The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene (pho A) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5'-terminal end of pho A. In the other construction a 42 base pair DNA fragment has been deleted from the 3'-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10-15 U/mg and 18-25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.     

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

Increased yield of fructose from glucose employing thermophilic xylose isomerase in water-ethanol mixtures

Summary The isomerization of D-glucose in mixed ethanol-water was studied at various reaction temperatures (40–70 °C), employing glucose isomerase fromStreptomyces phaeochromogenes andClostridium thermohydrosulfuricum, respectively. The thermophilicClostridium enzyme was considerably, more stable towards the combination of organic cosolvent and increased temperature and with this enzyme a 55% yield of fructose from glucose was obtained at relatively low concentration of ethanol (40 %).     

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli

We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit. Thus, immediate assay start up was possible.     

Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Role of hydrophilic bile acids and of sterols on cholelithiasis in the hamster

The effect of various dietary additions such as cholesterol, beta-sitosterol, bile acids, and bile acid analogs on gallstone formation was studied in the hamster. Gallstones were formed in 50% of the animals fed a high glucose, fat-free diet. Administration of 0.2% cholesterol or 1% beta-sitosterol had no effect on the incidence of gallstones. Ursodeoxycholic acid (0.5%) and its analog ursodeoxy-oxazoline [2-(3 alpha, 7 beta-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] were ineffective in preventing gallstones. Hyodeoxycholic acid and hyodeoxy-oxazoline [2-(3 alpha,6 alpha-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] at the same dosage effectively prevented gallstones, while the trihydroxy bile acid, hyocholic acid, was not effective. Of all the dietary regimens tested, only hyodeoxycholic acid significantly lowered serum cholesterol. The lithogenic diet produced a five-fold increase in hepatic HMG-CoA reductase activity; this activity was not affected by dietary cholesterol or beta-sitosterol. Hyodeoxycholic acid and hyocholic acid feeding increased the reductase activity by an additional 50% while the other bile acids had no effect. beta-Sitosterol doubled the cholesterol 7 alpha-hydroxylase activity whereas hyodeoxy-oxazoline lowered it. Hyodeoxycholic acid-fed animals had significantly lower cholesterol absorption than the animals on the lithogenic diet alone. Biliary cholesterol content increased dramatically in the animals fed the lithogenic diet and was increased still further by ursodeoxycholic acid, hyodeoxycholic acid, and hyodeoxy-oxazoline. These data show that hyodeoxycholic acid and hyodeoxy-oxazoline do not prevent gallstones by inhibiting hepatic cholesterol synthesis or biliary cholesterol secretion.     

Preparation of a NAD(H)-polymer matrix showing coenzyme function of the bound pyridine nucleotide

To a Sepharose gel the pyridine nucleotide NAD(H) has been bound using dicyclohexyl carbodiimide. In order to improve the steric availability of the nucleotide for added soluble enzymes such as dehydrogenases, a spacer molecule, ε-amino caproic acid, was inserted between the carbohydrate matrix and the nucleotide. The obtained preparation contained 56 μmoles NAD⁺/g dry polymer. The obtained matrix-bound NAD(H) was accepted as coenzyme by added lactate dehydrogenase. These preparations were still active after storage for several weeks at 4° C and could be used repeatedly without loss of activity. This represents the first necessary step taken in the preparation of compact closed systems consisting of “enzyme–coenzyme–coenzyme-regenerating enzyme” bound to individual polymer beads; such systems eliminate the need for continuous coenzyme addition.     

Isolation and Biochemical Characterization of a Neural Proteoglycan Expressing the L5 Carbohydrate Epitope

The monoclonal L5 antibody reacts with an N-glycosidically linked carbohydrate structure which is present on the neural cell adhesion molecule L1, neural chondroitin sulfate proteoglycans, and other not yet identified glycosylated proteins. Using this antibody, we isolated and characterized proteoglycans from adult mouse brain and cultured astrocytes biosynthetically labeled with Na2 35SO4 and a 3H-amino acid mixture. Our data suggest that the L5 proteoglycans of both sources are identical in their biochemical properties. The apparent molecular mass of the L5 proteoglycan is approximately 500 kDa. Digestion of the iodinated L5 proteoglycan from mouse brain and of the [35S]methionine-labeled L5 proteoglycan from cultured astrocytes with proteinase-free chondroitinases ABC and AC revealed three major core proteins with apparent molecular masses of approximately 380, 360, and 260 kDa. These represent molecularly distinct protein cores.     

Expression of Biologically Active Basic Fibroblast Growth Factor by Genetically Modified Rat Primary Skin Fibroblasts

Abstract: Basic fibroblast growth factor (FGF-2) is normally expressed as a cell-associated protein, and accordingly it is not clear how it exerts its action on target cells in vivo. It has been proposed that cells release, by death or other mechanisms, small amounts of FGF-2 that then acts in an autocrine manner. To address the question of whether it is necessary that FGF-2 remain cell associated or needs to be secreted from cells to have biological activity, we expressed the 18-kDa form of FGF-2 in primary fibroblasts as a cell-associated (FGF-2-B) or as a secreted (FGF-2-S) protein. FGF-2 protein is detected in cell lysates and membrane fractions of both cell types, whereas it is present in significant amounts only in the conditioned medium of FGF-2-S cells. No FGF-2 is detected in control (untransfected) cells. FGF-2-S cells also grow faster than the control or FGF-2-B cells. Yet, when evaluated for their ability to promote the survival of embryonic hippocampal neurons in vitro, both the cell types are active, establishing the activity of the transgene product. We conclude that FGF-2 is active when engineered to be expressed as a cell-associated form or secreted from cells.