce (Picea abies) seedlings measured by in vivo 14N-NMR

(14)N-NMR and (31)P-NMR have been used to monitor the in vivo pH in roots, stems, and needles from seedlings of Norway spruce, a typical ammonium-tolerant plant. The vacuolar and cytoplasmic pH measured by (31)P-NMR was found to be c. pH 4.8 and 7.0, respectively, with no significant difference between plants growing with ammonium or nitrate as the N-source. The (1)H-coupled (14) NH 4+ resonance is pH-sensitive: at alkaline pH it is a narrow singlet line and below pH 4 it is an increasing multiplet line with five signals. The pH values in ammonium-containing compartments measured by (14)N-NMR ranged from 3.7 to 3.9, notably lower than the estimated pH values of the P(i) pools. This suggests that, in seedlings of Norway spruce, ammonium is stored in vacuoles with low pH possibly to protect the seedlings against the toxic effects of ammonium ( NH 4+) or ammonia (NH3). It was also found that concentrations of malate were 3-6 times higher in stems than in roots and needles, with nitrate-grown plants containing more malate than plants grown with ammonium.     

Photosynthesis in ozone-exposed duckweed (Lemna gibba)

The photosynthetic light saturation curve in duckweed was lowered by 20–25% after ozone exposure (300 nmol mol⁻¹, 1 h). The light flux and oxygen concentration during ozone-exposure had no effect on reduction of net photosynthesis. Net photosynthesis and photorespiration were both depressed by about 40% after exposure for 1 h to 360 nmol mol⁻¹ ozone. We could not find any change in dark respiration after ozone exposure below 300 nmol mol⁻¹. When the concentration of ozone was doubled from 150 nmol mol⁻¹ to 300 nmol mol⁻¹, the uptake of ozone in duckweed changed from 100 nmol m⁻² s⁻¹ to 170 nmol m⁻² s⁻¹. We found no differences in fluorescence (pattern) between ozone treated plants and the control plants during a period of 150 min after ozone treatment, but there was an increase in synthesis of the Dl-protein and a significant reduction in degradation after ozone treatment (300 nmol mol⁻¹, 1 h). These results, together with fluorescence measurements, indicate that photochemical electron transport was not responsible for the ozone-induced reduction in net photosynthesis.     

Effect of Photon Fluence Rate and Specific Growth Rate on Geosmin Production of the Cyanobacterium Oscillatoria brevis (Kütz.) Gom

The production of geosmin from the cyanobacterium Oscillatoria brevis was studied as a function of the photon fluence rate and appears to be related to the chlorophyll content.     

Effect of Photon Fluence Rate and Specific Growth Rate on Geosmin Production of the Cyanobacterium Oscillatoria brevis (Kütz.) Gom

The production of geosmin from the cyanobacterium Oscillatoria brevis was studied as a function of the photon fluence rate and appears to be related to the chlorophyll content.     

Vegetation responses to rapid climatic changes during the last deglaciation 13,500–8,000?years ago on southwest And?ya, arctic Norway

The late-glacial vegetation development in northern Norway in response to climate changes during the Aller?d, Younger Dryas (YD), and the transition to the Holocene is poorly known. Here we present a high-resolution record of floral and vegetation changes at lake Lusvatnet, south-west And?ya, between 13500 and 8000 cal b.p. Plant macrofossil and pollen analyses were done on the same sediment core and the proxy records follow each other very closely. The core has also been analyzed using an ITRAX XRF scanner in order to check the sediment sequence for disturbances or hiatuses. The core has a good radiocarbon-based chronology. The Saksunarvatn tephra fits very well chronostratigraphically. During both the Aller?d and the Younger Dryas time-periods arctic vegetation prevailed, dominated by Salix polaris associated with many typically arctic herbs such as Saxifraga cespitosa, Saxifraga rivularis and Oxyria digyna. Both periods were cold and dry. Between 12450 and 12250 cal b.p. during the Younger Dryas chronozone, the assemblage changed, particularly in the increased abundance of Papaver sect. Scapiflora and other high-Arctic herbs, suggesting the development of polar desert vegetation mainly as a response to increased aridity. After 11520 cal b.p. a gradually warmer and more oceanic climate initiated a succession to dwarf-shrub vegetation and the establishment of Betula woodland after 1,000 years at c. 10520 cal b.p. The overall late-glacial aridity contrasts with oceanic conditions in southern Norway and is probably related to sea-ice extent.