The potato granule bound starch synthase chloroplast transit peptide directs recombinant proteins to plastids

The chloroplast targeting transit sequence from potato granule bound starch synthase (gbss) was used to direct the accumulation of recombinant proteins to the plastid stroma. The potato gbss transit sequence was fused to the N-terminus of the green fluorescent protein (GFP) and the Catharanthus roseus strictosidine synthase (Str1) enzyme. Fluorescence microscopy confirmed that the recombinant gbss-GFP fusion protein was exclusively targeted to the plastid stroma in tobacco suspension cells, demonstrating that the transit sequence was functional in vivo. The Str1 fusion protein accumulated to high levels in plastids isolated from transgenic plants. We conclude that the potato gbss transit sequence is functional and directs import of recombinant proteins into the chloroplast stroma.     

Socioeconomic Metabolism and Colonization of Natural Processes in SangSaeng Village: Material and Energy Flows, Land Use, and Cultural Change in Northeast Thailand

Conceptualizing environmental problems as sustainability problems contributing to local and global environmental change requires an understanding of how societies cope with their natural environment. Indicators for society–nature interactions are fairly well developed for national-level analyses. This study adapts some of these indicators to the local level and relates them to a qualitative assessment of economic and cultural change in a single community. Indicators are derived from material and energy flow accounting methods and address two major objectives: Firstly, to identify mutual influences between the global and the local level. Secondly, to assess future potentials of environmental pressures and impacts that can be expected to occur as such communities follow a path of further modernization. This study of a small rice-farming community in Northeast Thailand deals with physical as well as sociocultural aspects in order to produce a broad picture of society–nature relations. The indicators developed portray a society in the midst of transition and rapid modernization. This becomes apparent when comparing the results to those of similar studies in traditional and industrial societies. What we see is a community struggling to adapt to global influences, while at the same time maintaining subsistence with traditional coping mechanisms.     

The keystone role of leaf-removing crabs in mangrove forests of North Brazil

Principle factors which influence mangroveleaf litter turnover, in particular therole of leaf-removing crabs, were evaluatedin a riverine mangrove site nearBragança (Pará, North Brazil). Ourspecial interest was focussed on the roleof the leaf-removing crab Ucidescordatus. Leaf litter fluxes between themangrove forest and the adjacent estuarywere investigated by estimating the biomassand fate of leaf litter material and propagules. Vegetation is dominated by Rhizophora mangle, with Avicenniagerminans trees, both up to 25 m high,found intermittently. During 1997, Rhizophora trees produced around 1.40 gDW m^-2 d^-1 of leave fall and0.75 g DW m^-2 d^-1 of propagules.Leaf decomposition rates on the ground wereabout 0.06 g DW m^-2 d^-1,irrespective of species, habitat or siteexposure. This amount accounts for <3%of total leaf fall. Average leaf litterbiomass present on the ground was 0.01 gDW m^-2 d^-1. When the mangroveforest was flooded (on average 10 days permonth) the quantity of leaf litterand propagules washed out with the springtide was 10 and 17 times greater thanduring neap tide. Nevertheless, tidalexport and decomposition together made upless than 39 percent of annual leaf litterfall. The bulk of the remaining amount isapparently removed by Ucides. Eachcrab consumed about 1.30 g DW of leaflitter material and propagules per day.Since the average density of these crabswas 1.38 crabs m^-2, it is proposedthat Ucides is a keystone species inBragantinian mangroves.     

The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea

The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this “direct” pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: ΔBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene.     

Androgen receptor transactivation assay using green fluorescent protein as a reporter

For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpBUbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a fully functional transactivation assay system with beta-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a simplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and d-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC(50) values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity.     

Diagnostic value of unusual gross appearance of aspirated material from endoscopic ultrasound-guided fine needle aspiration of pancreatic and peripancreatic cystic lesions

OBJECTIVE: To evaluate the diagnostic value of the gross appearance of aspirated material from endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of pancreatic orperipancreatic cystic lesions. STUDY DESIGN: This study focuses on nonneoplastic (peri)-pancreatic cystic lesions on EUS-FNA. Eight cases were identified with distinct gross appearances of aspirated material by the authors' experience and by computerized review. Gross observations are evaluated together with clinical data, radiologic findings, laboratory markers and cytologic and surgical microscopy findings. RESULTS: Aspiration of cystic lesions often results in nonspecific cytologic findings. Gross and microscopic appearance of aspirated material can provide valuable information. We divide findings of 8 cystic lesions into 3 different patterns according to their gross appearance at on-site EUS-FNA: Pattern 1, 2 cases of biloma; Pattern 2, 1 case of lymphoepithelial cyst with sebaceous differentiation; Pattern 3, 3 cases of regular lymphoepithelial cysts and 2 cases ofpancreatic pseudocyst. CONCLUSION: The full value of FNA is achieved only with the integrated approach, including the gross appearance of the cytology specimens. Certain unusual gross appearances of the aspirated material can add valuable information toward a pathologic diagnosis.     

HLA-DRB1*0401 and HLA-DRB1*0408 are strongly associated with the development of antibodies against interferon-beta therapy in multiple sclerosis

The formation of antibodies to interferon-beta (IFN-beta), a protein-based disease-modifying agent for multiple sclerosis (MS), is a problem in clinical practice. These antibodies may neutralize the biological effects of the protein drug, potentially decreasing its therapeutic effects. By high-resolution HLA class I and II typing we identified two HLA class II alleles associated with the development of antibodies to IFN-beta. In two independent continuous and binary-trait association studies, HLA-DRB1*0401 and HLA-DRB1*0408 (odds ratio: 5.15)--but not other HLA alleles--were strongly associated with the development of binding and neutralizing antibodies to IFN-beta. The associated HLA-DRB1*04 alleles differ from nonassociated HLA-DRB1*04 alleles by a glycine-to-valine substitution in position 86 of the epitope-binding alpha-helix of the HLA class II molecule. The peptide-binding motif of HLA-DRB1*0401 and *0408 might promote binding and presentation of an immunogenic peptide, which may eventually break T cell tolerance and facilitate antibody development to IFN-beta. In summary, we identified genetic factors determining the immunogenicity of IFN-beta, a protein-based disease-modifying agent for the treatment of MS.     

Expression and function of the bile acid receptor TGR5 in Kupffer cells

Kupffer cells are resident macrophages in the liver and play a central role in the hepatic response to injury. Bile acids can impair macrophage function leading to decreased cytokine release. TGR5 is a novel, membrane-bound bile acid receptor, and it has been suggested that the immunosuppressive effect of bile acids can be mediated by TGR5. However, the function of TGR5 in Kupffer cells has not been studied and a direct link between TGR5 and cytokine production in macrophages has not been established. The present study demonstrates that TGR5 is localized in the plasma membrane of isolated Kupffer cells and is responsive to bile acids. Furthermore, bile acids inhibited LPS-induced cytokine expression in Kupffer cells via TGR5-cAMP dependent pathways. TGR5-immunoreactivity in Kupffer cells was increased in rat livers following bile-duct ligation, suggesting that TGR5 may play a protective role in obstructive cholestasis preventing excessive cytokine production thereby reducing liver injury.