Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3‐dioxygenase1

Although human amniotic fluid does contain different populations of foetal‐derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in vitro expanded, amniotic fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second‐trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon (IFN)‐γ, including induction of the immunomodulatory enzyme indoleamine 2,3‐dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN‐γ–treated fHASCs caused significantly decreased T‐cell proliferation and increased frequency in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells. Both effects required an intact IDO1 function and were cell contact‐independent. An unprecedented finding in our study was that purified vesicles from IFN‐γ–treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC‐like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4⁺ CD25⁺ Foxp3⁺ T cells in graft‐draining lymph nodes from recipient mice. Thus fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo.     

Functional plasticity of dendritic cell subsets as mediated by CD40 versus B7 activation

Murine dendritic cells (DCs) can present Ag in an immunogenic or tolerogenic fashion, the distinction depending on either the occurrence of specialized DC subsets or the maturation or activation state of the DC. Although DC subsets may be programmed to direct either tolerance or immunity, it is not known whether appropriate environmental stimulation can result in complete flexibility of a basic program. Using splenic CD8(-) and CD8(+) DCs that mediate the respective immunogenic and tolerogenic presentation of self peptides, we show that both the in vivo and in vitro activities of either subset can be altered by ligation of specific surface receptors. Otherwise immunogenic CD8(-) DCs become tolerogenic upon B7 ligation by soluble CTLA-4, a maneuver that initiates immunosuppressive tryptophan catabolism. In contrast, CD40 ligation on tolerogenic CD8(+) DCs makes these cells capable of immunogenic presentation. Thus, environmental conditioning by T cell ligands may alter the default function of DC subsets to meet the needs of flexibility and redundancy.     

The HCF136 protein is essential for assembly of the photosystem II reaction center in Arabidopsis thaliana

Hcf136 encodes a hydrophilic protein localized in the lumen of stroma thylakoids. Its mutational inactivation in Arabidopsis thaliana results in a photosystem II (PHII)-less phenotype. Under standard illumination, PSII is not detectable and the amount of photosystem I (PSI) is reduced, which implies that HCF136p may be required for photosystem biogenesis in general. However, at low light, a comparison of mutants with defects in PSII, PSI, and the cytochrome b(6)f complex reveals that HCF136p regulates selectively biogenesis of PSII. We demonstrate by in vivo radiolabeling of hcf136 that biogenesis of the reaction center (RC) of PSII is blocked. Gel blot analysis and affinity chromatography of solubilized thylakoid membranes suggest that HCF136p associates with a PSII precomplex containing at least D2 and cytochrome b(559). We conclude that HCF136p is essential for assembly of the RC of PSII and discuss its function as a chaperone-like assembly factor.     

Hydrolysis of grapefruit peel waste with cellulase and pectinase enzymes

Approximately 1 million metric tons of grapefruit were processed in the 2003/04 season resulting in 500,000 metric tons of peel waste. Grapefruit peel waste is usually dried, pelletized, and sold as a low-value cattle feed. This study tested different loadings of commercial cellulase and pectinase enzymes and pH levels to hydrolyze grapefruit peel waste to produce sugars. Pectinase and cellulase loadings of 0, 1, 2, 5, and 10mgprotein/g peel dry matter were tested at 45 degrees C. Hydrolyses were supplemented with 2.1mg beta-glucosidase protein/g peel dry matter. Five mg pectinase/g peel dry matter and 2mgcellulase/g peel dry matter were the lowest loadings to yield the most glucose. Optimum pH was 4.8. Cellulose, pectin, and hemicellulose in grapefruit peel waste can be hydrolyzed by pectinase and cellulase enzymes to monomer sugars, which can then be used by microorganisms to produce ethanol and other fermentation products.     

