Structure of the O-polysaccharide from the lipopolysaccharide from Vibrio cholerae O6

The O-polysaccharide from Vibrio cholerae O6 was isolated from the LPS by mild-acid hydrolysis and has been investigated by sugar and methylation analysis and NMR spectroscopy. The polysaccharide was also depolymerized with aqueous hydrofluoric acid to give the repeating unit and multiples thereof. The O-polysaccharide had the following tetrasaccharide repeating unit. Two O-acetyl groups are present, one of them making the GlcNAc residue fully substituted and the steric crowding considerable at the branching residue.     

The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites

BACKGROUND: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin-protease complex have been proposed. In the absence of a spatial structure of a serpin-protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial. RESULTS: We used a recently developed method for making precise distance measurements, based on donor-donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1') were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into beta sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease-inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker. CONCLUSIONS: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.     

The ecology and evolution of pollen odors

The literature is reviewed and new evidence presented that pollen produces odors, which serve multiple functions in pollination and defense. Pollen odor, which originates from pollenkitt, comprises volatiles that belong to the same chemical classes found in flower scents, that are in species-specific mixtures, and that contrast with odors of other floral parts. Pollen can also take up volatiles from surrounding floral odors, but this adsorption is selective and varies among species. Pollen odors are more pronounced in insect- than bird- or wind-pollinated plants, suggesting that volatile emission evolved in part under selection to attract pollinators. Pollen-feeding insects can perceive pollen odor and use it to discriminate between different pollen types and host plants. Pollen odor influences bee foraging, including the location of pollen sources, discrimination of flowers with different amounts of pollen, and hostplant recognition by pollen-specialist species. In the few wind-pollinated plants studied, odors of male flowers or pollen are comparatively high in -methyl alcohols and ketones; these volatiles may serve in pollen defense, with some known to repel insects. Pollen odor often includes chemicals with documented defense activity, which is probably aimed mainly at nonpollinator pollen-feeding insects and pathogens; an involvement in pollen allelopathy is also possible. Pollen volatiles comprise chemically diverse compounds that may play multiple roles, and their emission in pollen odor undoubtedly evolved under the principle, and often conflicting, selective pressures to both protect the male gametophyte and increase its dispersal by animals.     

Use of weak monoclonal antibodies for affinity chromatography

When using weakly interacting ligands in affinity chromatography, it is possible to take advantage of a true chromatographic process in the separation, as compared with traditional affinity chromatography which is rather an on/off process. In this work, weak monoclonal antibodies were immobilized on a silica and a perfusion-type support (POROS AL) and used for high-performance liquid affinity chromatography (HPLAC). Similar carbohydrate antigens were separated under isocratic conditions according to their weak interaction with the immobilized monoclonal antibody. The affinity of the antibodies was adjusted with temperature and pH to modify the separation. The productivity of the chromatographic system was increased with the immobilized perfusion support but at the expense of loss of plate numbers. This study clearly demonstrates the potential of weak affinity biological interactions as a basis for chromatographic separation.     

Intraspecific Autochthonous and Allochthonous Resource Use by Zooplankton in a Humic Lake during the Transitions between Winter,Summer and Fall

Seasonal patterns in assimilation of externally produced, allochthonous, organic matter into aquatic food webs are poorly understood, especially in brown-water lakes. We studied the allochthony (share biomass of terrestrial origin) in cladoceran, calanoid and cyclopoid micro-crustacean zooplankton from late winter to fall during two years in a small humic lake (Sweden). The use of allochthonous resources was important for sustaining a small population of calanoids in the water column during late winter. However, in summer the calanoids shifted to 100% herbivory, increasing their biomass several-fold by making efficient use of the pelagic primary production. In contrast, the cyclopoids and cladocerans remained at high levels of allochthony throughout the seasons, both groups showing the mean allochthony of 0.56 (range in mean 0.17-0.79 and 0.34-0.75, for the respective group, depending on model parameters). Our study shows that terrestrial organic matter can be an important resource for cyclopoids and cladocerans on an annual basis, forming a significant link between terrestrial organic matter and the higher trophic levels of the food web, but it can also be important for sustaining otherwise herbivorous calanoids during periods of low primary production in late winter.     

Derivation of 30 human embryonic stem cell lines—improving the quality

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process, we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal, but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked, and they are available for researchers.     

A morphological and molecular study of <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> transmission events at the time of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> tick bite

Background  

Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever (TBF) in ruminants. The bacterium invades and replicates in phagocytes, especially in polymorphonuclear granulocytes.     

Growth hormone receptor deficiency in mice results in reduced systolic blood pressure and plasma renin, increased aortic eNOS expression, and altered cardiovascular structure and function

To study the role of the growth hormone receptor (GHR) in the development of cardiovascular structure and function, female GHR gene-disrupted or knockout (KO) and wild-type (WT) mice at age 18 wk were used. GHR KO mice had lower plasma renin levels (12 +/- 2 vs. 20 +/- 4 mGU/ml, P < 0.05) and increased aortic endothelial NO synthase (eNOS) expression (146%, P < 0.05) accompanied by a 25% reduction in systolic blood pressure (BP, 110 +/- 4 vs. 147 +/- 3 mmHg, P < 0.001) compared with WT mice. Aldosterone levels were unchanged, whereas the plasma potassium concentration was elevated by 14% (P < 0.05) in GHR KO. Relative left ventricular weight was 14% lower in GHR KO mice (P < 0.05), and cardiac dimensions as analyzed by echocardiography were similarly reduced. Myograph studies revealed a reduced maximum contractile response in the aorta to norepinephrine (NE) and K(+) (P < 0.05), and aorta media thickness was decreased in GHR KO (P < 0.05). However, contractile force was normal in mesenteric arteries, whereas sensitivity to NE was increased (P < 0.05). Maximal acetylcholine-mediated dilatation was similar in WT and GHR KO mice, whereas the aorta of GHR KO mice showed an increased sensitivity to acetylcholine (P < 0.05). In conclusion, loss of GHR leads to low BP and decreased levels of renin in plasma as well as increase in aortic eNOS expression. Furthermore, GHR deficiency causes functional and morphological changes in both heart and vasculature that are beyond the observed alterations in body size. These data suggest an important role for an intact GH/IGF-I axis in the maintenance of a normal cardiovascular system.     

Antibodies against alpha-synuclein reduce oligomerization in living cells

Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson's disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP constructs to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. C-terminal and oligomer-selective α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.     

Contribution of Sediment Respiration to Summer CO2 Emission from Low Productive Boreal and Subarctic Lakes

We measured sediment production of carbon dioxide (CO₂) and methane (CH₄) and the net flux of CO₂ across the surfaces of 15 boreal and subarctic lakes of different humic contents. Sediment respiration measurements were made in situ under ambient light conditions. The flux of CO₂ between sediment and water varied between an uptake of 53 and an efflux of 182 mg C m⁻² day⁻¹ from the sediments. The mean respiration rate for sediments in contact with the upper mixed layer (SedR) was positively correlated to dissolved organic carbon (DOC) concentration in the water (r² = 0.61). The net flux of CO₂ across the lake surface [net ecosystem exchange (NEE)] was also closely correlated to DOC concentration in the upper mixed layer (r² = 0.73). The respiration in the water column was generally 10-fold higher per unit lake area compared to sediment respiration. Lakes with DOC concentrations <5.6 mg L⁻¹ had net consumption of CO₂ in the sediments, which we ascribe to benthic primary production. Only lakes with very low DOC concentrations were net autotrophic (<2.6 mg L⁻¹) due to the dominance of dissolved allochthonous organic carbon in the water as an energy source for aquatic organisms. In addition to previous findings of allochthonous organic matter as an important driver of heterotrophic metabolism in the water column of lakes, this study suggests that sediment metabolism is also highly dependent on allochthonous carbon sources.