Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.     

Biochemical and Functional Analysis of Two Plasmodium falciparum Blood-Stage 6-Cys Proteins: P12 and P41

The genomes of Plasmodium parasites that cause malaria in humans, other primates, birds, and rodents all encode multiple 6-cys proteins. Distinct 6-cys protein family members reside on the surface at each extracellular life cycle stage and those on the surface of liver infective and sexual stages have been shown to play important roles in hepatocyte growth and fertilization respectively. However, 6-cys proteins associated with the blood-stage forms of the parasite have no known function. Here we investigate the biochemical nature and function of two blood-stage 6-cys proteins in Plasmodium falciparum, the most pathogenic species to afflict humans. We show that native P12 and P41 form a stable heterodimer on the infective merozoite surface and are secreted following invasion, but could find no evidence that this complex mediates erythrocyte-receptor binding. That P12 and P41 do not appear to have a major role as adhesins to erythrocyte receptors was supported by the observation that antisera to these proteins did not substantially inhibit erythrocyte invasion. To investigate other functional roles for these proteins their genes were successfully disrupted in P. falciparum, however P12 and P41 knockout parasites grew at normal rates in vitro and displayed no other obvious phenotypic changes. It now appears likely that these blood-stage 6-cys proteins operate as a pair and play redundant roles either in erythrocyte invasion or in host-immune interactions.     

Taste difference thresholds for monosodium glutamate and sodium chloride in pigtail macaques (Macaca nemestrina) and spider monkeys (Ateles geoffroyi)

The purpose of this study was to determine taste difference thresholds for monosodium glutamate (MSG) and sodium chloride (NaCl) in pigtail macaques (Macaca nemestrina) and spider monkeys (Ateles geoffroyi). Using a two-bottle preference test of brief duration, three animals of each species were presented with four different reference concentrations of 50, 100, 200, and 400 mM of a tastant and tested for their ability to discriminate these from lower concentrations of the same tastant. The just noticeable differences (JNDs), expressed as Weber ratios (DeltaI/I), were found to range from 0.1 to 0.5 for MSG and 0.2 to 0.45 for NaCl in the pigtail macaques, with a significant tendency for higher Weber ratios with higher reference concentrations. In the spider monkeys, JNDs ranged from 0.15 to 0.4 for MSG and 0.1 to 0.25 for NaCl, with Weber ratios staying fairly constant across the reference concentrations tested. Thus, the JNDs were found to be generally similar in both species and to be at least as low as those found in humans for MSG and NaCl, as well as those found in spider monkeys for sucrose. The results support the assumption that both pigtail macaques and spider monkeys may use differences in perceived intensity of MSG and NaCl as a criterion for food selection.     

Is polycystic ovary syndrome associated with high sympathetic nerve activity and size at birth?

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disturbance among women of reproductive age and is proposed to be linked with size at birth and increased prevalence of cardiovascular disease. A disturbance in the sympathetic nervous system may contribute to the etiology of PCOS. This study evaluates sympathetic outflow in PCOS and its relation to size at birth. Directly recorded sympathetic nerve activity to the muscle vascular bed (MSNA) was obtained in 20 women with PCOS and in 18 matched controls. Ovarian ultrasonographic evaluation, biometric, hormonal, and biochemical parameters were measured, and birth data were collected. Women with PCOS had increased MSNA (30 +/- 8 vs. 20 +/- 7 burst frequency, P < 0.0005) compared with controls. MSNA was positively related to testosterone (r = 0.63, P < 0.005) and cholesterol (r = 0.55, P = 0.01) levels in PCOS, which, in turn, were not related to each other. Testosterone level was a stronger predictor of MSNA than cholesterol. Birth size did not differ between the study groups. This is the first study to directly address sympathetic nerve activity in women with PCOS and shows that PCOS is associated with high MSNA. Testosterone and cholesterol levels are identified as independent predictors of MSNA in PCOS, although testosterone has a stronger impact. The increased MSNA in PCOS may contribute to the increased cardiovascular risk and etiology of the condition. In this study, PCOS was not related to size at birth.     

UV effects that come and go: a global comparison of marine benthic community level impacts

Ambient UV radiation has substantially increased during the last decades, but its impact on marine benthic communities is hardly known. The aim of this study was to globally compare and quantify how shallow hard‐bottom communities are affected by UV during early succession. Identical field experiments in 10 different coastal regions of both hemispheres produced a consistent but unexpected pattern: (i) UV radiation affected species diversity and community biomass in a very similar manner, (ii) diversity and biomass were reduced to a larger extent by UVA than UVB radiation, (iii) ambient UV levels did not affect the composition of the communities, and (iv) any UV effects disappeared during species succession after 2–3 months. Thus, current levels of UV radiation seem to have small, predictable, and transient effects on shallow marine hard‐bottom communities.     

