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The aim of the study was to compare the bronchodilatory effect of 2x50 microg salbutamol inhaled via Turbuhaler(R) as two consecutive doses compared with two divided doses (50+50 microg) at different time intervals. The study was of a randomized, double-blind, crossover design. The patients inhaled two doses of 50 microg salbutamol immediately after each other and with a time interval between the doses of 1.5, 3, 5, or 10 mins. Forced expiratory volume in 1 s (FEV(1)) was measured before the first inhalation and at 1, 2. 5, 4.5, 9.5, 15, 20, 30, and 60 min after the first inhalation. Seventeen asthmatic patients (8 men) with a mean age of 32 years (range: 19-49 years), mean FEV(1) of 2.9 l (range: 1.7-3.9 l) and a mean FEV(1) in percentage of predicted normal value of 77% (range: 63-91%) participated. The mean reversibility 15 min after inhaling 100 microg salbutamol from pMDI was 23% (range: 16-35%). The mean maximum increase in FEV(1) from baseline ranged between 18.6% (consecutive doses) and 21.2% (1.5 min between doses), corresponding to a difference between the treatments of 0.06 l. There were no significant differences in maximum FEV(1) or time to reach maximum FEV(1) between the treatments. We were not able to show any clinically relevant differences in maximal bronchodilating effect, assessed as FEV(1), in stable asthmatics, when therapeutic doses of salbutamol were given via Turbuhaler either as two consecutive doses or as two divided doses separated by different time intervals.

α2-Adrenoreceptor mimics, prepared by molecular imprinting of yohimbine, were used to select ligands from a phage display hexapeptide library. Phages with affinity for the yohimbine imprinted methacrylic acid–ethylene glycol dimethacrylate copolymer were selected. Phage affinities were estimated using an enzyme immunoassay. The selected library showed three-fold higher affinity for the imprinted polymer compared with the primary library. Eighty-two of ninety characterized phage clones from the selected library showed low affinity for the polymer. The hexapeptides on eight of these low binding phage clones consisted of mainly hydrophobic amino acid residues, and four clones were identical. The hexapeptides on five of the eight high affinity phage clones contained positively charged amino acids. Identical hexapeptides were expressed on four of these five clones. The results of this study suggest that the majority of the selected phages form hydrophobic and/or ionic interactions with the polymer framework rather than specific interactions only with the yohimbine imprinted sites. Furthermore, a direct correlation can be seen between hexapeptide sequence affinity and the number of positively charged amino acid residues they contain.

A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against α-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with α-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions. The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of α-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions. The inhibition constant for α-chymotrypsin was estimated to be 10-6 M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified α-chymotrypsin than for natural α-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively prevented from binding to α-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of α-chymotrypsin and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature.

Molecularly imprinted polymers (MIPs) selective for the α2-adrenoreceptor antagonist yohimbine (1) have been prepared and studied as α2-adrenoreceptor mimics. Marked ligand stereoselectivity was demonstrated in radioligand binding and HPLC studies upon comparison to blank and corynanthine (2) MIPs. KD values in the nanomolar range have been shown for anti-1 MIP prepared in chloroform solutions upon rebinding in organic media.

We have constructed two different fusion proteins consisting of the C-terminal end of CS1 fused in-frame to the N-terminal end of MDH1 and HSA, respectively. The fusion proteins were expressed in mutants of Saccharomyces cerevisiae in which CS1 and MDH1 had been deleted and the phenotypes of the transformants characterized. The results show that the fusion proteins are transported into the mitochondria and that they restore the ability for the yeast mutants CS1-, MDH1-, and CS1-/MDH1- to grow on acetate. Determination of CS1 activity in isolated mitochondria showed a 10-fold increase for the strain that expressed native CS1, relative to the parental. In the transformant with CS1/MDH1 fusion protein, parental levels of CS1 were observed, while one-fifth this amount was observed for the strain expressing the CS1/HSA conjugate. Oxygen consumption studies on isolated mitochondria did not show any significant differences between parental-type yeast and the strains expressing the different fusion proteins or native CS1. [3(-13)C]Propionate was used to study the Krebs TCA cycle metabolism of yeast cells containing CS1/MDH1 fusion constructs. The 13C NMR study was performed in respiratory-competent parental yeast cells and using the genetically engineered yeast cells consisting of CS1- mutants expressing native CS1 and the fusion proteins CS1/MDH1 and CS1/HSA, respectively. [3(-13)C]Propionate is believed to be metabolized to [2(-13)C]succinyl-CoA before it enters the TCA cycle in the mitochondria. This metabolite is then oxidized through two symmetrical intermediates, succinate and fumarate, followed by conversion to malate, oxalacetate, and other metabolites such as alanine.(ABSTRACT TRUNCATED AT 250 WORDS)

