Search

The analytical procedures for determining plant constituents involve the application of sample preparation methods to fully isolate and/or pre-concentrate the analyzed substances. High-temperature liquid extraction is still applied most frequently for this purpose. The present paper shows that high-temperature extraction cannot be applied for the analysis of chlorogenic acids (CQAs) and their derivatives in plants as it causes the CQAs transformation leading to erroneous quantitative estimations of these compounds. Experiments performed on different plants (black elder, hawthorn, nettle, yerba maté, St John’s wort and green coffee) demonstrate that the most appropriate method for the estimation of CQAs/CQAs derivatives is sea sand disruption method (SSDM) because it does not induce any transformation and/or degradation processes in the analyzed substances. Owing to the SSDM method application we found that the investigated plants, besides four main CQAs, contain sixteen CQAs derivatives, among them three quinic acids. The application of SSDM in plant analysis not only allows to establish a true concentration of individual CQAs in the examined plants but also to determine which chlorogenic acids derivatives are native plant components and what is their concentration level. What is even more important, the application of SSDM in plant analysis allows to eliminate errors that may arise or might have arisen in the study of chlorogenic acids and their derivatives in plant metabolism. [ABSTRACT FROM AUTHOR]

The occurrence of protochlorophyllide b and protochlorophyllide b phytyl ester in green plants is described. The chemical structure of protochlorophyllide b phytyl ester was established by proton nuclear magnetic resonance, fast atom bombardment mass spectroscopic analysis, and chemical derivatization coupled to electronic spectroscopic analysis. The macrocycles of protochlorophyll(ide) b are identical to those of conventional protochlorophyll(ide) except for the presence of a formyl group instead of a methyl group at position 3 of the macrocycles. They differ from chlorophyll(ide) b by the presence of an oxidized double bond at positions 7 and 8 of the macrocycles. The trivial name protochlorophyll(ide) b is proposed to differentiate these two tetrapyrroles from conventional protochlorophyll(ide), which in turn will be referred to as protochlorophyll(ide) a. Protochlorophyll(ide) b appears to be widely distributed in green plants. Its molar extinction coefficients in 80% acetone and diethyl ether are reported. The impact of this discovery on the heterogeneity of the chlorophyll a and b biosynthetic pathways is discussed.

An inhibitor (BGIA) against an acidic amino acid-specific endopeptidase of Streptomyces griseus (Glu S. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. griseus protease with an inhibition constant (Ki) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:1 complex with this protease. However, two other acidic amino acid-specific endopeptidases, protease V8 from Staphylococcus aureus and Bacillus subtilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, barley subtilisin-chymotrypsin inhibitor CI-1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corresponding amino acid in BGIA is alanine.

To determine if atrial natriuretic peptides are present in plants as well as animals, where they are important for water and sodium metabolism, the leaves and stems of the Florida Beauty (Dracena godseffiana) were examined. The N-terminus consisting of amino acids (a.a.) 1-98 (i.e., pro ANF 1-98), the mid portion of the N-terminus (a.a. 31-67; pro ANF 31-67), and C-terminus (a.a. 99-126; ANF) of the 126 a.a. atrial natriuretic factor (ANF) prohormone were all present in the leaves and stems of this plant. The concentrations of pro ANF 1-98, pro ANF 31-67 and ANF-like peptides of 120 +/- 20, 123 +/- 21, and 129 +/- 20 ng/g of plant tissue in leaves and 109 +/- 20, 96 +/- 21, and 124 +/- 18 ng/g of tissue, respectively, in the stems were lower (P less than 0.05) than their concentrations in rat (Rattus norvegicus) heart atria of 196 +/- 40, 192 +/- 28, and 189 +/- 15 ng/g of tissue respectively, but higher (P less than 0.001) than their respective concentrations of 4.3 +/- 1.4, 4.1 +/- 1.2, and 3.9 +/- 1 ng/g of rat heart ventricular tissue. We conclude that the atrial natriuretic peptide-like hormonal system is present in the plant kingdom as well as in the animal kingdom.