DNA-bound proteins contribute much more than soluble intracellular compounds to the intrinsic protection against radiation-induced DNA strand breaks in human cells

To assess the role of soluble intracellular compounds and DNA-bound proteins in the intrinsic protection against radiation-induced DNA strand breaks, the alkaline unwinding technique was applied to cellular, nuclear, and nucleoid monolayers. It was found that, when the soluble intracellular compounds were removed from human fibroblasts by permeabilization (nuclear monolayers) and irradiated in a phosphate buffer containing 150 mM monovalent cations (Na+ and K+) and 0.8 mM MgCl2, the frequency of radiation-induced DNA strand breaks increased twofold. Removal of both soluble intracellular compounds and DNA-bound proteins from the cells by a pretreatment with 2 M NaCl (nucleoid monolayers) resulted in a 100-fold increase in the frequency of strand-break induction by gamma radiation. Expressed as percentage of total intrinsic protection against radiation-induced DNA strand breaks, DNA-bound protein contributed 99% compared to 1% by soluble intracellular compounds. Using a different experimental approach it was found that the radioprotective capacity of soluble intracellular compounds was equivalent to about 5 mM dimethyl sulfoxide (DMSO) and DNA-bound proteins to about 70 mM DMSO. It is concluded that DNA-bound proteins play a much greater role than soluble intracellular compounds in the intrinsic protection against radiation-induced DNA strand breaks in cultured human cells.     

Calanus the cannibal

Recent modelling efforts have suggested that cannibalism oneggs and young nauplii is an important factor controlling thepopulation dynamics of Calanus. We measured cannibalistic feedingrates on eggs and nauplii of Calanus helgolandicus in the laboratory.Clearance rates on nauplii were     

Life cycle assessment in road infrastructure planning using spatial geological data

Purpose

The purpose of the study was to outline and demonstrate a new geographic information system (GIS)-based approach for utilising spatial geological data in three dimensions (i.e. length, width and depth) to improve estimates on earthworks during early stages of road infrastructure planning.

Methods

This was undertaken by using three main methodological steps: mass balance calculation, life cycle inventory analysis and spatial mapping of greenhouse gas (GHG) emissions and energy use. The mass balance calculation was undertaken in a GIS environment using two assumptions of geological stratigraphy for two proposed alternative road corridors in Sweden. The estimated volumes of excavated soil, blasted rock and filling material were later multiplied with the GHG emission and energy use factors for these processes, to create spatial data and maps in order to show potential impacts of the studied road corridors. The proposed GIS-based approach was evaluated by comparing with actual values received after one alternative was constructed.

Results and discussion

The results showed that the estimate of filling material was the most accurate (about 9 % deviation from actual values), while the estimate for excavated soil and blasted rock resulted in about 38 and 80 % deviation, respectively, from the actual values. It was also found that the total volume of excavated and ripped soils did not change when accounting for stratigraphy.

Conclusions

The conclusion of this study was that more information regarding embankment height and actual soil thickness would further improve the model, but the proposed GIS-based approach shows promising results for usage in LCA at an early stage of road infrastructure planning. Thus, by providing better data quality, GIS in combination with LCA can enable planning for a more sustainable transport infrastructure.

     

Importance of Viral Lysis and Dissolved DNA for Bacterioplankton Activity in a P-Limited Estuary,Northern Baltic Sea

Through lysis of bacterioplankton cells, viruses mediate an important, but poorly understood, pathway of carbon and nutrients from the particulate to the dissolved form. Via this activity, nutrient-rich cell lysates may become available to noninfected cells and support significant growth. However, the nutritional value of lysates for noninfected bacteria presumably depends on the prevailing nutrient limitation. In the present study, we examined dynamics of dissolved DNA (D-DNA) and viruses along a transect in the phosphorus (P)-limited Ore Estuary, northern Baltic Sea. We found that viruses were an important mortality factor for bacterioplankton and that their activity mediated a significant recycling of carbon and especially of P. Uptake of dissolved DNA accounted for up to 70% of the bacterioplankton P demand, and about a quarter of the D-DNA pool was supplied through viral lysis of bacterial cells. Generally, the importance of viral lysates and uptake of D-DNA was highest at the estuarine and offshore stations and was positively correlated with P limitation measured as alkaline phosphatase activity. Our results highlight the importance of viral activity for the internal recycling of principal nutrients and pinpoints D-DNA as a particularly relevant compound in microbial P dynamics.