Alternative microbial testing: a novel DNA-based detection system for specified microorganisms in pharmaceutical preparations.

Fluorescence-coupled PCR technology was employed to quantify DNA segments specific for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. The PCR procedure is put forward as an alternative method for detecting microbial contaminations in pharmaceutical preparations and is compared to the tests for specified microorganisms described in European Pharmacopoeia (EP) 2, 2.6.13 and the USP, chapter 61. Data presented here describe the validation of this analytical method when used for proof of absence of specified microorganisms. The detection systems were specific for the microorganisms analyzed, and led to linear results over a wide range (more than 6-7 log intervals). The correlation coefficients lay above 0.99. The precision of replicate determinations within a single test was observed to be high, the relative standard deviation being between 0.39% and 1.53%. The precision between different tests was also high, with a relative standard deviation between 0.76% and 1.91%. The sensitivity without pre-enrichment amounted to 1-10 CFU. Since determination of the specified bacteria was performed following pre-enrichment, the limit of detection amounted to 1 CFU. Equivalent results were obtained in a study on nine batches of a milky hydrophilic cream (SH-No. M 440 A) with the conventional test for microbial contamination and the PCR procedure. The data presented here strongly indicate that the use of fluorescence-coupled PCR techniques can prove the absence of specified bacteria faster and more efficiently than conventional methods.

Mechanisms of interaction of Escherichia coli threonine synthase with substrates and inhibitors.

Threonine synthase (TS), the last enzyme of the threonine biosynthetic pathway, catalyzes L-threonine formation from L-homoserine phosphate (HSerP; Km = 0.5 mM, V = 440 min-1) and DL-vinylglycine. Furthermore, TS catalyzes beta-elimination reactions with L-serine (Km = 150 mM, V = 4.7 min-1), DL-3-chloroalanine, L-threonine, and L-allo-threonine as substrates to yield pyruvate or alpha-ketobutyrate, while L-alanine, L-2-aminobutanoic acid, and L-2-amino-5-phosphonopentanoic acid are substrates for half-transamination reactions to form the pyridoxamine form of the enzyme and the corresponding alpha-keto acid. Spectral analyses of all these reactions revealed the transient formation of strongly absorbing long-wavelength chromophores (lambda max = 440-445 nm), implying the accumulation of the corresponding pyridoxaldimine p-quinonoidal intermediates. HSerP turnover was competitively inhibited by L-3-hydroxyhomoserine phosphate 1 (Ki = 0.050 mM), L-2,3-methanohomoserine phosphate 2 (Ki = 0.010 mM), L-2-amino-3-[(phosphonomethyl)thio)]propanoic acid 5 (Ki = 0.011 mM) and DL-E-2-amino-5-phosphono-4-pentenoic acid 10 (Ki = 0.54 mM). 5 and 10 induced the formation of long-wavelength quinonoidal chromophores (lambda max = 458 and 460 mm, epsilon 47,000 and 30,000 M-1 cm-1), while incubation with either 1 or 2 induced only minor spectral changes. DL-2-Amino-3-[(phosphonomethyl)amino)]propanoic acid inactivated TS (Ki = 0.057 mM, kinact = 1.44 min-1) with 1:1 stoichiometry, transient formation of a 450-nm chromophore, and finally bleaching of any absorbance at wavelengths longer than 320 nm. Z-2-Amino-5-phosphono-3-pentenoic acid 8 is the unusual amino acid found in the peptide antibiotics of the plumbemicin and rhizocticin families. Racemic 8 irreversibly inhibited TS (Ki = 0.1 mM, kinact = 1.50 min-1) with 1:1 stoichiometry and the concomitant formation of a 482-nm chromophore (epsilon approximately 30,000 M-1 cm-1). DL-E-2-Amino-5-phosphono-3-pentenoic acid was a less potent irreversible inhibitor of TS (Ki = 0.4 mM, kinact = 0.25 min-1), inducing absorption maxima at 462 and 500 nm. The acetylenic amino acid DL-2-amino-5-phosphono-4-pentynoic acid 12 bound to TS (KD = 0.38 mM) forming a quinonoidal chromophore (lambda max = 452 nm, epsilon approximately 30,000 M-1 cm-1), but inhibition of the enzyme by 12 could not be detected under assay conditions even at high inhibitor concentrations. Mechanisms consistent with these observations are proposed.

Partial amino acid sequence of fructose-1,6-bisphosphatase from the blue-green algae Synechococcus leopoliensis.

Purified fructose-1,6-bisphosphatase from the cyanobacterium Synechococcus leopoliensis was S-carboxymethylated and cleaved with trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography and the amino acid sequence of six of the purified peptides was determined by gas-phase microsequencing. The results revealed sequence homology with other fructose-1,6-bisphosphatases. The obtained sequence data provides information required for the design of oligonucleotide hybridization probes to screen existing libraries of cyanobacterial DNA. The determination of the amino acid sequence of cyanobacterial proteins may yield important information with respect to the endosymbiotic theory of evolution.

Pharmacological profile of a new potent 5-hydroxytryptamine (5-HT2) alpha 1-receptor antagonist.

