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The main purpose of the present study was to identify B-cell epitopes on the S4 subunit of pertussis toxin (PT) by the synthetic peptide approach. Two strategies were followed: (i) screening of two series of overlapping peptides (12- and 25-residue peptides) covering the entire S4 sequence by a panel of murine monoclonal anti-PT antibodies and various polyclonal anti-PT antisera in an enzyme-linked immunosorbent assay (ELISA), and (ii) analysis of the S4 amino acid sequence by a predictive algorithm followed by synthesis and immunization of mice with the predicted peptides coupled to diphtheria toxoid. The anti-peptide conjugate antisera were tested in an ELISA for cross-reactivity with native PT, B oligomer, and S4. Screening of the free peptides in an ELISA by the PT antisera indicated the presence of six B-cell epitope-containing domains covered by residues 18 to 32, 33 to 46, 39 to 52, 51 to 65, 71 to 84, and 91 to 106. None of the peptides, however, were recognized by the monoclonal anti-PT antibodies in an ELISA. Immunization with six computer-predicted peptides (B1 to B6) and three potential T-cell epitopes (T1 to T3) gave rise to very high antibody responses towards the homologous conjugates. With the exception of the anti-T1/diphtheria toxoid antisera, all anti-peptide conjugate antisera cross-reacted with PT in an ELISA at different levels. None of these anti-peptide conjugate antisera, however, showed any PT-neutralizing effect as measured by the Chinese hamster ovary cell assay and the leukocytosis-promoting activity test. The results of the present study suggest that discontinuous epitopes are predominant in the S4 subunit of native PT.

The formation of three oxidant-derived products of 5-aminosalicylic acid (5-ASA) in vivo was demonstrated in patients with active ulcerative colitis as well as in healthy subjects. The products were isolated from faeces by preparative HPLC and their chemical structures were found to be oxidation products of 5-ASA using 1H-NMR spectroscopy and mass spectrometry. Reactions carried out in vitro between 5-ASA and oxidants suggested to be present in the inflamed bowel verified that the hypochlorite-mediated oxidation of 5-ASA as well as the haemoglobin-catalysed H2O2-dependent oxidation of 5-ASA resulted in the formation of a single oxidation product of 5-ASA. This product was similar to, but not identical to any of the products identified in faeces from patients receiving 5-ASA. Oxygen radical-mediated oxidation of 5-ASA gave several products, different from the products isolated. Finally, it was verified that the products formed in vivo are not formed as a result of autooxidation of 5-ASA either in faeces extract or in pharmaceuticals.

4-Methylimidazole (MeI) is formed when hay is ammoniated and it has been suspected of being the compound causing the 'crazy cow' syndrome. The aim of the present study was to describe the disposition of MeI in goats and heifers. The mean residence time was about five hours and the volume of distribution 0.9 litre kg-1 bodyweight in both goats and heifers. Goats metabolised MeI to a much higher extent than heifers which excreted the major part as the unchanged compound. MeI and its metabolites were excreted mainly in urine, but also in milk and faeces. In spite of administration of a dose (20 mg kg-1 bodyweight) which is much higher than the one intoxicated animals may have received through contaminated fodder none of the typical signs of intoxication were observed and it is concluded that MeI may not alone be the cause of the 'crazy cow' syndrome.

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1'-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1'-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.

A microsomal system catalyzing the in vitro synthesis of the aglycones of the two cyanogenic glucosides linamarin and lotaustralin has been isolated from young etiolated seedlings of cassava (Manihot esculenta Crantz). A prerequisite to obtain active preparations is the complete removal of the endosperm pellicle covering the cotyledons before seedling homogenization. The rates of conversion of the parent amino acids valine and isoleucine to their cyanohydrins are 19 and 6 nmol/h/mg protein, respectively. The conversion rates for the corresponding oximes (2-methylpropanal oxime and 2-methylbutanal oxime) are 475 and 440 nmol/h/mg protein and for the nitriles (2-methylpropionitrile and 2-methylbutyronitrile) 45 and 75 nmol/h/mg protein. With the exception of 2-cyclopentenylglycine, none of the additionally tested amino acids are metabolized, whereas a broad substrate specificity is observed using oximes and nitriles as substrates. The in vitro biosynthesis is photoreversibly inhibited by carbon monoxide, demonstrating the involvement of cytochrome P450 in the hydroxylation processes. All tissues of the cassava seedling contain cyanogenic glucosides. The microsomal enzyme system responsible for their synthesis is restricted to the cotyledons and their petioles. This demonstrates that the cyanogenic glucosides are actively transported to other parts of the seedling. The enzyme activity decreases with the height of the etiolated seedling and is barely detectable in seedlings above 75 mm.

