Thin-layer Chromatography Of Extracts Research Articles

Pyrrolizidine alkaloidosis of cattle associated with Senecio lautus.

Serious incidents of pyrrolizidine alkaloidosis of cattle in 10 herds exposed to the Australian native plant, Senecio lautus (Asteraceae), were seen in central Queensland during 1988-1992. The deaths of 226 cattle were recorded. A mean of 8% of cattle died in affected groups (range 2 to 58%). Sickness and deaths usually occurred some months after access to S lautus. Typically, affected cattle lost body condition to the point of emaciation before dying and had persistent diarrhoea. Some animals developed abnormal behaviour and died after a shorter illness. Liver specimens from affected cattle in all herds contained lesions consistent with pyrrolizidine alkaloidosis. Thin layer chromatography of extracts of blood and liver samples from cattle from 5 herds detected pyrrolic metabolites. The identity of these was confirmed by mass spectroscopy on samples from one herd. Unseasonal autumn and winter rain after a dry summer appeared to favour growth of S lautus at the expense of other pasture species. A subsequent dry period promoted consumption of S lautus and was followed by a cluster of poisoning incidents.

′CH-19甘′を片親としたトウガラシ (<i>Capsicum annuum</i> L.) の雑種におけるカプサイシノイドならびにカプサイシノイド様物質の含量

Thin layer chromatography of extracts of pepper (Capsicum annuum L.) fruit gave two capsaicinoid-like substances (tentatively named CLS-A and -B), which differed from capsaicinoid in their Rf values. Intervarietal crosses were made using as a parent the sweet variety, ′CH-19 sweet′ which had been selected and fixed from the hot variety, ′CH-19 hot′, and the formation of capsaicinoid and CLS-A and -B was studied.Sweet varieties such as ′California Wonder′ and ′Murasaki′ contained no CLS-A nor-B. Capsaicinoid content was notably high, and CLS-A and -B contents low, in F1 populations between these sweet varieties and ′CH-19 sweet′. In F1 populations between hot varieties and ′CH-19 sweet′, capsaicinoid content was either comparable to, or lower than the parent hot varieties. CLS-A was detected in the fruit 20 days after anthesis, and CLS-B 10 days after anthesis, that is, prior to capsaicinoid formation. Varieties differed in their CLS-A and -B contents. Several hot varieties were high in these contents, ′CH-19 sweet′ being the highest.These results suggest the possible role of CLS-A and -B in capsaisinoid formation. Crossing with ′CH-19 sweet′, and determining the contents of capsaicinoids and cap-saicinoid-like substances in F1 populations will reveal the genetic aspect of capsaicinoid formation in the variety used for crossing.

Enhanced thymidine uptake causes the lowered thymidine requirement of D. discoideum auxotroph HPS 401

Dictyostelium discoideum strain HPS 401 contains a spontaneous mutation that lowers the amount of thymidine required for cell growth relative to that of the auxotrophic parental strain HPS 400. Growth studies in defined medium show that as little as 8 μg thymidine/ml supports maximal growth of HPS 401, whereas 50 μg/ml is required by HPS 400. In contrast, both strains require over 40 μg thymidylate/ml to achieve maximal growth. HPS 401 exhibits thymidineless death when grown without thymidine; relative viability decreases to <0.01 % after 190 h incubation. Assays for enzymes related to thymidine metabolism reveal that none of the strains tested (HPS 401, HPS 400, and prototrophic HPS 83 cells) contain detectable thymidine phosphorylase activity and that the specific activity of thymidine kinase is the same in these three strains. Thin-layer chromatography of extracts from cells grown on radiolabeled thymidine shows that there is no detectable conversion of thymidine to thymine in any of these strains. These analyses show that HPS 401 has rapid intracellular accumulation of thymidine, while only slight uptake is observed with HPS 400 or wild-type strains. HPS 401 also shows greater uptake of uridine in comparison to HPS 400 and wild-type cells. Thymidylate uptake was the same for all three strains. Thus, the mutation giving rise to the HPS 401 phenotype selectively increases the uptake of thymidine into the cell, where it can be efficiently utilized for DNA synthesis by the “salvage” pathways of nucleotide metabolism.

A Rapid Screening Technique for Detection of Diosgenin ThroughIn SituCytophotometry

Costus speciosus (Koenig) Sm. contains diosgenin, an important drug in family planning programs in India and underdeveloped countries. A simple, rapid method has been developed for in situ quantitation of diosgenin in this plant. The method is based on the formation of a suitable colored product of diosgenin in frozen sections of fresh material followed by determination of its optical density by in situ cytophotometry. The staining reagent is a combination of anisaldehyde, sulfuric acid and acetic acid. A positive correlation has been observed between cytophotometric diosgenin estimates and those derived from thin layer chromatography of extracts. The method is convenient for routine screening of plants for steroidal sapogenins such as diosgenin.

