A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability

Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.     

Plants and human health in the twenty-first century

The concept of growing crops for health rather than for food or fiber is slowly changing plant biotechnology and medicine. Rediscovery of the connection between plants and health is responsible for launching a new generation of botanical therapeutics that include plant-derived pharmaceuticals, multicomponent botanical drugs, dietary supplements, functional foods and plant-produced recombinant proteins. Many of these products will soon complement conventional pharmaceuticals in the treatment, prevention and diagnosis of diseases, while at the same time adding value to agriculture. Such complementation can be accelerated by developing better tools for the efficient exploration of diverse and mutually interacting arrays of phytochemicals and for the manipulation of the plant's ability to synthesize natural products and complex proteins. This review discusses the history, future, scientific background and regulatory issues related to botanical therapeutics.     

Immunohistochemical localisation of ecdysteroids in the follicular epithelium of locust oocytes

Summary Frozen sections of growing terminal follicles of the locust ovary were incubated with an ecdysteroid-specific rabbit antibody and the bound antibody visualised by the use of FITC-labelled goat-anti-rabbit antiserum. A bright fluorescence was seen in the cytoplasm of the follicle cells in terminal follicles with a length between 4.0 and 6.0mm with a maximum intensity at 5.5mm, indicating the presence of ecdysteroids in these cells in this particular developmental stage.     

Catalytical oxidation of ecdysteroids to 3-dehydro products and their biological activities

Oxidation of ecdysone and ecdysterone with platinum as catalyst gives rise to several polar and 4–5 apolar substances, among them 3-dehydroecdysone and 3-dehydroecdysterone. Highest yields in the 3-dehydro products were reached after 4 to 8 hr of oxidation. 3-dehydroecdysone and 3-dehydroecdysterone are less active in inducing ecdysone specific puffs in Drosophila hydei salivary gland giant chromosomes but still contain a remarkable biological activity when compared with the original ecdysteroids. The activity of these two compounds is about one tenth that of ecdysone in the Calliphora assay.     

Synthesis of a compound soluble in organic solvents from deoxycytidine triphosphate in permeabilized normal human lymphocytes

Summary Normal human lymphocytes may be rendered permeable to deoxynucleoside triphosphates. When [³H]dCTP is furnished to permeabilized lymphocytes two compounds are formed: DNA and a compound soluble in organic solvents. [³H]dCTP incorporation is higher in stimulated lymphocytes than in unstimulated cells. Some characteristics of the system are reported.Abbreviations PHA Phytohemagglutinin - dNTP deoxyribonucleotide triphosphate - MEM minimal essential medium - PBS Phosphate buffered saline - SSC standard saline citrate This paper is dedicated to Dr.Luis F. Leloir on occasion of his 70th birthday.To whom reprints should be requested.     

Seasonal effects on cuticular permeability and epicuticular lipid composition inCentruroides sculpturatus ewing 1928 (Scorpiones: Buthidae)

Summary Third larval instar hemolymph of the fruitflyDrosophila hydei did not metabolize juvenile hormone (JH) at all developmental stages. In contrast, prepupal and pupal body fluid showed JH-esterase activity with a maximum at 4 h after puparium formation. In body wall and fat body of all developmental stages investigated, JH-metabolic activity was found. In both tissues JH catabolism was most active in the 120,000g supernatant and pellet. The 800g and 15,000g pellet showed a lower activity. In all subcellular fractions the JH-acid was identified as the predominant metabolite. There is evidence that JH-specific esterases are responsible for ester cleavage in the 120,000g supernatant. During mid and late third larval instar development in both body wall and fat body JH-esterase activity remains relatively constant.     

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust

Summary Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.     

Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent alpha-cyanoester and alpha-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC(50) values for a series of known inhibitors. The new IC(50) values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC(50) values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.     

Stimulation of gap junctional communication: comparison of acyclo-retinoic acid and lycopene

Carotenoids and retinoids stimulate gap junctional communication (GJC), thought to be related to cancer-preventive properties. Lycopene, a nonprovitamin A carotenoid and its possible oxidation product, acyclo-retinoic acid, were tested for their effect on GJC, on stabilization of connexin43 mRNA, and on the transactivation of the RAR-beta2-promoter in vitro. In human fetal skin fibroblasts, GJC was stimulated by lycopene and acyclo-retinoic acid. Lycopene was effective at a concentration of 0.1 microM, whereas higher amounts of acyclo-retinoic acid (1 microM) were needed for comparable stimulation. Stabilizing effects of acyclo-retinoic acid on the mRNA of connexin43 via elements located in the 3'-UTR were weak. In comparison to retinoic acid (0.1 microM), considerably higher concentrations of the acyclo analog (50 microM) were required for similar effects; lycopene (0.1 microM) was not active in this system. Likewise, unphysiologically high levels of acyclo-retinoic acid (50 microM) were necessary to transactivate the RAR-beta2 promoter. The data demonstrate that acyclo-retinoic acid is much less active than retinoic acid with respect to GJC and retinoid-related signaling. Therefore, we conclude that lycopene affects GJC independent of the formation of acyclo-retinoic acid.