We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA-. The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl. The fusion protein produced was isolated and purified. Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000. Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000). This is the expected M(r) for the fusion protein subunit. The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes. In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS reaction, was examined. It was observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are employed. In addition, the transient time for the coupled reaction sequence was less for the fusion protein than for the free enzymes.

A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.

Genetically prepared protein A fusion proteins, having retained antibody binding capacity, were used to design different well-defined standard molecular weight marker proteins for Western blotting. The blotted marker proteins are developed at the same time and with the same reagents as the protein sample of interest.

The purpose of this study was to estimate the relative dose potency of salbutamol Turbuhaler compared with salbutamol pressurized metered dose inhaler (pMDI) with respect to the protective effect against methacholine bronchoconstriction. Twenty-three asthmatic subjects with stable asthma participated in the study. Baseline forced expiratory volume in 1 s (FEV1) was 70% or more of predicted, and baseline methacholine provocative concentration causing a 20% fall in FEV1(PC20) was 4 mg/mL or less. The design was randomized, double-blind, double-dummy, crossover and placebo controlled and was conducted over seven study days. On each study day, the subjects inhaled 50 µg or 100 µg of salbutamol via Turbuhaler, 100 µg, 200 µg, 400 µg or 800 µg of salbutamol via pMDI, or placebo in randomized order. PC20was determined 30 mins after inhalation. Increasing doses of salbutamol pMDI increased the PC20in a dose-dependent fashion from 3.9 mg/mL after placebo to 13.3 mg/mL after pMDI 100 µg, 19.0 mg/mL after 200 µg, 32.6 mg/mL after 400 µg, and 35.1 mg/mL after 800 µg. The half-maximum response dose for pMDI (ED50) was 104 µg. Salbutamol Turbuhaler 50 µg increased the PC20to 10.0 mg/mL and 100 µg to 12.6 mg/mL. Salbutamol pMDI 200 µg provided significantly greater protection to methacholine than pMDI 100 µg or Turbuhaler 100 µg and significantly less protection than pMDI 400 µg (P

Several artificial bi- and polyfunctional enzymes have been prepared by gene fusion in vitro . These recombinant enzymes catalyze consecutive reactions and often exhibit favorable proximity effects due to the closeness of the active sites. This is indicated by channeling of the intermediate substrate and reduced lag phases. Applications can be found in enzymatic analysis and metabolic engineering. The potential of gene fusion as a conjugation method in enzyme immunoassay is also discussed.

Peptides with affinity for the surface of alpha-chymotrypsin (EC 3.4.21.1) were selected from a hexapeptide phage display library consisting of approximately 10(7) different clones. Seven selections were performed and five individual phage clones analysed. Compared to the primary library, the five peptide phage clones all interacted more strongly with alpha-chymotrypsin, and DNA sequencing of the phage clones revealed five different amino acid sequences: Gly-Ala-Val-Ile-Thr-His, Arg-Asp-Ile-Val-Val-Ala, Val-Tyr-Ser-His-Ala-Ser, Gly-Ser-Tyr-Ser-Ala-Gly and Leu-Asp-Ile-Val-Val-Ala. Two of the peptides exhibited 83% identity (i.e. a difference of just one amino acid). The chemically synthesized peptides competitively reduced the binding of the corresponding peptide phage clone to alpha-chymotrypsin. Binding of some of the selected peptide phage clones to alpha-chymotrypsin was also reduced by several of the other non-corresponding synthesized peptides, suggesting that these peptides have common recognition areas on the enzyme. Three of the synthesized peptides were poor substrates of alpha-chymotrypsin and they did not inhibit enzyme activity. Our results suggest that it is possible to select peptides from peptide phage display libraries with affinity for different surface structures on the enzyme, not involved in the biologically active site.