Nucleotide sequences encoding signal peptides from the precursors of alpha-amylase/trypsin inhibitors from cereals are homologous to those corresponding to the precursors of thaumatin II and of plastocyanins. Non-synonymous (KA) and synonymous (KS) rates of nucleotide substitutions have been calculated for all possible binary combinations. Extreme variation in KA/KS ratios has been observed; from the 0.167 average found within the plastocyanin family to an average of 1.90 calculated for the inhibitors/thaumatin II transition. A similar calculation has been carried out for the signal peptide sequences of thionins, which are unrelated to those of the alpha-amylase/trypsin inhibitor family, and an average KA/KS of 0.12 has been obtained. This variation can be largely explained in terms of an empirical index of stability related to amino acid composition and seems to be independent of functional constraints.

Twenty unsaturated sesquiterpene dialdehydes were tested for antimicrobial, algaecidal, cytotoxic, and mutagenic activity. In addition to the known antifungal activity, polygodial (1) also exhibited antibacterial and cytotoxic activity; epipolygodial (2) was slightly less active. The most active compounds were: isovelleral (7), isoisovelleral (8), velleral (20), and methylmarasmate (6). With the exception of velleral (20), they also exhibited mutagenic activity in the Salmonella/microsome assay. Derivatization to less polar compounds usually increased the antimicrobial and cytotoxic effects and reduced mutagenicity, while the introduction of hydroxyl groups had the reverse effect.

The composition of the essential oils of Bystropogon plumosus, B. origanifolius var. palmensis, B. wildpretii, B. maderensis, and B. canariensis var. smithianus were studied by GLC and GLC/MS. The first four species provide relatively similar oils characterized by monoterpene ketones as the main constituents while the last species is quite different with main constituents consisting of mono- and sesquiterpene hydrocarbons. In B. plumosus the ontogenetic variation of the monoterpene ketones was studied for a period of four years. During this time the behaviour of pulegone was inversely correlated with that of menthone and isomenthone. Pulegone was the most active compound of the monoterpene ketones in antimicrobial tests using three species of bacteria and fungi, respectively.

Four phloroglucinol derivatives, named mallotophenone (5-methylene-bis-2,6-dihydroxy-3-methyl-4-methoxyacetophenone), mallotochromene (8-acetyl-5,7-dihydroxy-6-(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-2,2-dimethylchromene), mallotojaponin (3-(3,3(dimethylallyl)5-(3(acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone) and mallotolerin (3-(3-methyl-2-hydroxybut-3-enyl)-5(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone), have been tested for their ability to inhibit the activity of human immunodeficiency virus (HIV)-reverse transcriptase. Under the reaction conditions with (rA)n.(dT)12-18 as the template.primer, the enzyme activity was inhibited by approximately 70% in the presence of 10 micrograms/ml mallotochromene or mallotojaponin, whereas mallotophenone and mallotolerin were much less inhibitory to the enzyme. The enzyme activity was also inhibited, though to lesser extent, by these compounds under similar conditions with initiated MS-2 phage RNA as the template.primer. The mode of inhibition was, as analyzed with mallotojaponin, competivite with respect to the template.primer, (rA)n.(dT)12-18, and non-competitive with respect to the triphosphate substrate, dTTP. The Ki value of mallotojaponin for HIV-reverse transcriptase was determined to be 6.1 microM.

Plasma membranes of oat root cells were isolated from intracellular membranes by subfractionation of the microsomal fraction using an aqueous polymer two-phase system. The plasma membranes originated from oat plants which were acclimated to dehydration by exposure to a repeated water-deficit stress program. Glucosylceramides was a major component of the plasma membrane lipids and amounted to 9% of the lipid of control plants and 5% of the lipid of acclimated plants. Structural analysis using FAB-MS showed only one type of glucosylceramides. The constituent monosaccharide was exclusively glucose and the sphingosine base was 4,8-sphingadienine. The fatty acid composition was determined to 24:1-OH, with only trace levels of non-hydroxy acids. The decrease in the level of glucosylceramides during acclimation to dehydration was accompanied by a corresponding decrease in phospholipids and increase in free sterols.

