A non-BRICHOS surfactant protein c mutation disrupts epithelial cell function and intercellular signaling

Background

Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient.

Results

The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing - thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing - thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior.

Conclusions

The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing - thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.     

Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics

Accurate profiling of lipidomes relies upon the quantitative and unbiased recovery of lipid species from analyzed cells, fluids, or tissues and is usually achieved by two-phase extraction with chloroform. We demonstrated that methyl-tert-butyl ether (MTBE) extraction allows faster and cleaner lipid recovery and is well suited for automated shotgun profiling. Because of MTBE's low density, lipid-containing organic phase forms the upper layer during phase separation, which simplifies its collection and minimizes dripping losses. Nonextractable matrix forms a dense pellet at the bottom of the extraction tube and is easily removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol delivers similar or better recoveries of species of most all major lipid classes compared with the "gold-standard" Folch or Bligh and Dyer recipes.     

Adipocyte-specific inactivation of Acyl-CoA synthetase fatty acid transport protein 4 (Fatp4) in mice causes adipose hypertrophy and alterations in metabolism of complex lipids under high fat diet

Fatp4 exhibits acyl-CoA synthetase activity and is thereby able to catalyze the activation of fatty acids for further metabolism. However, its actual function in most tissues remains unresolved, and its role in cellular fatty acid uptake is still controversial. To characterize Fatp4 functions in adipocytes in vivo, we generated a mouse line with adipocyte-specific inactivation of the Fatp4 gene (Fatp4(A-/-)). Under standard conditions mutant mice showed no phenotypical aberrance. Uptake of radiolabeled palmitic and lignoceric acid into adipose tissue of Fatp4(A-/-) mice was unchanged. When exposed to a diet enriched in long chain fatty acids, Fatp4(A-/-) mice gained more body weight compared with control mice, although they were not consuming more food. Pronounced obesity was accompanied by a thicker layer of subcutaneous fat and greater adipocyte circumference, although expression of genes involved in de novo lipogenesis was not changed. However, the increase in total fat mass was contrasted by a significant decrease in various phospholipids, sphingomyelin, and cholesteryl esters in adipocytes. Livers of Fatp4-deficient animals under a high fat diet exhibited a higher degree of fatty degeneration. Nonetheless, no evidence for changes in insulin sensitivity and adipose inflammation was found. In summary, the results of this study confirm that Fatp4 is not crucial for fatty acid uptake into adipocytes. Instead, under the condition of a diet enriched in long chain fatty acids, adipocyte-specific Fatp4 deficiency results in adipose hypertrophy and profound alterations in the metabolism of complex lipids.     

Sphingolipid profiling of human plasma and FPLC-separated lipoprotein fractions by hydrophilic interaction chromatography tandem mass spectrometry

Sphingolipids comprise bioactive molecules which are known to play important roles both as intracellular and extracellular signalling molecules. Here we used a previously developed hydrophilic interaction chromatography tandem mass spectrometry (HILIC-MS/MS) method to profile plasma sphingolipids. Method validation showed sufficient precision and sensitivity for application in large clinical studies. Sample stability testing demonstrated that immediate plasma separation is important to achieve reliable results. Analysis of plasma from 25 healthy blood donors revealed a comprehensive overview of free sphingoid base, sphingosylphosphorylcholine (SPC), hexosylceramide (HexCer), lactosylceramide (LacCer), and ceramide-1-phosphate (Cer1P) species level. Besides the major sphingoid base sphingosine (d18:1), we found d16:1 and d18:2 species in most of these lipid classes. Interestingly, pronounced differences were detected in the species profiles of HexCer and LacCer. Additionally, sphingolipids were quantified in lipoprotein fractions prepared by fast performance liquid chromatography (FPLC). HexCer and LacCer showed similar distributions with about 50% in LDL, 40% in HDL and less than 10% in the VLDL fraction. More than 90% of sphingoid base phosphates were found in HDL and albumin containing fractions. In summary, HILIC-MS/MS provides a valuable tool to profile minor sphingolipid species in plasma and in lipoprotein fractions. Comparing profiles from tissues or blood cells, these species profiles may help to address the origin of plasma sphingolipids.     

Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes

Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges. Biotechnol. Bioeng. 2013;110: 2928–2937. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Biological activity of some conjugated gibberellins

Summary The biological activity of several gibberellin (GA) conjugates was studied and compared with that of the corresponding free GAs. The following conjugates were included: O(3)--d-glucopyranosides of GA₁, GA₃ and GA₄; O(13)--d-glucopyranosides of GA₁, GA₃ and GA₅; O(13)--d-glucopyranosyl-GA₅--d-glucopyranosyl ester; GA₃--d-glucopyranosyl ester and GA₃--d-glucopyranosyl ester; N-GA₃-oyl-glycine, its methyl ester, N-GA₃-oyl-glycylglycine, and N-GA₃-oyl-proline. All compounds were synthesized chemically but some of them are known to occur as endogenous plant products, or to be formed in plants upon application of a free GA. Activity was determined in the dwarf pea, dwarf corn, dwarf rice, and lettuce hypocotyl bioassays. The GA conjugates were found to posses different relative activities depending on the chemical structure, the bioassay system, and the site of application (shoot or roots). It is concluded that the activity of GA conjugates as measured in different bioassays is based upon the ability of plant enzymes and possibly of certain microorganisms to hydrolyze glucosidic, glucosyl ester, and amide-like linkages.Gibberellins-XLVIII. Part XLVII=Adam et al. (1976b)     

The yeast acyltransferase Sct1p regulates fatty acid desaturation by competing with the desaturase Ole1p

The degree of fatty acid unsaturation, that is, the ratio of unsaturated versus saturated fatty acyl chains, determines membrane fluidity. Regulation of expression of the fatty acid desaturase Ole1p was hitherto the only known mechanism governing the degree of fatty acid unsaturation in Saccharomyces cerevisiae. We report a novel mechanism for the regulation of fatty acid desaturation that is based on competition between Ole1p and the glycerol-3-phosphate acyltransferase Sct1p/Gat2p for the common substrate C16:0-CoA. Deletion of SCT1 decreases the content of saturated fatty acids, whereas overexpression of SCT1 dramatically decreases the desaturation of fatty acids and affects phospholipid composition. Whereas overexpression of Ole1p increases desaturation, co-overexpression of Ole1p and Sct1p results in a fatty acid composition intermediate between those obtained upon overexpression of the enzymes separately. On the basis of these results, we propose that Sct1p sequesters C16:0-CoA into lipids, thereby shielding it from desaturation by Ole1p. Ta-king advantage of the growth defect conferred by overexpressing SCT1, we identified the acyltransferase Cst26p/Psi1p as a regulator of Sct1p activity by affecting the phosphorylation state and overexpression level of Sct1p. The level of Sct1p phosphorylation is increased when cells are supplemented with saturated fatty acids, demonstrating the physiological relevance of our findings.     

Lipid Alterations in Experimental Murine Colitis: Role of Ceramide and Imipramine for Matrix Metalloproteinase-1 Expression

Background

Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD). Therefore, we analysed alterations of bioactive lipids in experimental models of colitis and examined the functional consequence of the second messenger ceramide in inflammatory pathways leading to tissue destruction.

Methodology/Principal Findings

Chronic colitis was induced by dextran-sulphate-sodium (DSS) or transfer of CD4⁺CD62L⁺ cells into RAG1^−/−-mice. Lipid content of isolated murine intestinal epithelial cells (IEC) was analysed by tandem mass spectrometry. Concentrations of MMP-1 in supernatants of Caco-2-IEC and human intestinal fibroblasts from patients with ulcerative colitis were determined by ELISA. Imipramine was used for pharmacologic inhibition of acid sphingomyelinase (ASM). Ceramide increased by 71% in chronic DSS–induced colitis and by 159% in the transfer model of colitis. Lysophosphatidylcholine (LPC) decreased by 22% in both models. No changes were detected for phosphatidylcholine. Generation of ceramide by exogenous SMase increased MMP-1-protein production of Caco-2-IEC up to 7-fold. Inhibition of ASM completely abolished the induction of MMP-1 by TNF or IL-1β in Caco-2-IEC and human intestinal fibroblasts.

