Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

Market Affections: Moral Encounters with Kenyan Fairtrade Flowers

This paper explores commodity exchange as a morally inflected practice, one that mediates competing tensions of greed and generosity, the sacred and profane, and affection and estrangement through the fairtrade flower. Using the UK–Kenya fairtrade flower commodity chain to examine the cultural economy of fairtrade, I suggest that like the charity business and the international development industry, fairtrade complicates the distinction between the sacred and secular and the gift and commodity as Northern consumers and NGOs weave webs of obligation through the medium of the market. Further, I argue that while fairtrade is predicated on values of partnership and interdependence, it also operates within commodity chains that advance liberal ethics as a mode of ‘governmentality’ over African producers, translating consumers' sympathy-based humanism into new technologies of regulation and surveillance.     

Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals

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Sex Differences in the Association of Thigh Fat and Metabolic Risk in Older Adults

We have previously shown a favorable association of subcutaneous leg fat with markers of insulin resistance and dyslipidemia in postmenopausal women. It is not known whether there is a sex dimorphism in the association of lower‐body adiposity with reduced metabolic risk. Thus, our primary aim was to determine whether the favorable association of thigh subcutaneous fat, independent of abdominal fat, is also observed in older men. Mid‐thigh and abdominal fat areas were measured by computed tomography (CT) in 108 older men and postmenopausal women (mean ± s.d.; 69 ± 7 years). Additionally, trunk and leg fat mass (FM) were measured by dual‐energy X‐ray absorptiometry (DXA). Markers of insulin resistance and dyslipidemia were determined from oral glucose tolerance tests and lipid and lipoprotein measurements, respectively. Outcomes were fasted and postchallenge (area under the curve, AUC) insulin (INS_AUC) and glucose (GLU_AUC), product of the insulin and glucose AUC (INS_AUC × GLU_AUC), triglycerides (TG), and high‐density lipoprotein (HDL)‐cholesterol. Consistent with our previous findings in postmenopausal women, adjusting for DXA trunk FM revealed a favorable association of DXA leg FM with the metabolic risk outcomes in both older men and postmenopausal women. Likewise, adjusting for CT abdominal visceral fat generally revealed a favorable association of CT thigh fat with metabolic risk outcomes in women, but not men. The discordance between the DXA and CT results in men is unclear but may be due to sex differences in visceral fat accrual. The mechanisms underlying the protective effect of thigh fat on metabolic risk factors need to be elucidated.     

Expression of the human atypical kinase ADCK3 rescues coenzyme Q biosynthesis and phosphorylation of Coq polypeptides in yeast coq8 mutants

Coenzyme Q (ubiquinone or Q) is a lipid electron and proton carrier in the electron transport chain. In yeast Saccharomyces cerevisiae eleven genes, designated COQ1 through COQ9, YAH1 and ARH1, have been identified as being required for Q biosynthesis. One of these genes, COQ8 (ABC1), encodes an atypical protein kinase, containing six (I, II, III, VIB, VII, and VIII) of the twelve motifs characteristically present in canonical protein kinases. Here we characterize seven distinct Q-less coq8 yeast mutants and show that unlike the coq8 null mutant, each maintained normal steady-state levels of the Coq8 polypeptide. The phosphorylation states of Coq polypeptides were determined with two-dimensional gel analyses. Coq3p, Coq5p, and Coq7p were phosphorylated in a Coq8p-dependent manner. Expression of a human homolog of Coq8p, ADCK3(CABC1) bearing an amino-terminal yeast mitochondrial leader sequence, rescued growth of yeast coq8 mutants on medium containing a nonfermentable carbon source and partially restored biosynthesis of Q(6). The phosphorylation state of several of the yeast Coq polypeptides was also rescued, indicating a profound conservation of yeast Coq8p and human ADCK3 protein kinase function in Q biosynthesis.     

Effect of indomethacin on prostaglandin content of several rabbit tissues

The prostaglandin (PG) content of several tissues and fluids from 6 day pregnant rabbits was evaluated following treatment with indomethacin or vehicle . PGE and PGF were measured by radioimmunoassay. More complete depletion of PGE and PGF was accomplished by 3 injections of indomethacin (s.c.) given during the 18 h before sacrifice at a dose of 10 mg indomethacin per kg body weight than was accomplished by 1 injection of the same amount of indomethacin (i.v.) 1.5 h before sacrifice. Levels of PGF were more easily depressed by indomethacin than were those of PGE. PG levels in the kidney and blastocysts were depressed to a greater extent by indomethacin than were those in the uterus, uterine fluid or peritoneal fluid. Evaluation of the effect of indomethacin on a particular physiological function should be interpreted with caution unless the extent of PG depletion in that tissue is also measured.     

Heparan sulfate 2-O-sulfotransferase (Hs2st) and mouse development

Heparan sulphate 2-O-sulphotransferase (Hs2st) acts at an intermediate stage in the pathway of biosynthesis of heparan sulphate (HS), catalysing the transfer of sulphate from 3-phosphoadenosine-5-phosphosulfate (PAPS) to the C2-position of selected hexuronic acid residues within the maturing HS chain. It is well established that 2-O-sulphation within HS, particularly of iduronate residues, is essential for HS to participate in a variety of high-affinity ligand-binding interactions. HS plays a central role in embryonic development and cellular function, modulating the activities of an extensive range of growth factors. Interestingly, in contrast to the early failure of embryos entirely lacking HS, Hs2st ^–/– mice survive until birth, but die perinatally due to a complete failure of kidney formation. The phenotype of Hs2st ^–/– mutant kidneys suggests that signalling between two tissues, ureteric bud and metanephric mesenchyme, is disrupted. We discuss candidate signalling molecules that may mediate this interaction. The HS generated by these mice lacks 2-O-sulphate groups but is extensively modified above wild type levels by O-sulphation at C-6 of glucosamine-N-sulfate (GlcNS) residues. We will discuss the potentially altered role of this atypical HS in growth factor signalling. Published in 2003.     

Diversity surveys and evolutionary relationships of aoxB genes in aerobic arsenite-oxidizing bacteria

A new primer set was designed to specifically amplify ca. 1,100 bp of aoxB genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny. Alphaproteobacteria-, Betaproteobacteria-, and Gammaproteobacteria-related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic As-contaminated soils. Our study underlines the usefulness of the aoxB gene as a functional marker of aerobic As(III) oxidizers.