Measurement of parameters of cholic acid kinetics in plasma using a microscale stable isotope dilution technique: application to rodents and humans.

A stable isotope dilution method is described that allows measurement of cholic acid (CA) kinetics, that is, pool size, fractional turnover rate (FTR), and synthesis rate in mice, rats, and humans. Decay of administered [2,2,4,4-2H4]CA enrichment was measured in time in 50-microl plasma samples by gas-liquid chromatography/electron capture negative chemical ionization-mass spectrometry, applying the pentafluorobenzyl-trimethylsilyl derivative. The kinetic data expressed species-dependent differences. The CA pool sizes were 16.8 +/- 2.1, 10.6 +/- 1.2, and 2.4 +/- 0.7 micromol/100 g body weight for mice, rats, and humans, respectively. The FTR values were 0.44 +/- 0.03, 0.88 +/- 0.10, and 0.46 +/- 0.14 per day for mice, rats, and humans. The corresponding synthesis rates were 7.3 +/- 1.6, 9.3 +/- 0.1, and 1.0 +/- 0.2 micromol/100 g body weight per day. The human data agreed well with literature data obtained by conventional isotope dilution techniques. For rats and mice these are the first reported isotope dilution data. The method was validated by confirmation of isotopic equilibrium between biliary CA and plasma CA in the rat. Its applicability was demonstrated by the observation of increased CA FTR and CA synthesis rate in rats fed cholestyramine, which is known to increase fecal bile acid excretion. The presented stable isotope dilution method enables the determination of CA kinetic parameters in small plasma samples. The method can be applied in unanesthetized rodents with an intact enterohepatic circulation and may also be valuable when studying the development of human neonatal bile acid kinetics.     

Carotenoids and fatty acids in red yeasts Sporobolomyces roseus and Rhodotorula glutinis

Rhodotorula glutinis and Sporobolomyces roseus, grown under different aeration regimes, showed differential responses in their carotenoid content. At higher aeration, the concentration of total carotenoids increased relative to biomass and total fatty acids in R. glutinis, but the composition of carotenoids (torulene > beta-carotene > gamma-carotene > torularhodin) remained unaltered. In contrast, S. roseus responded to enhanced aeration by a shift from the predominant beta-carotene to torulene and torularhodin, indicating a biosynthetic switch at the gamma-carotene branch point of carotenoid biosynthesis. The overall levels of total carotenoids in highly aerated flasks were 0.55 mol-percent and 0.50 mol-percent relative to total fatty acids in R. glutinis and S. roseus (respectively), and 206 and 412 microg g(-1) dry weight (respectively).     

A general system for generating unlabelled gene replacements in bacterial chromosomes

 A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species. Received: 2 April 1996 / Accepted: 15 July 1996     

Tripeptidase gene (pepT) of Lactococcus lactis: molecular cloning and nucleotide sequencing of pepT and construction of a chromosomal deletion mutant.

The gene encoding a tripeptidase (pepT) of Lactococcus lactis subsp. cremoris (formerly subsp. lactis) MG1363 was cloned from a genomic library in pUC19 and subsequently sequenced. The tripeptidase of L. lactis was shown to be homologous to PepT of Salmonella typhimurium with 47.4% identity in the deduced amino acid sequences. L. lactis PepT was enzymatically active in Escherichia coli and allowed growth of a peptidase-negative leucine-auxotrophic E. coli strain by liberation of Leu from a tripeptide. Using a two-step integration-excision system, a pepT-negative mutant of L. lactis was constructed. No differences between the growth of the mutant and that of the wild-type strain in milk or in chemically defined medium with casein as the sole source of essential amino acids were observed.     

Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.

To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.     

Small oligomers of galacturonic acid are endogenous suppressors of disease resistance reactions in wheat leaves

Plants infected by a phytopathogenic fungus appear to recognize the presence of the pathogen by the molecular recognition of fungal cell wall fragments, termed 'elicitors', or of breakdown products of their own cell walls, termed 'endogenous elicitors'. Successful pathogens are thought to counteract this elicitation of active resistance reactions by the production of 'suppressors'. Evidence is presented here that fragments of the host cell wall, presumably produced enzymatically during fungal penetration, may act as 'endogenous suppressors' of resistance reactions in wheat. Pectic fractions were extracted from wheat cell walls by a variety of methods: Ca²⁺-chelators (CDTA and imidazole), a commercial mixture of pectic enzymes (Pectolyase Y23), a highly purified recombinant endopolygalacturonase (EPG), and solvolyses of the cell walls in anhydrous hydrogen fluoride at low temperatures followed by imidazole extraction. All of these fractions suppressed elicitor-induced activities of phenylalanine ammonia-lyase and peroxidases when co-injected with a glycoproteogalactan-elicitor, isolated from germ tubes of the wheat stem rust fungus, into the intercellular spaces of wheat leaves. Suppressor activity was correlated with the content of galacturonic acid in the extracts. Of the oligogalacturonides tested (monomer to hexamer), the dimer and trimer proved to be most active. This was not only true for suppression of elicitor-induced responses, but also for suppression of the hypersensitive resistance reaction in infected, genetically resistant host plants. As a consequence of reduced host cell necrosis in suppressor-treated leaves, the fungus developed larger colonies than in water-treated control leaves. Small oligomers of galacturonic acid, thus, are endogenous suppressors of resistance reactions in wheat leaves.     

Fate of peptides in peptidase mutants of Lactococcus lactis

The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepN pepC pepO pepX and pepT were deleted. Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu–Gly–Gly, Gly–Phe–Leu, Leu–Gly–Pro, Ala–Pro–Leu and Gly–Leu–Gly–Leu, respectively, indicating that no other peptidases are present to release the essential amino acid Leu. In these mutants, peptides accumulate intracellularly, demonstrating that peptides are translocated as whole entities prior to degradation. The mutant lacking all five peptidases could still grow on Gly–Leu and Tyr–Gly–Gly–Phe–Leu, which confirmed the presence of a dipeptidase and led to the identification of an unknown PepO-like endopeptidase. These studies have also shown that the general aminopeptidases PepN, PepC and PepT have overlapping but not identical specificities and differ in their overall activity towards individual peptides. In contrast, PepX has an unique specificity, because it is the only enzyme which can efficiently degrade Ala–Pro–Leu. The concerted action of peptidases in the breakdown of particular peptides revealed how these substrates are utilized as sources of nitrogen.