HPLC methods for the determination of bound and free doxorubicin, and of bound and free galactosamine, in methacrylamide polymer-drug conjugates

Using insertional inactivation of the different genes of the dlt operon in Bacillus subtilis, we searched for metabolic and morphological changes caused by D-alanine ester deprivation of lipoteichoic acid and wall teichoic acid. There were no alterations of cell growth, basic metabolism, cellular content of phosphorus-containing compounds, ultrastructure, cell separation, and surface charge. The only alteration observed was an enhancement of endogenous and beta-lactam-induced cell lysis. Since this enhancement is doubtless correlated with the D-alanine ester deprivation of the teichoic acids, the present view based on in vitro experiments, that negatively charged LTA is inhibitory to autolysins, may be questioned. We propose that negatively charged lipoteichoic acid and/or wall teichoic acid serve in vivo to fix the cationic autolysins within the cell wall-membrane complex by electrostatic interaction. Positively charged D-alanine ester substituents decrease the binding capacity of the teichoic acids for autolysins by charge compensation.     

Role reversal for substrates and inhibitors. Slow inactivation of D-amino acid transaminase by its normal substrates and protection by inhibitors

D-Amino acid transaminase, which catalyzes the synthesis of D-alanine and D-glutamate for the bacterial cell wall, is a candidate for the design of specific inhibitors that could be novel antimicrobial agents. Under the experimental conditions usually employed for enzyme assays, kinetic parameters for its substrates were determined for short incubation periods, when intermediates and products do not accumulate and the enzyme activity is linear with time. Such kinetic analyses indicate that the enzyme accepts most D-amino acids but D-aspartate and D-glutamate are the best substrates. Under a different type of experimental conditions when the enzyme is exposed to D-alanine, intermediates, and products for periods of hours, it slowly becomes inactivated (Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., and Manning, J. M. (1992) Biochemistry 31, 6018-6023). We now report that D-aspartate, D-glutamate, and L-alanine also lead to slow inactivation. Methylation or amidation of the alpha-COOH group of D-alanine prevents inactivation, indicating that decarboxylation is required for inactivation; the slow release of CO2 from substrate is demonstrated. The alpha-methyl analog of D-alanine, D-aspartate, and D-glutamate do not lead to inactivation, showing that the alpha-hydrogen of the substrate is required, i.e. that some processing is required. Lys145, which binds pyridoxal 5'-phosphate in the wild-type enzyme, is not involved in the inactivation since two active site mutant enzymes, K145Q and K145N, are also inactivated. Reactivation of the inactive enzyme at acidic pH is accompanied by the release of ammonia corresponding to 1 mol/mol of dimeric enzyme. Competitive inhibitors, amine-containing buffers, and thiols effectively impede the inactivation. This reversal in the roles of substrates and inhibitors, i.e. when a substrate can be an inactivator and an inhibitor can act as a protector, occurs during a time period not usually used to measure steady-state kinetics or initial velocities of enzyme reactions and could have physiological relevance in cells.     

Inactivation of bacterial D-amino acid transaminase by beta-chloro-D-alanine

Purified D-amino acid transaminase from Bacillus sphaericus catalyzes an alpha,beta elimination from the D isomer of beta-chloroalanine to yield equivalent amounts of pyruvate, chloride, and ammonia; the L isomer of chloroalanine is not a substrate for this transaminase. During the beta elimination there is a synchronous loss in enzyme activity; the Kinact for beta-chloroalanine was estimated to be about 10 micrometers. The alpha-aminoacrylate-Schiff base intermediate formed after beta elimination of chloride ion is probably the key intermediate that partitions between one inactivation event for every 1500 turnovers. In the presence of D-alanine and alpha-ketoglutarate, which are good substrates for the transaminase activity of this enzyme, beta-chloroalanine is a potent, competitive inhibitor (K1 = 10 micrometers) with D-alanine and a weak, uncompetitive inhibitor with alpha-ketoglutarate.     