Expression of Coxsackie-Adenovirus receptor (CAR) in the developing mouse olfactory system

Interest in manipulating gene expression in olfactory sensory neurons (OSNs) has led to the use of adenoviruses (AdV) as gene delivery vectors. OSNs are the first order neurons in the olfactory system and the initial site of odor detection. They are highly susceptible to adenovirus infection although the mechanism is poorly understood. The Coxsackie-Adenovirus receptor (CAR) and members of the integrin family have been implicated in the process of AdV infection in various systems. Multiple serotypes of AdV efficiently bind to the CAR, leading to entry and infection of the host cell by a mechanism that can also involve integrins. Cell lines that do not express CAR are relatively resistant, but not completely immune to AdV infection, suggesting that other mechanisms participate in mediating AdV attachment and entry. Using in situ hybridization and western blot analyses, we show that OSNs and olfactory bulbs (OB) of mice express abundant CAR mRNA at embryonic and neonatal stages, with progressive diminution during postnatal development. By contrast to the olfactory epithelium (OE), CAR mRNA is still present in the adult mouse OB. Furthermore, despite a similar postnatal decline, CAR protein expression in the OE and OB of mice continues into adulthood. Our results suggest that the robust AdV infection observed in the postnatal olfactory system is mediated by CAR and that expression of even small amounts of CAR protein as seen in the adult rodent, permits efficient AdV infection and entry. CAR is an immunoglobulin domain-containing protein that bears homology to cell-adhesion molecules suggesting the possibility that it may participate in organization of the developing olfactory system.     

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon

Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.     

Consequential LCA and LCC using linear programming: an illustrative example of biorefineries

Purpose

This paper aims to demonstrate how LCA can be improved by the use of linear programming (LP) (i) to determine the optimal choice between new technologies, (ii) to identify the optimal region for supplying the feedstock, and (iii) to deal with multifunctional processes without specifying a certain main product. Furthermore, the contribution of LP in the context of consequential LCA and LCC is illustrated.

Methods

We create a mixed integer linear program (MILP) for the environmental and economic assessment of new technologies. The model is applied in order to analyze two residual beech wood-based biorefinery concepts in Germany. In terms of the optimal consequences for the system under study, the principle of the program is to find a scaling vector that minimizes the life cycle impact indicator results of the system. We further transform the original linear program to extend the assessment by life cycle costing (LCC). Thereby, two multi-objective programming methods are used, weighted goal programming and epsilon constraint method.

Results and discussion

The consequential case studies demonstrate the possibility to determine optimal locations of newly developed technologies. A high number of potential system modifications can be studied simultaneously without matrix inversion. The criteria for optimal choices are represented by the objective functions and the additional constraints such as the available feedstock in a region. By combining LCA and LCC targets within a multi-objective programming approach, it is possible to address environmental and economic trade-offs in consequential decision-making.

Conclusions

This article shows that linear programming can be used to extend standard LCA in the field of technological choices. Additional consequential research questions can be addressed such as the determination of the optimal number of new production plants and the optimal regions for supplying the resources. The modifications of the program by additional profit requirements (LCC) into a goal program and Pareto optimization problem have been identified as promising steps toward a comprehensive multi-objective LCSA.

     

Optimumβ-D-glucosidase supplementation of cellulase for efficient conversion of cellulose to glucose

Summary To assess optimal saccharification performance, -cellulose and dilute acid pretreated aspen (DAA) wood meal were subjected to various loadings of commercial cellulase and -D-glucosidase preparations. Fifteen international filter paper units (IFPU)/g cellulose content and 30 IFPU/g cellulose content were required to digest 95% of the available cellulose in -cellulose and pretreated aspen, respectively. The optimal supplementation ratios, based on Genencor GC 123 cellulase and -D-glucosidase from Novo SP 188 for the -cellulose and DAA digestions range from 0.25 to 0.5 and 0.12 to 0.25, respectively.     

Separation of the complementary strands of DNA fragments on polyacrylamide gels.

32P-labeled (in vivo) phiX174 RFI DNA was restricted by Hinc II. Three aliquots of the same digest: a) nondenatured, b) heat denatured, and c) denatured by 5 mM Me-HgOH were analyzed on 3-15% acrylamide gel gradients or on 3% gels with reduced N,N'-methylene-bis-acrylamide. The autoradiography of the gels showed that the nondenatured sample migrates two times faster than the denatured samples. After denaturation each original fragment appeared as a doublet. Using in vitro synthesized RFI DNA labeled only in negative strand with 32P we could identify the position of the negative strand in each denatured doublet. The single strand DNA fragments could be recovered from the gel slices on a semi-preparative scale by electrophoresis into dialysis tubing.