A hexapeptide phage library was used to affinity select peptides which interact with single stranded heptaoligonucleotides consisting of cytosine (oligo-C). Selections were performed in two different buffer systems, 50 mM 2[N-Morpholino]ethanesulphonic acid(MES)-buffer, pH 5.5, and 50 mM Tris(hydroxymethyl)-aminomethane, 150 mM NaCl, pH 7.5, respectively. Selection was more successful in 50 mM MES-buffer and three clones with affinity for oligo-C were further investigated. The peptides, Pro-Pro-Pro-Leu-Tyr-Phe, Arg-Phe-Cys-Asp-Thr-Ser and Arg-Ser-Arg-Leu-Ile-Trp, all interacted more strongly with oligo-C compared to the original peptide phage library and wildtype phage. The selected clones also showed different specificity in the interaction with oligo-C, -G, -A and -T.

Genetically prepared enzyme conjugates were used in soluble enzyme assays for determination of lactose and galactose with spectrophotometric, fluorimetric, and bioluminometric detection. The use of a bifunctional enzyme in biosensors based on calorimetric detection is also presented. Due to proximity of the enzymes to one another several advantages in the analyses were achieved. The lag phase of coupled enzymatic reactions was reduced and the steady-state rates were increased, which could increase the sensitivity of the assays and decrease the time of analysis.

Enzyme immunoassay, using enzymes crosslinked to either antibodies or antigens, has proved a valuable immunological tool for many years. Recently, gene fusion techniques have been used to prepare these enzyme conjugates. This method may be especially advantageous in cases where (1) the antigen is difficult and costly to obtain in large quantities or (2) when the activity of the marker enzyme or the affinity of the antibody or antigen is severely reduced or even destroyed by use of conventional linking methods such as chemical crosslinking. This article uses specific examples to illustrate the potential of gene fusion as a conjugation method.

The two structural genes encoding galactose dehydrogenase (Pseudomonas fluorescens) and the beta subunit of luciferase (Vibrio harveyi) were fused in-frame in order to prepare and subsequently characterize an artificial bifunctional enzyme complex. This hybrid enzyme exhibited both galactose dehydrogenase activity and bioluminescence when expressed in Escherichia coli together with the alpha subunit of luciferase. The purified conjugate was used to study possible proximity effects in a sequential three-enzyme reaction with the bifunctional enzyme catalyzing the first and the last reaction. The intermediate enzyme, diaphorase, was added separately. The engineered enzyme system, comprising the galactose dehydrogenase/luciferase conjugate, could display a twofold higher bioluminescence in the overall enzyme reaction compared to a corresponding reference system with separate native enzymes. The increased bioluminescence obtained for the engineered enzyme system is proposed to be due to an improved organization of the enzyme in solution.

The genes encoding staphylococcal protein A and bacterial luciferase (Vibrio harveyi) were fused in-frame in order to obtain a general marker enzyme for bioluminescent immunoassays. Two constructs were made where protein A was ligated to the first and the 12th amino acid residue, respectively, of the N terminus of the beta subunit of luciferase. Only the first fusion protein encoding the entire beta subunit was able to form an enzymatically active luciferase complex when expressed together with the alpha subunit. The fusion of protein A to luciferase did not notably alter the emitted wavelength spectrum or its stability to urea treatment. The fusion protein was found to retain at least 50% of the specific bioluminescent activity compared to native luciferase. In preliminary tests, this hybrid protein was shown to be useful in bioluminescent immunoassays.

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.

The formation of a D-Ala-D-Ala containing tripeptide in aqueous solution, catalysed by muramoylpentapeptide carboxypeptidase (E.C. 3.4.17.8), is described.