Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry.

This work demonstrates that amino acid analysis based on isotope dilution mass spectrometry (IDMS) can be applied to quantify proteins having different complexities and natures. Five proteins and one decapeptide were selected for the study: C-reactive protein (CRP), beta-2-microglobulin (B2M), cystatine C (CysC), human serum albumin (HSA), Ara h1, and angiotensin I. The quantification was based on the determination of four amino acids, proline (Pro), isoleucine (Ile), valine (Val), and phenylalanine (Phe) within a working range between 5 and 100 pmol/injection of each amino acid, after 60 min digestion with HCl at 150°C. The amino acids were selected taking into account their abundance in the protein sequence and to include the more difficult to break peptide bonds. Quantification of the protein amounts calculated from each amino acid is consistent, indicating that the method is working reliably. This consistency points to a complete hydrolysis of the proteins. The trueness of the method was proven when dry mass determination after dialysis was applied to HSA and CRP and the results were compared to those from amino acid analysis. Traceability to SI was assured by extensive characterisation of the amino acid calibrants by nuclear magnetic resonance, neutron activation analysis, and Karl Fischer titration.

Stereospecificity and stereoselectivity of flobufen metabolic profile in male rats in vitro and in vivo: phase I of biotransformation.

Flobufen (F) is the original nonsteroidal antiinflammatory drug (NSAID) containing two enantiomers. The aim of this investigation was to elucidate the biotransformation pathway of F at chiral level in phase I of biotransformation. Stereoselectivity and stereospecificity of the respective enzymes were studied in male rats in vitro (microsomal and cytosolic fractions, hepatocytes suspension) and in vivo. The rac-F, (+)-R-F and (-)-S-F were used as substrates. Amounts of F enantiomers, 4-dihydroflobufen diastereoisomers (DHF) and other metabolites (M-17203, UM) were determined with a chiral HPLC method in two chromatographic runs on R,R-ULMO and allyl-terguride bonded columns. Stereoselective biotransformation of the two enantiomers of F was observed at all tested levels and significant bidirectional chiral inversion of enantiomers of F was observed in hepatocytes. Mean enantiomeric ratios of F concentrations (S-/R-), after rac-F incubations, ranging from 1.09 in cytosolic fraction to 18.23 in hepatocytes. Stereospecificity of the respective F reductases was also observed. (2R;4S)-DHF and (2S;4S)-DHF are the principal metabolites of F in microsomes and hepatocytes. Neither DHF diastereoisomers nor M-17203 were found in cytosolic fraction. Only the nonchiral metabolite, M-17203, was found in all urine and feces samples after oral administration of F.

Biotransformation of flobufen enantiomers in ruminant hepatocytes and subcellular fractions.

Flobufen (F), a new antiinflammatory drug, has one chiral and one prochiral center in its structure. Reduction of rac-F, the principal biotransformation pathway, leads to the formation of four diastereoisomers of 4-dihydroflobufen (DHF). F was chosen as a model substrate for interspecies comparison of activity, stereospecificity, and stereoselectivity of biotransformation enzymes in fallow bucks, red deer stags, and roe bucks in vitro. Formation of F metabolites was examined in hepatocyte suspension and in subcellular fractions of liver homogenate. (+)-R-F, (-)-S-F and rac-F were used as substrates. After incubation of substrates, the amounts and ratios of DHF diastereoisomers and F enantiomers were assessed by HPLC, with (R,R)-ULMO and terguride-bonded columns. Considerable interspecies differences in stereoselectivity and stereospecificity of F reductases were found at the cellular and subcellular levels, although these ruminants are closely related. Chiral inversion of F enantiomers to their antipodes was detected in vitro in all ruminants tested, but individual species also differed in the direction and rate of this inversion.

Activity, stereospecificity, and stereoselectivity of microsomal enzymes in dependence on storage and freezing of rat liver samples.

Liver microsomes are now one of the most widely used in vitro test systems for biotransformation studies of drugs, toxins, and other xenobiotics. The standard procedure of preparation of microsomes from fresh liver taken immediately after death of the animal is impossible in experiments with liver samples from human or wild animals and the choice of a relatively optimal way of liver storage is necessary in these cases. We studied the possibility of using the stereoselectivity and stereospecificity of biotransformation enzymes for evaluation of the changes in enzyme function dependent on tissue handling. Activity, stereospecificity, and stereoselectivity of several enzymes in microsomes prepared from fresh liver, frozen liver in liquid nitrogen, or ice-cooled liver were compared. The effect of storage period (2, 3, 5 h) on these parameters were also tested. Both freezing and cooling of liver change the native function of enzyme systems and could result in incorrect stereospecificity data for the microsomal metabolism. All parameters observed also differ in their dependence on period of ice cooled storage. As it is difficult to hold strictly to the same storage period, we recommend freezing liver in liquid nitrogen if the storage of liver is necessary. In projects comparing enzyme activities in human and laboratory animals the same freezing procedure of liver should be maintained before preparation of microsomes from all species.

Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system.