From bioactivity-directed fractionation of the EtOH extract of Endlicheria dysodantha, dysodanthin A and dysodanthin B, which are new hexahydrobenzofuranoid neolignans, have been isolated. In addition, the known neolignans, compound 4, which is a burchellin analogue, and megaphone acetate [1] were isolated. All four neolignans showed activities in the brine shrimp lethality test; compounds 1-3 also inhibited the growth of crown gall tumors on potato discs and were cytotoxic to human tumor cells in culture. This is the first report of these neolignans isolated from the genus Endlicheria and of their completely assigned 1H-and 13C-nmr data.

We established a highly sensitive immunofluorescence procedure for localizing bound atrazine in the aquatic macrophytes Elodea canadensis and E. densa. The technique included biotin-labeled anti-rabbit IgG as a first enhancement step and R-phycoerythrin (R-PE) coupled to streptavidin for fluorescent labeling as a second improvement on the procedure. A comparison with the conventional indirect immunofluorescence method confirmed the superior results of the R-PE approach. The use of atrazine-free plants (grown in charcoal-filtered water) and a variety of other controls excluded both contaminating atrazine and nonspecific incubation constituents as sources of tissue staining. Pre-incubations to block nonspecific binding sites proved to be unnecessary in this system. The highly sensitive procedure described here might be a useful tool for the localization of tissue-bound pesticides in general and possibly of other haptens as well.

Affinity chromatography was used to separate two components A and B of the crystalline arrowhead proteinase inhibitor. Both A and B are double-headed and multifunctional proteinase inhibitors. Inhibitor A is capable of inhibiting equimolarly trypsin and chymotrypsin simultaneously, and has a weak inhibitory activity toward kallikrein; whereas inhibitor B can inhibit two molecules of trypsin simultaneously, and shows rather higher inhibitory activity toward kallikrein than inhibitor A, but its inhibitory activity toward chymotrypsin is much weaker than that of inhibitor A. The results of chemical modification and the competitive binding of trypsin and chymotrypsin with inhibitor A showed that the two reactive sites of both inhibitors A and B are Lys and Arg residues. Among them the Lys reactive site is specific for inhibiting mainly trypsin, whereas the active domain composed of the Arg reactive site appears to be multifunctional and capable of inhibiting many different Ser proteinases. Based on the structural characteristics of inhibitors A and B, it was predicated that the two reactive sites should be located in the positions Lys-Ser (44-45) and Arg-Tyr-Lys (76-78), respectively. In inhibitor A, there exists another hydrophobic residue involved in inhibiting chymotrypsin. This residue might be situated in the reactive region composed of the Arg reactive site.

High-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 9-13) separates aminosaccharides, neutral saccharides and glycuronic acids based upon their molecular size, saccharide composition and glycosidic linkages. Carbohydrates were extracted by utilizing 0.5 M H2SO4 (neutral monosaccharides), 0.25 M H2SO4 coupled with enzyme catalysis (glycuronic acids) and 3 M H2SO4 (aminosaccharides). Solid-phase extraction with strong cation and strong anion resins was used to partition the cationic aminosaccharides and anionic glycuronic acids and to deionize acid extracts for neutral saccharides. Separation was conducted on a medium-capacity anion-exchange column (36 mequiv.) utilizing sodium hydroxide (5-200 mM and sodium acetate (0-250 mM) as the mobile phase. The saccharides were detected by oxidation at a gold working electrode with triple-pulsed amperometry. HPAEC-PAD was found superior to high-performance liquid chromatography with refractive index (RI) detection for neutral monosaccharides and aminosaccharides and to low-wavelength UV detection for glycuronic acids in terms of resolution and sensitivity. HPAEC-PAD was not subject to interferences as was the case for low UV detection (210 nm) or RI analyses and was highly selective for mono- and aminosaccharides and glycuronic acids. The use of HPAEC-PAD was applied for the determination of the saccharide composition of organic materials (plant residues, animal wastes and sewage sludge), microbial polymers and soil.

Antileishmanial activity is reported for the first time for saponins of ivy, Hedera helix L., in vitro on promastigote and amastigote forms of Leishmania infantum and Leishmania tropica. The compounds tested were an extract containing 60% of saponic complex (CS 60), the bidesmosides hederasaponin B, C, and D (saponin K10), their corresponding monodesmosides alpha-, beta-, and delta-hederin, and hederagenin. CS 60 and bidesmosides have shown no effect. Monodesmosides were found to be as effective on promastigote forms as the reference compound (pentamidine). Against amastigote forms only hederagenin exhibited a significant activity which was equivalent to that of the reference compound (N-methylglucamine antimonate).