A cyclic AMP antigen was prepared utilizing a stable thioether linkage from C8 of the purine moiety. Monoclonal antibodies were raised toward the cyclic AMP-C8-diphtheria toxoid antigen and found to be of high specificity, with only very low cross-reactivity against related compounds, less than 0.03% against cGMP being the most significant cross-reaction. The stability of the thioether linkage enabled the preparation of an enzyme tracer, which was used in an ELISA. The assay had an effective working range from 0.1 to 100 pmol cAMP. Due to the structure of the antigen, the need for sample derivatization was eliminated.

Toxin production in a large number of Pyrenophora teres isolates have been investigated. During fungal growth, the pH of the medium decreases from 6.5 to about 3.0. Aspergillomarasmine A is the major toxin excreted into the culture medium. Nonenzymatic acid-catalyzed conversion of aspergillomarasmine A to anhydroaspergillomarasmine A proceeds at low pH and is prevented by repeated titration of the culture medium to pH 6.5. The four possible stereoisomers of N-(2-amino-2-carboxyethyl)aspartic acid have been chemically synthesized and their absolute configuration determined. From circular dichroism and NMR spectroscopy and by measurements of specific optical rotation, the LL-form is identified as the stereoisomer produced by P. teres. Biosynthetic experiments using radioisotopes demonstrate that the LL-isomer of N-(2-amino-2-carboxyethyl) aspartic acid is a direct precursor of aspergillomarasmine A. Consequently, L-configuration is assigned to the two corresponding asymmetric carbon atoms of aspergillomarasmine A. This is in contrast to earlier reports which had indicated D configuration. The phytotoxicity of anhydroaspergillomarasmine A is comparable with that of L-aspartic acid, whereas LL-N-(2-amino-2-carboxyethyl)aspartic acid exerts strong phytotoxicity in the bioassays as shown previously for aspergillomarasmine A. The amount of LL-N-(2-amino-2-carboxyethyl)aspartic acid which accumulates in the P. teres cultures is low, indicating that aspergillomarasmine A is the toxin which plays the major role in the pathological changes associated with the barley net-spot blotch disease.

Four peptides and serum albumin have been derivatized with the bicyclic anydride of diethylenetriamine-pentaacetic acid. Procedures were developed to isolate the labelled species and determine the degree of derivatization. By using perturbed angular correlation of gamma-ray spectroscopy it is possible, through the determination of rotational correlation times, to decide whether labelled peptides interact with other molecules (receptors). In the case of the peptide cholecystokinin it is shown that the interaction between the peptide and its corresponding polyclonal antibody can be determined down to 1 pmol hormone. Experiments on 111In- and 111mCd-labelled Gly-Trp showed that, where the 111Cd PAC spectrum directly reflected the rotational motion of the molecule, the 111In PAC spectrum was affected by the nuclear transitions to 111mCd.

Mid-trimester therapeutic abortion is performed routinely in several clinics by using a combination of intra-amniotic instillation of prostaglandin F2 alpha, followed by intravenous infusion of oxytocin in 5% glucose. In doses as low as 10 mU/min, oxytocin has an antidiuretic action. Two cases of grand mal-like seizure in connection with this procedure of therapeutic abortion are reported and the background discussed.

Chromatographic and functional properties of tyrosine isoaccepting transfer ribonucleic acids (tRNAs) from placenta and HeLa cells were analyzed and compared. RPC-5 chromatography separated four major isoacceptors from each source, with those from HeLa cells eluting generally later than those from placenta. There was some overlap: HeLa tRNA1Tyr eluted in a position between placenta tRNA3Tyr and tRNA4Tyr; and HeLa tRNA2Tyr and placenta tRNA4Tyr eluted in similar positions with the HeLa isoacceptor eluting slightly later than the placental isoacceptor. Thus there are no isoacceptors common to both sources. The function of the individual isoacceptors was compared in a rabbit-reticulocyte, cell-free, protein-synthesizing system for both the rate of incorporation of tyrosine into the polypeptide chain and the site of incorporation in alpha-globin. Two isoacceptors were compared simultaneously in the same reaction, and overlapping comparisons were made to relate each isoacceptor to all the others. There were no significant differences in the rates of incorporation among the isoacceptors, nor were there any differences in the sites of incorporation. All eight isoacceptors donated tyrosine equally well into the three tyrosine containing tryptic peptides of alpha-globin. Whatever the structural differences among the tyrosine isoacceptors are, they do not affect the function of the tRNA in this protein-synthesizing system.