612 DIFFERENCES IN PROCESSING OF ORALLY ADMINISTERED PROSTAGLANDIN F2α IN THE GASTROINTESTINAL TRACT AND LIVER OF SUCKLING AND WEANLING RATS

The presence of prostaglandin F2α (PGF) in milk led us to explore developmental differences in its metabolism between suckling and weanling rats. Disposition of orally administered 3H-PGF (20 μCi/kg/b.w.) to 12-day-old suckling (Su) and 30-day-old weanling (We) rats was evaluated quantitatively by determination of total radioactivity (TRA) recovery; and qualitatively by column and thin layer chromatography of extracts of liver (L), gastric wall and lumen (G), and small intestinal wall and lumen (I) two hours after administration. Recovery of TRA in these organs was 32.6% ± 2.1,8 (mean ± SE, N) of counts administered in Su, and 22.7% ± 4.8,5 in We (p < 0.03). In Su, G values tended to be higher than in We (11.1% ± 1.3,8 vs 8.5% ± 3.2,5); I values were 15.9% ± 0.7 vs 10.3% ± 1.8 (p < 0.01). Despite the presence of higher nonabsorbed TRA in G and I of Su, increased TRA was present in L [9.1% ± 1.7 vs 3.4% ± 0.6 (p < 0.05)]. Qualitatively, there was no substantial difference in distribution of TRA (un-metabolized parent PGF vs metabolites) in G and I. However, L of Su when compared to L of We, exhibited more unmetabolized PGF (12% ± 0.9 vs 7% ± 1.1 of TRA in L (p < 0.01) and less of the polar metabolites (21% ± 3.7 vs 35% ± 3.1 (p < 0.02). Conclusions. Qualitative and quantitative differences in metabolism of orally administered PGF were found between suckling and weanling rats. Despite slower absorption of PGF from the gastrointestinal tract of suckling rats, a larger proportion of prostaglandin was found in the liver in an unmetabolized form.

Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets.

The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.

Absorption and translocation of metobromuron and chlorbromuron

Abstract Tomato (Lycopersicum esculentum Mill., ‘Heinz 1350'), a crop plant susceptible to both herbicides used, readily absorbed root‐fed 3‐(p‐bromophenyl)‐l‐methoxy‐l‐methylurea (metobromuron) and 3‐(4‐bromo‐3‐chlorophenyl)‐l‐methoxy‐l‐methylurea (chlorbromuron) and translocated them to the foliage. The umbelliferous crop plants, carrot (Daucus carota L., ‘Gold Pak') and coriander (Coriandrum sativum L.)., both moderately susceptible and resistant to metobromuron and chlorbromuron respectively, absorbed and translocated metobromuron to the foliage but chlorbromuron, although found in the roots of these plants in high concentration, was not translocated upward appreciably. Neither herbicide applied to the leaves was translocated away from the area of application in significant amounts in either tomato or coriander. The thin layer chromatography of extracts from the various plant parts indicated that neither compound was metabolized appreciably by the resistant coriander. Extracts from roots and tops of t...

Synthesis of ecdysone-14C and ecdysterone-14C from cholesterol-14C in cockroaches (Periplaneta americana) without molting glands.

The synthesis of molting hormone of cockroaches of which the prothoracic glands had previously been extirpated was investigated after injection of cholesterol-14C. It could be proved by means of radio thin-layer chromatography of extracts that ecdysterone and in small amounts ecdysone are synthesized in Periplaneta americana larvae without prothoracic glands. The results demonstrate that other tissues are also able to perform the molting hormone besides the molting gland itself.

Residue analyses on 2-amino-4-phenylthiazole, a piscine anesthetic, in fishes. III. Metabolism in rainbow trout and carp.

The major biotransformation product of 2-amino-4-phenylthiazole in rainbow trout (salmo gairdneri irideus) was isolated from water following exposure of fish to the anesthetic. The isolated crystalline metabolite was shown by means of ultraviolet, infrared and optical rotatory dispersion spectroscopy and gas chromatography to be identical to 2-amino-4-pphenylthiazole-2-N-β-mono-D-glucopyranosiduronic acid, the major biotransformation product previously found in medaka (killifish, Oryzias latipes). The major biotransformation product in carp (Cyprinus carpio) was also identified as 2-amino-4-phenylthiazole-2-N-ββ-mono-D-glucopyranosiduronic acid by molecular sieve, thin layer and gas chromatography. Conversion of 2-amino-4-phenylthiazole to the N-glucuronyl conjugate was 8 and 12%, respectively, in rainbow trout and carp as shown by thin layer chromatography of extracts from fish treated with 3H-labeled anesthetic. In addition, a minor metabolite of the anesthetic in rainbow trout was isolated as a yellowish-white crystalline powder and identified as 2-acetamido-4-(4'-hydroxyphenyl)-thiazole by means of ultraviolet and infrared spectroscopy, NMP and mass spectrometry. Chromatography suggested that this same metabolite was also formed in carp but in concentrations to low for isolation and definitive identification.