The F concentrations of precipitate dust, agricultural products, and fingernail and hair at the surrounding Al factory were investigated. The F content of dust ranged from 15400 to 42500 micrograms/g dry weight, 190,000 to 380,000 micrograms/g Al. Rice grain contained about 3.4 times more F than that in the control area, but some kinds of agricultural products, egg plants (S. melongena L.), mulberry plants (M. japonica Bailey non Sieb.), and soy beans (G. max (L.) Merrill) were almost equal to that of controls. Also, the high F concentration in the hair and nails of some workers was affected by available F contents in the emission from the factory as well as food and water surrounding the aluminum factory compared with those of control area.

An Arabidopsis thaliana library constructed in the pBluescript expression vector lambda ZAP II (Stratagene) was screened with three affinity-purified antibodies raised against (i) rat liver connexin 32, (ii) a polypeptide from soybean root cells that migrates with a molecular mass of 29 kDa in SDS/polyacrylamide gels and is immunologically related to rat liver connexin, and (iii) a synthetic peptide corresponding to a sequence in rat liver connexin 32. A single clone was obtained whose gene product demonstrated immunological crossreactivity with all three reagents. The cDNA from this clone contained 1171 base pairs and coded for a protein of 280 amino acids with a calculated molecular mass of 32,339 Da (migrates as a 29-kDa polypeptide in SDS/polyacrylamide gels). The sequence homology observed between the 32-kDa polypeptide of Arabidopsis and connexin 32 from rat liver, in conjunction with observed similarities in predicted number and distribution of hydrophobic domains, sites for posttranslational modification, and basic pI, provides strong evidence that the biological range for connexin-type proteins may now be considered to include the plant kingdom.

Bovine, porcine and sheep adrenodoxin, and the trypsin-resistant form of bovine adrenodoxin have been studied by one- and two-dimensional 1H-NMR spectroscopy. Assignment of the resonances for all the aromatic amino acids with resolved aromatic resonances have been made by correlating NMR spectra with the amino acid sequences from various species. Slowly exchanging amide protons and downfield shifted alpha-protons of His10 and Phe11 suggest possible involvement in beta-sheet structure. The effects on the assigned resonances due to the specific spin-label with a nitroxide radical at Cys95 have been analyzed on a two-dimensional 1H-NMR spectrum. The present results provide evidence for a structural similarity with a model for the structure of adrenodoxin based on a sequence alignment with that of Spirulina platensis ferredoxin, for which X-ray crystallographic data is available. epsilon-Methyl groups of Met120 and Met122 have been assigned by comparing 1H-NMR spectra of adrenodoxin with those of the trypsin-resistant form of adrenodoxin which is specifically cleaved at Arg115. epsilon-Methyl groups of Met120 and Met122 have an exceptionally long longitudinal relaxation time compared with those of valyl and leucyl methyl groups, suggesting that the COOH-terminal peptide spanning over 13 amino acids rotates rather freely in the solvent.

A sensitive method for the accurate determination of Se in agricultural products at sub-ppm levels is described. The proposed procedure involves the wet oxidation of samples by using a mixture of nitric, sulphuric and perchloric acids, co-extraction of Se and added Pd with diethylammonium N,N-diethyldithiocarbamate in chloroform, and electrothermal atomic absorption spectrometric determination of Se in the organic extract. Atomization and extraction conditions are discussed. Special attention is given to the wet oxidation step, and its advantages in speed and simplicity over conventional heating have been evaluated using an automated microwave digestion system. The results reported, obtained from several reference materials, confirm the accuracy of the method with which a detection limit of 0.002 micrograms g-1 of Se can be achieved.

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.

Angiosperms, fungi (including lichens), and bacteria are the main sources of natural quinones. Small numbers are present in algae, ferns, conifers, sponges, echinoderms, other marine animals, and arthropods. In angiosperms quinones have some chemotaxonomic value at the genus and family level but more surveys are required.