Fractionation of venom from an agelenid spider, Tegenaria agrestis, resulted in the isolation of a family of three peptides with potent insecticidal activity. These peptide toxins, TaITX-1, -2, and -3, whose sequences were revealed from cloned cDNAs, each consist of 50 amino acid residues, six of which are cysteines. They appear to be amidated at their C-termini and exhibit greater than 90% sequence identity. Unlike other reported spider toxins, the TaI toxins are processed from precursors containing no propeptide sequences. In lepidopteran larvae and corn rootworm beetles, the insecticidal Tegenaria toxins caused an unusual excitatory symptomatology with 50% paralytic doses ranging from 0.23 to 2.6 nmol/g. In a series of electrophysiological experiments performed in house fly larvae, these toxins caused an elevated rate of firing from central nervous system neurons. No significant effects were found when any peripheral sensory or motor systems were examined. Thus, it appears that the TaI toxins may act in a fashion not previously reported for insecticidal peptide toxins; they may act directly on the insect central nervous system.

Characterization and cloning of insecticidal peptides from the primitive weaving spider Diguetia canities.

Three potent insecticidal peptide toxins were purified from the venom of the primitive weaving spider, Diguetia canities. The toxins share significant homology (> 40%) in their amino acid sequences and are of related size (masses of 6371-7080 Da). In lepidopteran larvae, the toxins cause a progressive spastic paralysis, with 50% paralytic doses (PD50S) ranging from 0.38 to 3.18 nmol/g, suggesting them to be among the most potent insecticidal compounds yet described from arthropod venoms. The most potent of these toxins, DTX9.2, was cloned using a reverse transcription-polymerase chain reaction (RT-PCR). The cDNA encodes a 94 amino acid precursor which is processed to the active 56 amino acid peptide by removal of a signal and propeptide sequence. The gene encoding DTX9.2 was isolated and characterized. The transcriptional unit spans 5.5 kilobases and is segregated into five exons. DNA sequences upstream from the first exon contain a TATA box and two palindromic sequences (one with homology to a CAAT consensus) which together may constitute a functional promoter. The highly segmented gene structure observed for this small peptide suggests that a mechanism such as exon shuffling may have played a role in the evolution of this toxin family.

Concentrations of cadmium, lead and copper in atmospheric precipitation in Czechoslovakia.

The concentrations of Cd, Pb and Cu in wet and dry atmospheric precipitation were measured in 13 regions of Czechoslovakia. The Pb and Cd contents of rainwater were found to decrease with increasing rainfall intensity. The highest enrichment of Cd, Pb and Cu was observed in ice-accretion deposits; increasing amounts of metals were found at higher altitudes, i.e. 700-1000 m above sea level. The electrostatically separated fractions are rather high in Cu and Pb (approximately 90%), whereas Cd contributes less than 50%.

Background concentrations of lead and cadmium in the lichen Hypogymnia physodes at different altitudes.

The background concentrations of lead and cadmium in the epiphytic lichen Hypogymnia physodes were determined and found to depend on altitude. The lead content increases linearly with increasing altitude, whereas the cadmium content increases up to altitudes of 900-1100 m, and then decreases above this height.

Lead and cadmium in fruiting bodies of macrofungi in the vicinity of a lead smelter.

The concentrations of lead and cadmium in the fruiting bodies of mycorrhizal and edible macrofungi (Basidiomycetes, Agaricales) were measured in the vicinity of a lead smelter. Very high concentrations of both metals were found 1 km from the stack, and elevated concentrations were observed up to a distance of 4.5 km. The dependence of the concentrations on the distance from the stack were approximated by an exponential function, the extrapolation of which showed that only at a distance of 6 km did the concentrations approach those reported from an area in the Krusné Hory Mountains with a medium level of air pollution. The concentrations of lead were slightly higher in the caps than in the stems of the fungal fruiting bodies from the sites close to the smelters; at a distance of 1.7 km they were the same, and at greater distances the lead concentrations in the caps were slightly lower than those in the stems. The cap/stem concentration ratio for cadmium does not exhibit any change with distance. The different behaviour of the two metals can be explained in terms of the lower mobility of Pb2+ ions. Because of their lead and cadmium contents, edible mycorrhizal fungi picked within a distance of 0.6 km of the stack can be toxic to man if eaten often in quantities that are usual in Czechoslovakia.

Field recognition of eastern poison oak: with emphasis on plants in Alabama.

Eastern poison oak, Toxicodendron toxicarium, is a nonclimbing shrub whose leaves have three leaflets. The leaflets with their numerous round lobes resemble somewhat the leaves of some oaks in the white oak groups. The plant grows in nutritionally poor, sandy soil, and especially in dry pine-oak woodland. Deposits resembling black enamel paint can often be seen where plants have been injured. During winter, the U- or V-shaped leaf scars, the naked fruit stalks in female plants, and the sparse upright branches all aid in recognition. Other features can be useful in spring and fall. While poison oak and poison ivy are so closely related that the sensitivity of individuals and resulting dermatitis are not markedly different, accurate recognition is important in avoiding contact.