Identification of 5-keto-(7E,9E,11Z,14Z)-eicosatetraenoic acid as a novel nonenzymatic rearrangement product of leukotriene A4

Leukotriene A4 (LTA4), the reaction product of 5-lipoxygenase in human polymorphonuclear (PMN) leukocytes, is transformed both to LTB4 and a mixture of 5,6- and 5,12-dihydroxy-eicosatetraenoic acids (diHETE) via nonenzymatic hydrolysis. Evidence has been obtained that LTA4 is also converted to 5-keto-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE). The compound was isolated from the products of the 5-lipoxygenase reaction and its structure elucidated by UV spectroscopy, LC-MS, two-dimensional [1H]NMR spectroscopy and chemical reduction to the corresponding alcohol. The 5-oxo-ETE represented about 14% of the LTA4 hydrolysis products as compared to 72 and 14% for the 5,12-diHETE and 5,6-diHETE, respectively. A similar profile of hydrolysis products was obtained after incubation of synthetic LTA4 in aqueous buffer. Human PMN leukocytes produced 5-oxo-ETE in an arachidonic acid-dependent and MK-886-inhibitable manner. The 5-oxo-ETE caused 50% inhibition of 5-lipoxygenase activity at 1 microM. These results demonstrate that the nonenzymatic conversion of LTA4, in addition to the previously described hydrolysis products, yields 5-oxo-ETE during both the 5-lipoxygenase reaction and arachidonic acid oxidation by human PMN leukocytes. They indicate that allylic epoxides can rearrange in aqueous media at physiological pH to spontaneously form beta,gamma-unsaturated ketones.     

Lipoproteins inhibit macrophage activation by lipoteichoic acid

Regulation of lipid metabolism during infection is thought to be part of host defense, as lipoproteins neutralize endotoxin (LPS) and viruses. Gram-positive infections also induce disturbances in lipid metabolism. Therefore, we investigated whether lipoproteins could inhibit the toxic effects of lipoteichoic acid (LTA), a fragment of gram-positive bacteria. LTA activated RAW264.7 macrophage cells, stimulating production of tumor necrosis factor (TNF) in a dose-dependent matter, but produced less TNF than that seen after LPS activation. High density (HDL) or low density lipoprotein (LDL) alone inhibited the ability of LPS to stimulate TNF production, but had little effect on the activation by LTA. When a maximally effective dose of LTA was mixed with lipoproteins and 10% lipoprotein-depleted plasma (LPDP), the ability of LTA to stimulate macrophage production of TNF was inhibited. HDL, LDL, and the synthetic particle, Soyacal, when mixed with LPDP, were able to inhibit the ability of LTA to activate macrophages. Lipopolysaccharide-binding protein (LBP) substituted for LPDP in catalyzing lipoprotein neutralization of LTA by HDL. Antibody to LBP inhibited the ability of LPDP to induce LTA neutralization by HDL.Thus, lipoproteins can prevent macrophage activation by fragments from both gram-positive and gram-negative microorganisms.-Grunfeld, C., M. Marshall, J. K. Shigenaga, A. H. Moser, P. Tobias, and K. R. Feingold. Lipoproteins inhibit macrophage activation by lipoteichoic acid.     

Mechanism-based inactivation of serine transhydroxymethylases by D-fluoroalanine and related amino acids

Serine transhydroxymethylase, from lamb or rabbit liver, is known to catalyze slow transamination of D-alanine, but not of L-amino acids, in a tetrahydrofolate-independent reaction. Both enzymes will process the D-isomer of beta-fluoroalanine for alpha, beta-elimination of HF to yield an aminoacrylate-pyridoxal-P-enzyme intermediate. This intermediate partitions between harmless hydrolysis to pyruvate, NH4+, and active enzyme-pyridoxal-P (catalytic turnover) and suicidal enzyme alkylation by covalent modification with an average partition ratio of 40-60 turnovers/inactivation event/monomer unit of this tetrameric enzyme. Enzyme inactivation occurs with stoichiometric incorporation of radioactive label from D-[1,2-14C]fluoroalanine. Titration of enzymic cysteinyl --SH groups with 5,5'-dithiobis(2-nitrobenzoate) indicates loss of 1 --SH group on inactivation. Acid hydrolysis of radioactive-inactive enzyme confirms cysteine residue modification. Treatment of inactive enzyme with 6 M urea, then KBH4, followed by acid hydrolysis yields two radioactive compounds, lanthionine and S-carboxyhydroxyethylcysteine, in about equal amounts. The addition of tetrahydrofolate stimulates both pyruvate production and inactivation to equal extents with about a 200-fold rate acceleration at 0.5 mM tetrahydrofolate to turnover numbers of approximately 120 min-1. The Km for D-fluoroalanine is high, 10-60 mM, and this low substrate affinity suggests D-fluoroalanine will not be a useful in vivo agent for selective inactivation of liver cell serine transhydroxymethylases.     