Conclusions/Significance

Mucosal inflammation leads to accumulation of ceramide and decrease of LPC in the intestinal epithelium. One aspect of ceramide generation is an increase of MMP-1. Induction of MMP-1 by TNF or IL-1β is completely blocked by inhibition of ASM with imipramine. Therefore, inhibition of ASM may offer a treatment strategy to reduce MMP-1 expression and tissue destruction in inflammatory conditions.     

Apo AI/ABCA1-dependent and HDL3-mediated lipid efflux from compositionally distinct cholesterol-based microdomains

We have investigated whether a raft heterogeneity exists in human monocyte-derived macrophages and fibroblasts and whether these microdomains are modulated by lipid efflux. Triton X-100 (Triton) or Lubrol WX (Lubrol) detergent-resistant membranes from cholesterol-loaded monocytes were associated with the following findings: (i) Lubrol-DRM contained most of the cellular cholesterol and at least 75% of Triton-detergent-resistant membranes. (ii) 'Lubrol rafts', defined by their solubility in Triton but insolubility in Lubrol, were enriched in unsaturated phosphatidylcholine and showed a lower cholesterol to choline-phospholipid ratio compared to Triton rafts. (iii) CD14 and CD55 were recovered in Triton- and Lubrol-detergent-resistant membranes, whereas CD11b was found exclusively in Triton DRM. ABCA1 implicated in apo AI-mediated lipid efflux and CDC42 were partially localized in Lubrol- but not in Triton-detergent-resistant membranes. (iv) Apo AI preferentially depleted cholesterol and choline-phospholipids from Lubrol rafts, whereas HDL₃ additionally decreased the cholesterol content of Triton rafts. In fibroblasts, neither ABCA1 nor CDC42 was found in Lubrol rafts, and both apo AI and HDL₃ reduced the lipid content in Lubrol- as well as in Triton-detergent-resistant membranes. In summary, we provide evidence for the existence of compositionally distinct membrane microdomains in human cells and their modulation by apo AI/ABCA1-dependent and HDL₃-mediated lipid efflux.     

The satiety factor apolipoprotein A-IV modulates intestinal epithelial permeability through its interaction with alpha-catenin: implications for inflammatory bowel diseases.

INTRODUCTION: Apolipoprotein A-IV (apoA-IV), an intestinally and cerebrally synthesized satiety factor and anti-atherogenic plasma apolipoprotein, was recently identified as an anti-inflammatory protein. In order to elucidate whether intestinal apoA-IV exerts similar repair function as its hepatic homologue apolipoprotein A-V (apoA-V), apoA-IV-interactive proteins were searched and in vitro functional studies were performed with apoA-IV overexpressing cells. ApoA-IV was also analyzed in the intestinal mucosa of patients with inflammatory bowel diseases (IBD), together with other genes involved in epithelial junctional integrity. METHODS: A yeast-two-hybrid screening was used to identify apoA-IV-interactors. ApoA-IV was overexpressed in Caco-2 and HT-29 mucosal cells for colocalization and in vitro epithelial permeability studies. Mucosal biopsies from quiescent regions of colon transversum and terminal ileum were subjected to DNA-microarray analysis and pathway-related data mining. RESULTS: Four proteins interacting with apoA-IV were identified, including apolipoprotein B-100, alpha1-antichymotrypsin, cyclin C, and the cytosolic adaptor alpha-catenin, thus linking apoA-IV to adherens junctions. Overexpression of apoA-IV was paralleled with a differentiated phenotype of intestinal epithelial cells, upregulation of junctional proteins, and decreased paracellular permeability. Colocalization between alpha-catenin and apoA-IV occurred exclusively in junctional complexes. ApoA-IV was downregulated in quiescent mucosal tissues from patients suffering from IBD. In parallel, only a distinct set of junctional genes was dysregulated in non-inflamed regions of IBD gut. CONCLUSIONS: ApoA-IV may act as a stabilizer of adherens junctions interacting with alpha-catenin, and is likely involved in the maintenance of junctional integrity. ApoA-IV expression is significantly impaired in IBD mucosa, even in non-inflamed regions.