Calycanthine, the principal alkaloid of the order Calycanthaceae, has been isolated from a species of the genus Psychotria (Rubiaceae) occurring in some Pacific islands. The drug is considered as a very powerful convulsant poison but very little is known about its mechanism of action. The symptoms observed were similar to those of some neuropoisons such as strychnine. The properties of this alkaloid have been investigated on the genesis, conduction, and transmission of the nerve impulse, using giant axons of the cockroach (Periplaneta americana). Calycanthine hydrochloride (10(-5) M), which did not alter nervous conduction in pre- and post-synaptic fibers, significantly reduced the efficacy of the synaptic transmission.

A sensitive spectrophotometric method for the determination of paraquat using ascorbic acid (an easily available reducing agent) is described. Paraquat is reduced with ascorbic acid in alkaline solution to give a blue radical ion with an absorbance maximum at 600 nm. Beer's law is obeyed in the range 12-96 micrograms of paraquat in 10 ml of the final solution (1.2-9.6 ppm). The important analytical parameters and the optimum reaction conditions were evaluated. The method was applied successfully to the determination of paraquat in water, grain and plant materials.

A review is given of contemporary ethnobotany. Emphasis is placed on the interdisciplinary nature of the subject and most progress is made when botanists, anthropologists, ecologists, chemists, etc. work in close collaboration. Ethnoecology, the study of the management systems of indigenous peoples, is particularly important for its application to the creation of sustainable use systems in the tropics. This must include the study of peasant agriculturalists who have adapted many techniques from cultures that have long been extinct. Quantitative studies of the extent to which rainforest Indians use the forest have provided many data useful for conservation. The study of indigenous medicines is leading to the discovery of new medicines and agrochemicals. When commercial benefit is gained from products derived from information obtained from indigenous peoples, it is essential to ensure that they and their countries benefit from the royalties. There is a great urgency for further ethnobotanical research before indigenous cultures and natural habitats are destroyed.

4-Hydroxy-2-cyclopentenone is responsible for the anti-bacterial activity of an extract of leaves from Passiflora tetrandra with minimum inhibitory doses (MID) of ca. 10 micrograms/disk against Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa. 4-Hydroxy-2-cyclopentenone is also cytotoxic to P388 murine leukemia cells (IC50 of less than 1 microgram/ml).

The distribution and behavior of Cs in the soil-plant systems at some upland sites in Northeastern Italy, Scotland, and Norway have been investigated. From the limited range of samples taken, there appears to be no dominant physicochemical control on the plant availability of Cs. The presence of micaceous minerals or illitic clays does not significantly inhibit Cs uptake, either because of recycling in the organic surface horizons or because of clay-organic complex formation. Lower plants (bryophytes and lichens) show the highest Cs accumulation. Of the higher plants, ericaceous species take up Cs more than the others.

Several kinds of loline-type alkaloids, norloline, loline, N-acetylnorloline, N-acetylloline, N-formylnorloline, N-formylloline and N-methylloline were detected in the urine of race-horses. Furthermore, a new compound of the alkaloids, N-senecioylnorloline, was also found and identified. These compounds were mainly identified by means of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-fourier transform-infrared spectrometry (GC-FT-IR). A certain plant of Gramineae containing four kinds of loline-type alkaloids was found in a bale of hay used for the horse forage. The taxonomic feature of the plant was different from known plants containing loline-type alkaloids. The common fragmentation of loline-type alkaloids under electron ionization was briefly discussed.

Two formerly-uncharacterized subunits of 9 kDa and 14 kDa were found in spinach PSI complex. The 9 kDa subunit was released upon removal of antenna chlorophyll complex, whereas the 14 kDa subunit was tightly bound to the core complex. We determined the N-terminal amino acid sequence of the 9 kDa, and an internal sequence of the 14 kDa subunit after protease treatment, since the N-terminus of the latter protein was blocked. These partial sequences suggested that both subunits are new PSI components.