Benzodiazepines probably exert their anxiolytic, hypnotic, and anticonvulsant effects by interacting with brain-specific high-affinity benzodiazepine receptors. In searching for possible endogenous ligands for these receptors we have purified a compound 10(7)-fold from human urine by extractions, treatment with hot ethanol, and column chromatography. The compound was identified as beta-carboline-3-carboxylic acid ethyl ester (IIc) by mass spectrometry, NMR spectrometry, and synthesis; IIc was also isolated from brain tissues (20 ng/g) by similar procedures. Very small concentrations of IIc displaced [3H]diazepam completely from specific cerebral receptors, but not from liver and kidney binding sites; the concentration causing 50% inhibition of specific [3H]diazepam binding (IC50) was 4-7 nM compared to ca. 5 nM for the potent benzodiazepine lorazepam. Specific binding sites for quinuclidinyl benzilate, naloxone, spiroperidol, serotonin, muscimol, and WB 4101 were not affected by IIc. In contrast to benzodiazepines, IIc exhibits "mixed type" competitive inhibition of forebrain benzodiazepine receptors (negative cooperativity). We surmise that an endogenous ligand for benzodiazepine receptors may be a derivative of beta-carboline-3-carboxylic acid.

Second trimester abortion was induced in a randomized investigation using a single dose of prostaglandin F2alpha (PGF2alpha) in combination with oxytocin in one group of patients, and hypertonic saline (20%) in combination with oxytocin in another group of patients. Oxytocin was administered in order to induce a rapid abortion, and was given intravenously in a glucose drip (100 I.U. oxytocin added to 1 litre of 5% glucose). Administration rate was 10 I.U./hour. The PG-group consisted of 16 patients. The mean induction-abortion interval for this group was 14.2 hours, and all patients aborted within 24 hours. In three cases the bleeding exceeded 200 ml. In one of these cases infection occurred. Only mild side effects occurred. The saline group also consisted of 16 patients. The mean induction-abortion interval in this group was 21.5 hours. Eleven patients aborted within 24 hours. In two cases the bleeding exceeded 200 ml. No side effects occurred. Intraamniotic instillation of PGF2alpha seems to be a good alternative to hypertonic saline for termination of second trimester pregnancies.

The change of extracellular space volume of rat brain cortex during ischemia and cortical spreading depression, CSD (Leão 1944) was evaluated by a new method. The cortical surface was irrigated with isotonic CSF containing the extracellular markers 50 mM cholin or 50 mM trimethyltris(hydroxymethyl)methyl ammonium ion (N-TRIS), and their extracellular concentrations were monitored by ion-selective microelectrodes. When steady-state for the concentration of these markers was attained, CSD evoked a reversible increase of the concentration of the markers, indicating shrinkage of the interstitial volume of distribution. During ischemia an initial slow rate of concentration increase was observed, followed a few minutes later by a rapid increase concomitant with the sharp rise in extracellular potassium concentration. During CSD and ischemia, the maximal increases of choline and N-TRIS concentration reflected a shrinkage of the extracellular space amounting to about 50% of the initial volume.

The pharmacokinetics of sulphadiazine was studied in newborn, 1 week, and 8 weeks old piglets after intravenous administration of 60 mg/kg. Kinetic parameters were calculated using a two compartment open model. Steady state volume of distribution averaged 0.62, 0.56, and 0.48 1/kg at birth, 1 week, and 8 weeks, respectively. Elimination half-life decreased from 455 min. at birth to 322 min. at 1 week and 157 min. at 8 weeks leading to a rise in body clearance from 0.99 to 2.20 ml/min./kg during the same age period. Urinary excretion data indicated that the increase in body clearance reflects maturational changes in both renal function and metabolic capacity. Although renal clearance increased several times more than metabolic clearance, metabolism remained the main contributor to elimination of SDZ at all ages. Metabolism of SDZ involves two important pathways - acetylation and aromatic hydroxylation; the former being well developed at birth, while the latter increased markedly during the age period studied.

The production of collagenase (EC 3.4.24.3) by endotoxin-stimulated macrophages was significantly inhibited by indomethacin, indicating that prostaglandins (PGs) mediate this effect. Inhibitions of collagenase production by indomethacin was overcome by addition of exogenous PGE2 at 10 nM whereas the addition of 0.1 and 1.0 micrometer PGE2 increased the enzyme production to 3 times that achieved by endotoxin. Although the addition of prostaglandin alone to macrophage cultures did not stimulate collagenase production, the simultaneous addition of PGE1 or PGE2 and endotoxin enhanced collagenase activity 2- to 10-fold. This increase was detectable at PGE concentrations of 10 nM and was optimal at 0.1-1.0 micrometer. PGF2alpha had little effect on either the enhancement of collagenase production by endotoxin-stimulated macrophages or its restoration in cultures inhibited by indomethacin. Radioimmunoassay of prostaglandins in the culture media revealed that macrophages exposed to endotoxin secreted 40-fold more PGE2 than did unstimulated cells. The increase in PGE2 was detected 4 hr after exposure to endotoxin and was maximal at 14 hr. The peak PGE2 concentrations in the culture media were similar to those of exogenous PGE2 (about 10 nM) needed to restore collagenase production in indomethacin-treated cultures. These findings demonstrate the involvement of PGE in the endotoxin-activation of macrophages with resultant production of collagenase.