Taxonomy and physiology of probiotic lactic acid bacteria

The current taxonomy of probiotic lactic acid bacteria is reviewed with special focus on the genera Lactobacillus, Bifidobacterium and Enterococcus. The physiology and taxonomic position of species and strains of these genera were investigated by phenotypic and genomic methods. In total, 176 strains, including the type strains, have been included. Phenotypic methods applied were based on biochemical, enzymatical and physiological characteristics, including growth temperatures, cell wall analysis and analysis of the total soluble cytoplasmatic proteins. Genomic methods used were pulsed field gel electrophoresis (PFGE), randomly amplified polymorphic DNA-PCR (RAPD-PCR) and DNA-DNA hybridization for bifidobacteria. In the genus Lactobacillus the following species of importance as probiotics were investigated: L. acidophilus group, L. casei group and L. reuteri/L. fermentum group. Most strains referred to as L. acidophilus in probiotic products could be identified either as L. gasseri or as L. johnsonii, both members of the L. acidophilus group. A similar situation could be shown in the L. casei group, where most of the strains named L. casei belonged to L. paracasei subspp. A recent proposal to reject the species L. paracasei and to include this species in the restored species L. casei with a neotype strain was supported by protein analysis. Bifidobacterium spp. strains have been reported to be used for production of fermented dairy and recently of probiotic products. According to phenotypic features and confirmed by DNA-DNA hybridization most of the bifidobacteria strains from dairy origin belonged to B. animalis, although they were often declared as B. longum by the manufacturer. From the genus Enterococcus, probiotic Ec. faecium strains were investigated with regard to the vanA-mediated resistance against glycopeptides. These unwanted resistances could be ruled out by analysis of the 39 kDa resistance protein. In conclusion, the taxonomy and physiology of probiotic lactic acid bacteria can only be understood by using polyphasic taxonomy combining morphological, biochemical and physiological characteristics with molecular-based phenotypic and genomic techniques.     

An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport

The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J. B. Ward, Jr., and S. A. Zahler, J. Bacteriol. 116:727-735, 1973). In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport. The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon. Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins.     

Effects of a streptococcal lipoteichoic acid on complement activation in vitro

This study describes activation of serum complement by lipoteichoic acid (LTA) from Streptococcus mutans OMZ 176, while in solution. Serum from 16 healthy students was taken. Test samples were incubated with increasing doses (1-5,000 micrograms/ml) of LTA or lipopolysaccharide (LPS) from Escherichia coli 0111:B4 for 1 h at 37 degrees C; then assayed for degradation of C3, C4 or factor B by crossed immunoelectrophoresis. Each preparation caused a significant (p < 0.05) dose-dependent conversion of C3. The response curves obtained were not statistically different. LPS was a stronger activator of the alternative pathway than LTA, as judged from analysis of C3 degradation in the presence of Mg2+/EGTA, and from their effects on factor B cleavage. LTA caused, however, pronounced alterations in the shape of C4 precipitation in the gels. Functional (hemolytic) assays showed that, when tested at 200 micrograms/ml, LTA and LPS triggered significant (p < 0.05) consumptions of both classical and alternative pathway proteins. LPS was a significantly (p < 0.05) stronger activator than LTA. Apparently, the C3 degradation found for this LTA involved the alternative pathway to a small extent; thus some other mechanism of fluid-phase C3 cleavage seemed also to be operative.     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.