Differential tolerance of peas to prometryne and terbutryn

The phytotoxicity of 2,4-bis(isopropyl)-6-(methylthio)- s-triazine (prometryne) and 2-( tert-butylamino)-4-(ethylamino)-6-(methylthio)- s-triazine (terbutryn) to peas ( Pisum sativum L. var. Perfection 3040) was studied. No differences were found when the herbicides were applied to the roots of intact plants in nutrient solution or directly to leaf discs. However, prometryne was much more toxic when uptake was from soil. Absorption and translocation of 14C-labeled prometryne and terbutryn showed that the majority of terbutryn accumulated in the roots, whereas prometryne was uniformly distributed between the roots and the shoot. Thin layer chromatography of extracts from prometryne-treated peas showed that only 20% of the absorbed compound was metabolized to produce one breakdown product. Extracts of terbutryn-treated plants contained three different metabolites. After 120 hr of exposure to terbutryn, about half of the absorbed herbicide was metabolized. The results show that the main factors responsible for the differential toxicity of the herbicides to peas were availability from the soil, translocation pattern and initial detoxication.

Absorption and Translocation of Terbutryn and Propazine

The absorption and translocation patterns of terbutryn [2-(tert-butylamino)-4-(ethylamino)-6-(methylthio)-s-triazine] and propazine [2-chloro-4,6-bis(isopropylamino)-s-triazine] were determined in wheat (Triticum vulgareVill. ‘Kaw’) and sorghum (Sorghum bicolorL. ‘RS 612’). Bioassay studies showed sorghum to be tolerant and wheat susceptible to terbutryn. Absorption and translocation of uniform14C-labeled terbutryn from 1 to 24 hr after initial treatment showed the roots of sorghum and the foliage of wheat to be the major sites of radioactivity accumulation. Foliage was the major site for propazine accumulation with both plant species. Thin-layer chromatography of extracts from terbutryn-treated plants showed that sorghum contained a higher percentage of metabolic breakdown products in the foliage than did the root systems, and wheat contained a high percentage of intact terbutryn in the foliage and roots. High temperature (32 C) enhanced translocation of terbutryn and propazine in both sorghum and wheat. The susceptibility of wheat and sorghum to terbutryn appears to be correlated with a combination of the factors of accumulation within the foliage of the plant and the ability to degrade the terbutryn molecule.

Factors in the measurement of pineal acetylserotonin methyltransferase activity in the lizard Sceloporus occidentalis

Pineal acetylserotonin methyltransferase (ASMT) activity was studied in western fence lizards ( Sceloporus occidentalis) collected in May in Napa Co., California. Although pineal dry weight differed in the two age (size) classes of animals present (adults, 25.1 ± 1.6 μg; subadults, 16.1 ± 1.3 μg), pineal ASMT activity per unit weight did not differ. Thin-layer chromatography of extracts from enzyme incubations with N-acetylserotonin showed a single methylated product comparable to melatonin or 6-hydroxymelatonin. Different apparent ASMT activities were obtained in incubations with different buffer systems over the same pH range. Results suggest that phosphate buffer coupled with excess substrate concentrations and incubation temperature of 37°C is inhibitory to pineal ASMT in this species. Tris-HCl is shown to be the preferred buffer, and saturation concentrations of substrate and methyl donor are demonstrated.

Distribution and metabolism of alpha-ecdysone in pupae of the silkworm antheraea polyphemus.

1. The distribution of inulin, water, and α-ecdysone was studied in pupae of the silkworm Antheraea polyphemus. All three compounds reach equilibrium within two hours after injection.2. Inulin is restricted to the extracellular space, which amounts to 0.49 ml per g live weight.3. The ecdysone space is slightly larger than the water space. About 50% of the radioactivity derived from α-ecdysone remains in the blood for at least 36 hours.4. The other 50% is associated with cells in a form which cannot be removed by extensive washing of the intact tissues with saline.5. Transport of ecdysone to the tissues does not involve a rate-limiting carrier system that can be saturated by 10 µg α-ecdysone or 100 µg β-ecdysone.6. There is no evidence that ecdysone is accumulated by any one tissue or excretory organ.7. The metabolism of α-ecdysone was studied by thin layer chromatography of extracts of blood after injection of radioactive α-ecdysone. α-ecdysone is rapidly converted to β-ecdysone and the latter to several more polar metabolites.8. β-ecdysone either inhibits the conversion of α-ecdysone to β-ecdysone or is itself converted to α-ecdysone at a significant rate.