Two types of indoleacetic acid (IAA) antigens have been described in the literature which show marked differences with respect to antibody specificity. In this communication an alternative antigen design is described. In the so-called IAA-C5-BSA conjugate, both the acetic acid group and the pyrrole moiety are presented free, as the covalent linkage between IAA and the carrier molecule is introduced in the benzene moiety. Antibodies were elicited in rabbits against this novel antigen. Using cross-reactivity data and the strategy of successive approximation for the measurements of auxin levels in the internodes of light-grown broad bean (Vicia faba L. cv Superfine), the three types of antibodies are compared.

Kidney stones from a plaice, Pleuronectes platessa, have been shown to consist of the sodium salt of orotic acid. Precipitation of orotic acid in human kidneys and urine samples has previously been reported but the precipitates must have been salts, most likely the sodium salt, of orotic acid and not the free acid. This reinterpretation is based on the acid strength of orotic acid and on data for the solubilities of sodium orotate and orotic acid. Sodium orotate is therefore a member on the list of compounds present in human urinary deposits and calculi. X-ray powder diagrams and d-values and IR-spectra of the sodium salt are recorded to facilitate future identifications.

The pharmacokinetics and metabolism of metioprim (MTP) have been studied after intravenous administration of a single dose of 5 mg/kg b.wt. to pigs (n = 4) and 10 mg/kg b.wt. to goats (n = 5). Kinetic parameters were calculated using a two-compartment open model. The elimination half-life was much shorter in goats (23 +/- 4 min.) than in pigs (169 +/- 17 min.). The apparent volume of distribution exceeded 1.0 1/kg b.wt. in both species indicating accumulation in tissues. Pigs excreted the major part of the dose (86 +/- 9%) in urine and only a small part (9 +/- 3%) in faeces, while the goats excreted almost equal amounts in urine (55 +/- 5%) and faeces (38 +/- 4%). Metabolism played the major role in elimination of MTP. Of the 80% of the dose excreted in urine within 24 hrs in pigs unchanged MTP made up 8%, metioprim sulfoxide 64% and O-desmethyl-MTP 8%. Metioprim sulfoxide was also the major metabolite in goats, but in this species another demethylated metabolite--metioprim sulfonic acid--was formed. Goats excreted 49 +/- 7% of the dose in urine within 24 hrs of which unchanged MTP made up less than 1%, while metioprim sulfoxide accounted for about 35% and metioprim sulfonic acid for approximately 14%. Metabolites isolated from urine were identified by NMR-spectroscopy combined with EI, FAB or 252Cf plasma desorption mass spectrometry.

A prospective study on the value of routine HPL determination as a supplement to clinical antenatal care is presented. The HPL values in the apparently normal pregnant women were not known to the clinicians. The material comprised 1 660 pregnancies and deliveries, in 87% of which at least one HPL value was available. The results are expressed as the ability of a low HPL value to predict the birth of a distressed infant and/or an infant of low birth weight, a normal HPL value the birth of a non-distressed infant and/or an infant of normal birth weight. Moreover, the results show has many of the distressed infants and infants of low birth weight were born to mothers with low HPL values. Like a number of other authors, we found that HPL screening increases the prognostic reliability in pregnancies with complications. On the other hand, its value in normal pregnancies could not be confirmed.

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin has been studied with a microsomal preparation obtained from etiolated seedlings of sorghum. The biosynthetic pathway involves tyrosine, N-hydroxytyrosine, and p-hydroxyphenylacetaldehyde oxime as early intermediates (Møller, B. L. and Conn, E. E. (1980) J. Biol. Chem. 254, 8575-8583). The use of deuterium-labeled tyrosine and mass spectrometric analyses demonstrate that the alpha-hydrogen atom of tyrosine is retained in the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime. This excludes p-hydroxyphenylpyruvic acid oxime as intermediate in the pathway. A high pressure liquid chromatography method was developed to separate the (E)- and (Z)-isomers of p-hydroxyphenylacetaldehyde oxime. The microsomal enzyme system was found to produce initially the (E)-isomer of p-hydroxyphenylacetaldehyde oxime. An isomerase then converts the (E)-isomer to the (Z)-isomer, which is the isomer preferentially utilized by the microsomal enzyme system in the subsequent biosynthetic reactions. The (E)-isomer produced in situ is more efficiently converted to the (Z)-isomer than exogenously added (E)-isomer and may thus be metabolically channeled.