The effects of I- ions on O2 evolution by photosystem 2 particles, which were depleted of the 18-kDa and the 23-kDa extrinsic proteins of the O2 evolution complex by NaCl washing (dPS2 particles) were examined. In the absence of Cl- (incompetent dPS2) I- stimulated O2 evolution up to 3-6 mM, depending on the associated cation, and inhibited it at higher concentrations. In the presence of Cl- (competent dPS2), I- was inhibitory at all concentrations. The inhibition was reversible, it occurred at a site preceding Tyrz (Tyr residue mediating electron transfer from H2O to photosystem 2), and it interfered noncompetitively with the reactivation of incompetent dPS2 with Cl-. Furthermore, the organic salts tetrabutyl ammonium iodide and tetraphenyl phosphonium iodide proved to be stronger inhibitors than the inorganic NaI. This is interpreted as an indication of a negatively charged surface, situated behind a hydrophobic permeability barrier. Permeant organic cations, being better compensators of the inner surface charge than Na+, are also more apt in facilitating access of the I- ions to the inhibitory site in the vicinity of Tyrz.

Omega-3 Fatty acids (omega-3FAs) are found in seafoods, some plants, and some livestock rations. Fish oils are the only concentrated source of eicosapentaenoic acid (EPA; 20:5 omega-3) and docosahexaenoic acid (DHA; 22:6 omega-3). The major omega-3FA in plants is alpha-linolenic acid (LNA; 18:3 omega-3). LNA must be converted to EPA before it exerts biological effects similar to EPA, such as reduced platelet aggregation. Human beings convert LNA to EPA to a small extent only. LNA may be more readily oxidized than incorporated into tissues. The effects of consuming LNA-rich oils are more modest than the effects of EPA-rich oils. Evidence suggests that omega-3FAs are essential and highly desirable for brain and eye development and heart health. LNA is the only source of omega-3FAs for vegetarians. Because LNA and EPA are not biologically equivalent, food composition data or product claims mentioning total omega-3FA content must clarify the individual omega-3FAs present.

A series of eight polyines, isolated from several plants and containing various furanyl, thiophene, and spiro groups, were evaluated for antibiotic, antiviral, and cytotoxic activities in the presence and absence of long wavelength ultraviolet light, UVA. One compound, a furanopolyine, had impressive activity against a range of microorganisms, and membrane-containing viruses, as well as cytotoxic activity. All of these properties were UVA-dependent. Three other similar compounds had different patterns of UV-dependent activity against the various organisms, but some of these could also be of use in therapy of infectious diseases. In contrast, the dehydromatricaria esters and the two spiro compounds had relatively little biological activity.

Two anti-H(O) lectins were separated from extracts of Cytisus sessilifolius seeds by successive affinity chromatographies on columns of di-N-acetylchitobiose- and galactose-Sepharose 4B. One was found to be inhibited most by di-N-acetylchitotriose or tri-N-acetylchitotriose [Cytisus-type anti-H(O) lectin designated as Cytisus sessilifolius lectin I (CSA-I)] and the other anti-H(O) lectin was inhibited by galactose or lactose and designated as Cytisus sessilifolius lectin II (CSA-II). These two anti-H(O) lectins were further purified by gel filtration on TSK-Gel G3000SW. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular masses of the purified lectins I and II were found to be 95,000 and 68,000 Da, respectively, by gel filtration on TSK-Gel G3000SW. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol, both lectins gave a single component of molecular masses of 27,000 +/- 2,000 and 34,000 +/- 2,000 Da, respectively, suggesting that the lectins I and II were composed of four and two apparently identical subunits, respectively. Lectins I and II contain 38% and 13% carbohydrate, respectively, and only very small amounts of cysteine and methionine, but they are rich in aspartic acid, serine and glycine. The N-terminal amino-acid sequences of these two lectins were determined and compared with those of several lectins already published.

A new type lactose-binding lectin was purified from extracts of Ulex europaeus seeds by affinity chromatography on a column of galactose-Sepharose 4B, followed by gel filtration on Sephacryl S-300. This lectin, designated as Ulex europaeus lectin III (UEA-III), was found to be inhibited by lactose. The dimeric lectin is a glycoprotein with a molecular mass of 70,000 Da; it consists of two apparently identical subunits of a molecular mass of 34,000 Da. Compositional analysis showed that this lectin contains 30% carbohydrate and a large amount of aspartic acid, serine and valine, but no sulfur-containing amino acids. The N-terminal amino-acid sequences of L-fucose-binding Ulex europaeus lectin I (UEA-I) and di-N-acetylchitobiose-binding Ulex europaeus lectin II (UEA-II), both of which we have already purified and characterized, and that of UEA-III were determined and compared.