In in vitro binding studies ZK 33.839 (4-(3-[3-(4-(4-fluorobenzoyl)-1-piperidinyl)-propoxy]-4-methoxyphenyl)- 2-pyrrolidone) showed highly specific binding affinity for 5-hydroxytryptamine (5-HT2) and alpha 1-receptors. With 2.0 nmol/l and 5.2 nmol/l both Ki-values occur in the same concentration range. The pharmacodynamic profile of ZK 33.839 has been investigated under in vitro and in vivo conditions. In human platelets, in rat vascular smooth muscle and in guinea pig tracheal smooth muscle 5-HT-induced proaggregatory and contractile effects were inhibited dose-dependently with IC50-values ranging from 1.85 x 10(-8) mol/l to 9 x 10(-9) mol/l. 5-HT-induced amplification of the response of rabbit femoral artery to different vasoconstrictors (angiotensin II, histamine, norepinephrine, and prostaglandin F2 alpha) and 5-HT-mediated increase of microvascular permeability in hamster cheek pouch preparation were also inhibited by ZK 33.839. ZK 33.839 was found to be a potent alpha 1-receptor antagonist, the pA2-value in rat aortic strips determined against phenylephrine was 9.16. In blood-perfused hindquarters of anaesthetized rats, pretreated with reserpine, pressor dose-response curves to norepinephrine and 5-HT were shifted to a higher dose range. ZK 33.839 lowered blood pressure in conscious Dahl-S-rats and in anaesthetized rabbits. Decrease of blood pressure was due to a decrease of peripheral vascular resistance. Cardiac output and heart rate were not significantly altered. ZK 33.839 is a potential antihypertensive compound which combines vasodilatatory effects due to selective alpha 1-receptor antagonistic action and platelet antiaggregatory, antivasospastic, and vasoprotective properties due to selective 5-HT2-receptor blockade.

Fructose 1,6-Bisphosphatase Form B from Synechococcus leopoliensis Hydrolyzes both Fructose and Sedoheptulose Bisphosphate.

The substrate specificity of purified fructose bisphosphatase form B from Synechococcus leopoliensis (EC 3.1.3.11; cf. K-P Gerbling, M Steup, E Latzko 1985 Eur J Biochem 147: 207-215) has been investigated. Of the phosphate esters tested only fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate were hydrolyzed by the enzyme. Both sugar bisphosphates were cleaved at the carbon 1-ester. Fructose- and sedoheptulose bisphosphate stabilized the activated (i.e. tetrameric) state of the enzyme and prevented a slow inactivation that is observed in the absence of sugar bisphosphates. With the activated enzyme, kinetic constants (half-saturating substrate concentrations, maximal reaction velocity, and the catalytical constant) were similar for both fructose- and sedoheptulose bisphosphate. The data suggest that fructose bisphosphatase form B from Synechococcus leopoliensis can catalyze both bisphosphatase reactions within the reductive pentose phosphate cycle.

Fructose-1,6-bisphosphatase from Synechococcus leopoliensis. Substrate-dependent dimer-tetramer interconversion.

Extracts of Synechococcus leopoliensis (Anacystis nidulans) contain two forms of D-fructose-1,6-bisphosphatase (EC 3.1.3.11) previously designated as forms A and B [Gerbling, K.-P., Steup, M., and Latzko, E. (1984) Arch. Microbiol. 137, 109-114]. Form B, which probably represents the major part of the total extractable fructose-1,6-bisphosphatase activity, has been purified to apparent homogeneity. Gel filtration, non-denaturing polyacrylamide gel electrophoresis, and cross-linking with bis(sulfosuccinimidyl)suberate revealed that the fructose-1,6-bisphosphatase B exists in either a dimeric or in a tetrameric subform, depending upon the absence or presence of fructose-1,6-bisphosphate and Mg2+. The dimer--tetramer interconversion was readily reversible. The results provide evidence for a two-step activation of fructose-1,6-bisphosphatase B involving the reduction of the dimeric subform and the subsequent substrate-dependent conversion of the reduced dimer to a reduced tetramer, which is the only catalytically active state. In contrast to form B, no substrate-dependent interconversion was detected with form A from S. leopoliensis.

Partial purification and properties of soluble ascorbate peroxidases from pea leaves.

One nonenzymic and two enzymic forms of ascorbate peroxidase were found in pea leaves, and designated A, B and C. Form A was due to a low molecular weight, heat-stable component, and could be separated from the enzymic forms by gel filtration. Forms B and C were soluble proteins with an apparent molecular weight of 57,000. These two forms could be separated by cation-exchange chromatography on CM-Sephadex C-50. This technique was incorporated into a procedure for their partial purification. Several properties of B and C were found to be similar: they were active over a wide pH range (5 to 8), they displayed very high affinities for H(2)O(2) (Km<5 µM), and Km values for ascorbate (6.5 mM and 2.9 mM, respectively) were comparable to physiological concentrations of this substrate. These properties are considered conducive to the proposed physiological role of ascorbate peroxidase, viz prevention of H(2)O(2) accumulation.

Multiple forms of amylase in leaf extracts: electrophoretic transfer of the enzyme forms into amylose-containing polyacrylamide gels.

A method for the analysis of multiple forms of glucan-degrading enzymes is described. The procedure consists of the separation of the proteins by electrophoresis or isoelectric focusing in glucan-free polyacrylamide gels followed by the nondenaturing electrophoretic transfer into a second polyacrylamide layer which contains immobilized glucans. The method combines the resolving power of electrophoretic separations in glucan-free media with the sensitivity of amylase activity detection in amylose-containing polyacrylamide gels. The procedure is especially useful when samples containing low amylase activity, but a large number of multiple enzyme forms, are to be analyzed.