The assembly and secretion of apoB 100 containing lipoproteins in Hep G2 cells. Evidence for different sites for protein synthesis and lipoprotein assembly

Pulse-chase studies combined with subcellular fractionation indicated that LpB 100 (i.e. the apoprotein B (apoB) 100 containing lipoproteins) was released to the lumen of the secretory pathway in a subcellular fraction enriched in smooth vesicles, and referred to as SMF (the smooth membrane fraction). The migration of SMF during gradient ultracentrifugation as well as kinetic studies indicated that the fraction was derived from a pre-Golgi compartment, probably the smooth endoplasmic reticulum (ER). Only small amounts of LpB 100 could be detected during these pulse-chase experiments in the subcellular fractions derived from the rough endoplasmatic reticulum (RER). SMF contained the major amount of the diacylglycerol acyltransferase activity present in the ER, while the major amount of membrane bound apoB 100 was present in the RER. Pulse-chase studies of the intracellular transfer of apoB 100 demonstrated the formation of a large membrane-bound preassembly pool in the ER, while no significant amount of apoB 100 radioactivity was present in the membrane of the Golgi apparatus. The maximal radioactivity of LpB 100, recovered from the ER or the Golgi lumen, was small compared with the radioactivity recovered from the ER membrane, indicating that the assembled LpB 100 rapidly leaves the cells. This in turn indicates that the rate-limiting step in the secretion of apoB 100 was the transfer of the protein from the ER membrane to the LpB 100 in the lumen. A portion of the intracellular pool of apoB 100 was not secreted but underwent posttranslational degradation.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.     

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.

     

Surface Display of the Receptor-Binding Region of the Lactobacillusbrevis S-Layer Protein in Lactococcuslactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

Global change‐driven effects on dissolved organic matter composition: Implications for food webs of northern lakes

Northern ecosystems are experiencing some of the most dramatic impacts of global change on Earth. Rising temperatures, hydrological intensification, changes in atmospheric acid deposition and associated acidification recovery, and changes in vegetative cover are resulting in fundamental changes in terrestrial–aquatic biogeochemical linkages. The effects of global change are readily observed in alterations in the supply of dissolved organic matter (DOM)—the messenger between terrestrial and lake ecosystems—with potentially profound effects on the structure and function of lakes. Northern terrestrial ecosystems contain substantial stores of organic matter and filter or funnel DOM, affecting the timing and magnitude of DOM delivery to surface waters. This terrestrial DOM is processed in streams, rivers, and lakes, ultimately shifting its composition, stoichiometry, and bioavailability. Here, we explore the potential consequences of these global change‐driven effects for lake food webs at northern latitudes. Notably, we provide evidence that increased allochthonous DOM supply to lakes is overwhelming increased autochthonous DOM supply that potentially results from earlier ice‐out and a longer growing season. Furthermore, we assess the potential implications of this shift for the nutritional quality of autotrophs in terms of their stoichiometry, fatty acid composition, toxin production, and methylmercury concentration, and therefore, contaminant transfer through the food web. We conclude that global change in northern regions leads not only to reduced primary productivity but also to nutritionally poorer lake food webs, with discernible consequences for the trophic web to fish and humans.     

Maturation of Oral Microbiota in Children with or without Dental Caries

Background

The aim of this longitudinal study was to evaluate the oral microbiota in children from age 3 months to 3 years, and to determine the association of the presence of caries at 3 years of age.

Methods and findings

Oral biofilms and saliva were sampled from children at 3 months (n = 207) and 3 years (n = 155) of age, and dental caries was scored at 3 years of age. Oral microbiota was assessed by culturing of total lactobacilli and mutans streptococci, PCR detection of Streptococcus mutans and Streptococcus sobrinus, 454 pyrosequencing and HOMIM (Human Oral Microbe Identification Microarray) microarray detection of more then 300 species/ phylotypes. Species richness and taxa diversity significantly increased from 3 months to 3 years. Three bacterial genera, present in all the 3-month-old infants, persisted at 3 years of age, whereas three other genera had disappeared by this age. A large number of new taxa were also observed in the 3-year-olds. The microbiota at 3 months of age, except for lactobacilli, was unrelated to caries development at a later age. In contrast, several taxa in the oral biofilms of the 3-year-olds were linked with the presence or absence of caries. The main species/phylotypes associated with caries in 3-year-olds belonged to the Actinobaculum, Atopobium, Aggregatibacter, and Streptococcus genera, whereas those influencing the absence of caries belonged to the Actinomyces, Bergeyella, Campylobacter, Granulicatella, Kingella, Leptotrichia, and Streptococcus genera.

Conclusions

Thus, during the first years of life, species richness and taxa diversity in the mouth increase significantly. Besides the more prevalent colonization of lactobacilli, the composition of the overall microbiota at 3 months of age was unrelated to caries development at a later age. Several taxa within the oral biofilms of the 3-year-olds could be linked to the presence or absence of caries.     

Activation of VPAC1 receptors aggravates early atherosclerosis in hypercholesterolemic apolipoprotein E-deficient mice

Objective: Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide widely expressed in the body and binding three types of receptors: VPAC₁-R, VPAC₂-R and PAC₁-R. Based on beneficial effects of VIP and VPAC₁-R agonists in mouse models of several chronic inflammatory disorders, we hypothesized that activation of VIP receptors would prevent atherosclerosis development in apolipoprotein E-deficient mice.Methods and results: Contrary to our hypothesis, administration of a VPAC₁-R agonist, (Ala^11,22,28)-VIP aggravated atherosclerotic lesion development in the aortic root of these mice compared to control mice. This was accompanied by a significant increase in the expression of MHC class II protein I-A^b, and suggests enhanced inflammatory activity in the vessel wall. The amount of macrophage-specific CD68 staining as well as serum cholesterol and triglyceride levels did not change as a result of the (Ala^11,22,28)-VIP treatment, i.e. the treatment resulted in significant changes in lipid accumulation in the lesions without changing the number of macrophages or systemic lipid levels. Interestingly, administration of VIP did not alter the course of the disease.Conclusion: Despite beneficial effects in murine models of several inflammatory disorders, VPAC₁-R activation aggravates atherosclerotic lesion formation in apolipoprotein E-deficient mice through enhanced inflammatory activity in the vessel wall.     

Changes in the pelagic microbial food web due to artificial eutrophication

The effect of nutrient enrichment on the structure and carbon flow in the pelagic microbial food web was studied in mesocosm experiments using seawater from the northern Baltic Sea. The experiments included food webs of at least four trophic levels; (1) phytoplankton–bacteria, (2) flagellates, (3) ciliates and (4) mesozooplankton. In the enriched treatments high autotrophic growth rates were observed, followed by increased heterotrophic production. The largest growth increase was due to heterotrophic bacteria, indicating that the heterotrophic microbial food web was promoted. This was further supported by increased growth of heterotrophic flagellates and ciliates in the high nutrient treatments. The phytoplankton peak in the middle of the experiments was mainly due to an autotrophic nanoflagellate, Pyramimonas sp. At the end of the experiment, the proportion of heterotrophic organisms was higher in the nutrient enriched than in the nutrient-poor treatment, indicating increased predation control of primary producers. The proportion of potentially mixotrophic plankton, prymnesiophyceans, chrysophyceans and dinophyceans, were significantly higher in the nutrient-poor treatment. Furthermore, the results indicated that the food web efficiency, defined as mesozooplankton production per basal production (primary production + bacterial production − sedimentation), decreased with increasing nutrient status, possibly due to increasing loss processes in the food web. This could be explained by promotion of the heterotrophic microbial food web, causing more trophic levels and respiration steps in the food web.