A survey of the inhibitory effects of various plant polysaccharides on DNA restrictions (HindIII and EcoRI) revealed that neutral polysaccharides (arabino-galactan, dextran, gum guar, gum locust bean, beta-glucan, inulin, laminaran, mannan and starch) were not very inhibitory. In contrast, acidic polysaccharides (carrageenan, dextran sulfate, gum ghatti, gum karaya, pectin and xylan) were very inhibitory, even at low concentrations. The Elutip-d (RPC-5 type resin) was evaluated for removal of the inhibitory polysaccharides. Used alone or in combination with a phenol/chloroform wash, it proved effective in removing the polysaccharide so that HindIII digestion was possible, except in the cases of carrageenan and dextran sulfate. In addition, the genomic DNA extracts from live oak (Quercus virginiana) and magnolia (Magnolia grandiflora) were sufficiently purified so that the DNAs could be restricted with both EcoRI and HindIII.

The furanoacridone alkaloid isogravacridonchlorine has been isolated from the roots of Ruta graveolens, the structure was elucidated by spectroscopic means. Its mutagenic activity and mode of action was characterized without as well as with metabolic activation using different Salmonella typhimurium strains.

Saponins were purified from Quillaja saponaria Molina bark by silica and reverse phase chromatography. The resulting purified saponins were tested for adjuvant activity in mice. Several distinct saponins, designated QS-7, QS-17, QS-18, and QS-21, were demonstrated to boost antibody levels by 100-fold or more when used in mouse immunizations with the Ag BSA and beef liver cytochrome b5. These purified saponins increased titers in all major IgG subclasses. To determine optimal dose in mice for adjuvant response, QS-7 and QS-21 were tested in a dose-response study in intradermal immunization with BSA in mice; for both of these purified saponins, adjuvant response (determined by stimulation of ELISA titers to BSA) neared maximum at doses of 5 micrograms and was shown to plateau up to the highest dose tested, 80 micrograms. These purified saponins vary considerably in their toxicity, as assessed by lethality in mice; the main component, QS-18, being the most toxic. Saponins QS-7 and QS-21 showed no or very low toxicity in mice, respectively. None of these saponins stimulated production of reaginic antibodies. The monosaccharide composition of these saponins showed similar but distinct compositions with all four containing fucose, xylose, galactose and glucuronic acid. Predominant differences were observed in the quantities of rhamnose, arabinose, and glucose. Monomer m.w. (determined by size exclusion HPLC) were determined to range from 1800 to 2200.

An extract of the seed of Sandoricum koetjape has yielded two new limonoids, sandoricin [1] and 6-hydroxysandoricin [2]. Both compounds are effective antifeedants when incorporated into artificial diets and fed to fall armyworm (Spodoptera frugiperda) or European corn borer (Ostrina nubilalis) larvae. Reduced growth rates and increased times to pupation were evident at lower dose levels while significant mortality was noted at higher dose levels. Structures of both compounds were determined by 1H nmr, 13C nmr, and ms and confirmed by X-ray crystallography.

Three flavonoids and three saponins have been isolated from Oxytropis species. Their structures were determined as isorhamnetin-3-O-beta-D-glucoside, rhamnetin-3-O-beta-D-galactoside, apigenin, 3-O-[alpha-L-rhamnopyranosyl (1----2)-beta-D-glucopyranosyl(1----4)-beta-D-glucuronopyranosyl]+ ++soyasapogenol B, 3-O-[beta-D-glucopyranosyl(1----2)-beta-D-glucuronopyranosyl] azukisapogenol and a new saponin 3-O-[beta-D-glucopyranosyl(1----2)-beta-D-glucopyranosyl]-25-O-alpha-L- rhamnopyranosyl-(20S,24S)-3 beta,16 beta, 20,24,25-pentahydroxy-9,19-cycloanostane.

The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin.