Detection of cytokinins in a seaweed extract

trans-Zeatin, trans-zeatin riboside, their dihydro derivatives, isopentenyladenine and isopentenyladenosine have been identified and quantified in Seasol, a commercial extract of Tasmanian Giant Bull kelp, Durvillea potatorum.     

Responses of Phaseolus lunatus callus tissues to cytokinin-active ureidopurines

Three ureidopurine derivatives were tested for their ability to promote the cytokinin-dependent growth of callus tissues derived from three genotypes of Phaseolus lunatus (cv. Jackson Wonder, cv. Kingston and P.I. 260415). In all three test systems, the ureidopurines exhibited the same relative cytokinin activities (6-phenylureidopurine > 6-p-tolylureidopurine > 6-cyclohexylureidopurine) and were less active than the aminopurine derivatives, N⁶-benzyl-adenine and N⁶-cyclohexylmethyladenine. Callus tissues of Jackson Wonder, which become cytokinin-autonomous if grown on cytokinin-active urea derivatives, retained cytokinin-dependence on media containing optimal concentrations of ureidopurines. In this respect, the biological activity of the ureidopurines was similar to that of the aminopurine derivatives and differed from that of cytokinin-active urea derivatives tested in this system.     

Endogenous Cytokinins in Bougainvillea ;San Diego Red': I. OCCURRENCE OF CYTOKININ GLUCOSIDES IN THE ROOT SAP

Using chromatographic, chemical, and enzymic techniques, 11 compounds capable of stimulating the division of soybean callus cells were tentatively identified in the root sap of Bougainvillea `San Diego Red.' These cytokinin-like compounds included phosphorylated and glucosylated forms of zeatin, ribosylzeatin, and their dihydro derivatives. In addition, isopentenyl adenosine and isopentenyl adenine were apparently also present. The occurrence of glucosylated derivatives in the root sap of plants has not been substantiated previously.     

Fatty acid changes in Hibiscus esculentus tissues during growth

Lipids were isolated from roots, stems, cotyledons, leaves, buds, flowers, pods and seeds of okra (Hibiscus esculentus) at different stages of plant growth from germination to seed formation and their fatty acid compositions analysed. The lipid contents of roots and stems were 1–3%, cotyledons 3.7–9%, leaves 2.5–5.1% and seeds 2.2–20.2%. Palmitic, linoleic and linolenic were the main fatty acids present in all tissues at all stages, but their relative proportions varied. Cyclopropene fatty acids (CFA) were present at some stages in roots and seeds. In the roots their formation coincided with bud formation (35 days after sowing) and their content reached a maximum (12.8%) seven days after flowering. CFA were present in maturing seeds from 31 days after flowering and occurred as dihydro derivatives throughout. Dihydro derivatives of the CFA were absent in all other tissues. Heptadecenoic acid was present (0.4–1.3%) in root lipids at all stages and in the stem lipids (0.4–1.2%) in the initial stages and after flowering.     

Cytokinins in the Leaves of Ginkgo biloba: I. The Complex in Mature Leaves

Cytokinin conjugates of zeatin, ribosylzeatin, and their respective dihydro derivatives tentatively have been identified as the major cytokinins present in mature Ginkgo biloba L. leaves. Ribosylzeatin was present in higher levels than zeatin and dihydrozeatin. No evidence could be found that 6-(2,3,4-trihydroxy-3-methylbutylamino)purine occurs as a metabolite in the mature leaves. From the available evidence, it is concluded that cytokinin conjugates are probably the major metabolites formed in the leaves of this deciduous gymnosperm.     

Determination of pyrrolidone carboxylate and gamma-glutamyl amino acids by gas chromatography.

Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and γ-glutamyl amino acids are described. These intermediates of the γ-glutamyl cycle were separated by ion exchange chromatography and converted to their N-acyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several γ-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.     

Simultaneous Analysis of Anthocyanin and Non-Anthocyanin Flavonoid in Various Tissues of Different Lotus (Nelumbo) Cultivars by HPLC-DAD-ESI-MSn

A validated HPLC-DAD-ESI-MSⁿ method for the analysis of non-anthocyanin flavonoids was applied to nine different tissues of twelve lotus genotypes of Nelumbo nucifera and N. lutea, together with an optimized anthocyanin extraction and separation protocol for lotus petals. A total of five anthocyanins and twenty non-anthocyanin flavonoids was identified and quantified. Flavonoid contents and compositions varied with cultivar and tissue and were used as a basis to divide tissues into three groups characterized by kaempferol and quercetin derivatives. Influences on flower petal coloration were investigated by principal components analyses. High contents of kaempferol glycosides were detected in the petals of N. nucifera while high quercetin glycoside concentrations occurred in N. lutea. Based on these results, biosynthetic pathways leading to specific compounds in lotus tissues are deduced through metabolomic analysis of different genotypes and tissues and correlations among flavonoid compounds.     

Specificity of thrombin: I. Esterolytic properties of thrombin,plasma, trypsin,and chymotrypsin with Nβ-substituted guanidino derivatives of p-nitrophenyl-p′-guanidinobenzoate

Thrombin exhibits a restricted specificity, relative to plasmin, trypsin, and chymotrypsin, for a series of derivatives of the titrant substrate p-nitrophenyl-p′-guanidinobenzoate (NPGB). Substitution on the beta guanidino nitrogen of NPGB with an n-butyl, n-hexyl, cyclo-hexyl, or benzyl residue does not prevent the esterolytic cleavage of these derivatives but does markedly alter their substrate properties with the four enzymes investigated.All four enzymes cleave NPGB at equivalent concentrations by releasing p-nitrophenol as pre-steady-state burst reactions followed by its steady-state production. Both chymotrypsin and trypsin similarly display burst reactions with the derivatives at corresponding concentrations. The acyl-enzyme intermediates formed with chymotrypsin, however, are more stable for the derivatives than for NPGB, and those formed with trypsin are less stable. In contrast, plasmin and thrombin exhibit incomplete burst reactions with the derivatives at these concentrations. Except for the cyclo-hexyl derivative, with which plasmin does not react, the derivatives relative to NPGB were cleaved faster by plasmin than by thrombin. These cleavages with thrombin, moreover, were competitively inhibited by benzamidine. Kinetic data obtained for thrombin further indicated that the substituent groups of derivatives hindered the initial formation of enzyme-substrate complexes. These results suggest that thrombin and, most likely, plasmin have restricted primary binding-site regions for small molecule substrates which do not readily accommodate bulky substituent groups.In addition, increasing concentrations of glycerol were found to greatly alter the esterolytic properties of thrombin for the compounds studied. This effect was demonstrated by increased deacylation rates with NPGB and by decreased cleavage rates with the n-butyl derivative.     

Dianthramides A and B,two N-benzoylanthranilic acid derivatives from elicited tissues of Dianthus caryophyllus

Two new phenolic derivatives, dianthramide A and B, were isolated from Dianthus caryophyllus tissues elicited with mycelial extracts of Phytophthora parasitica. The purified substances were identified on the basis of their spectral data and were characterized as N-salicyl-4-methoxyanthranilic acid (dianthramide A) and N-salicyl-4-hydroxyanthranilic acid methyl ester (dianthramide B). Dianthramides A and B co-occur in carnation tissues with the known phytoalexin dianthalexin.     

Requirement for Twist1 in frontonasal and skull vault development in the mouse embryo

Using a Cre-mediated conditional deletion approach, we have dissected the function of Twist1 in the morphogenesis of the craniofacial skeleton. Loss of Twist1 in neural crest cells and their derivatives impairs skeletogenic differentiation and leads to the loss of bones of the snout, upper face and skull vault. While no anatomically recognizable maxilla is formed, a malformed mandible is present. Since Twist1 is expressed in the tissues of the maxillary eminence and the mandibular arch, this finding suggests that the requirement for Twist1 is not the same in all neural crest derivatives. The effect of the loss of Twist1 function is not restricted to neural crest-derived bones, since the predominantly mesoderm-derived parietal and interparietal bones are also affected, presumably as a consequence of lost interactions with neural crest-derived tissues. In contrast, the formation of other mesodermal skeletal derivatives such as the occipital bones and most of the chondrocranium are not affected by the loss of Twist1 in the neural crest cells.     

Effect of nutritional factors on shikonin derivative formation in Lithospermum callus cultures

Some nutritional factors affecting the biosynthesis of shikonin derivatives in callus cultures of Lithospermum erythrorhizon were examined. High sucrose concentrations increased the content of shikonin derivatives, but neither glucose nor fructose was effective for shikonin derivative formation. High concentrations of nitrogen sources inhibited or retarded shikonin derivative formation and streptomycin sulphate stimulated their biosynthesis. Addition of ascorbic acid increased the content of shikonin derivatives. Among some precursors tested only l-phenylalanine had a positive effect. At high concentrations, Ca²⁺ and Fe²⁺ inhibited the biosynthesis of shikonin derivatives.     

Radioimmunoassay for biopterin in body fluids and tissues

Specific antibodies against l-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l-erythro-biopterin distinguished among l-erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l-erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-(l-erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with ¹²⁵I as the labeled ligand for the radioimmunoassay. BP-¹²⁵I-TYRA had similar binding affinity as the natural l-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP-¹²⁵I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H⁺ column was required for serum, cerebrospinal fluid, and brain tissues.     

Synthesis and properties of lipophilic derivatives of 5-fluorouracil

A series of N ¹-acyl derivatives of 5-fluorouracil (5-FU) bearing the residues of palmitic, p-myristoylaminobenzoic, p-oleoylaminobenzoic, and adamantane-1-carboxylic acids have been synthesized. The relative hydrolysis rates for the derivatives under physiological conditions (pH 7.2 and 37°C) have been determined, and it has been shown that the resistance of these compounds to hydrolysis increases as the steric accessibility of the amide group at residue N ¹ of 5-FU decreases. The derivatives easily incorporate into the lipid bilayer; their liposomal preparations show a marked cytostatic activity on human breast lymphoma cells (LD50 ～1 μM) and are of interest as potential antitumor preparations. In addition, a fluorescent analogue of the above derivatives, 1-[8-(3-perylenyl)octanoyl]-5-fluorouracil, has been synthesized, which is intended for studying the behavior of 5-FU derivatives in cells and tissues by instrumental methods.     

Synthesis of Two 3-(7′-Theophyllyl)glycals, 3-Deoxy-3-(7′-theophyllyl)-d-xylo-hex-1-enopyranose and Methyl 3-Deoxy-3-(7′-theophyllyl)-d-xylo-hex-1-enopyranuronate,and their Derivatives

Two 3-(7′-theophyllyl)glycals, (IV) and (V), were synthesized by fusion of theophylline and the appropriate glycals in the presence of p-toluenesulfonic acid. The structure and stereochemistry of the glycals were determined mainly from NMR analysis of their dihydro and 1,6-anhydro derivatives.     

Characterization of polyphenols in cell walls of cultured Populus trichocarpa tissues

The amount and composition of cell wall-bound polyphenol (lignin) in cultured Populus trichocarpa tissues which formed numerous xylem elements (xylogenic) or no xylem (non-xylogenic) were compared. Polyphenol accounted for ca 15% of the dry wt of the cell wall and did not differ significantly in amount in xylogenic and non-xylogenic tissues. The syringic acid derivatives, 3,4.5-trimethoxybenzoic acid, was identified as one of the oxidation products of methylated cell walls and was recovered in similar amounts irrespective of xylem formation. In contrast, lignin from xylogenic cultures contained more p-coumaryl alcohol derivatives and less coniferyl alcohol derivatives than lignin from non-xylogenic cultures. In this respect the lignin composition of xylogenic tissues closely resembled that from stems.     

Evidence for Host Genome Involvement in Cytokinin Metabolism by Male and Female Cells of Mercurialis annua Transformed by Strain 15,955 of Agrobacterium tumefaciens

When male and female individuals of a dioecious species Mercurialis annua L. were inoculated with the same strain of Agrobacterium tumefaciens (15,955), the corresponding tumor tissues of each sex clearly differed in their endogenous cytokinin content; only the male tumors had a morphogenetic feminizing effect on male flowers.

In male tumor tissues, zeatin (Z) in higher quantity than ribosyl-zeatin (RZ) became the major metabolite in contrast with the general situation for crown-galls; the female tumor tissues were characterized by an increase of total endogenous cytokinins and by the appearance of some specific metabolites such as a methyl-thio-Z and several glycosylated Z derivatives that had not been detected in healthy apices.

In both male and female tumor tissues, the cis form of RZ, present in healthy apices as 30% of trans-RZ form, was no longer detectable.

Quantitative and qualitative differences characterize male and female tumor tissues (host genes expression) but since differences also appeared between healthy male and female apices and their corresponding tumor tissues (TDNA gene expression), it can be tentatively concluded that a complex interaction between host cytokinin genes and those of TDNA control the endogenous metabolism of tumor tissues.

     

Interaction between adenosine generated endogenously in neocortical tissues,and homocysteine and its thiolactone

[¹⁴C]Adenine derivatives in normal guinea pig or rat neocortical tissues maintained by superfusion included ATP, ADP and AMP collectively forming some 98% of the acid-extracted ¹⁴C; adenosine, inosine and hypoxanthine each at less than 0.5% and S-adenosylhomocysteine at about 0.1%. l-Homocysteine and/or its thiolactone increased only a little the S-adenosylhomocysteine. The superfusion fluid carried from the tissue per minute about 0.1% of its acid-extractable [¹⁴C]adenine derivatives. Electrical stimulation of the superfused tissue increased 10-fold its output of [¹⁴C]adenine derivatives and diminished the 5′-nucleotides in the tissue to 94% of the acid-extractable [¹⁴C]adenine derivatives, the remainder being adenosine, inosine and hypoxanthine with little change in S-adenosylhomocysteine. Homocysteine in the superfusion fluids now caused large increases in tissue S-adenosylhomocysteine, which became the preponderant non-nucleotide ¹⁴C-derivative when homocysteine was 0.1 mM or greater. The total [¹⁴C]adenine conversion to non-nucleotide derivatives then increased and the 5′-nucleotides fell to 88% of the total. It is concluded that concentration relationships observed in the action of homocysteine make it feasible that convulsive conditions and mental changes associated with administered homocysteine and with homocystinuria are due to cerebral adenosine concentrations being diminished through formation of S-adenosylhomocysteine. Adenosine is preponderantly depressant in cerebral actions; effects of the S-adenosylhomocysteine produced may also be relevant.     

Synthesis and metabolism of prostaglandins E2, F2α and D2 by the rat gastrointestinal tract. Stimulation by a hypertonic environment in vitro

Whole cell preparations of rat stomach corpus, jejunum, and colon were incubated and the released prostaglandin E₂ (PGE₂), PGF_2α, PGD₂, 15 keto-13,14 dihydro PGE₂, and 15 keto-13,14 dihydro PGF_2α were measured by combined gas chromatography-mass spectrometry. All regions made PGD₂ and possessed a high capacity for producing 15 keto-13,14 dihydro derivatives of both PGE₂ and PGF_2α. Hypertonic sucrose solutions resulted in concentration-dependent increases in prostaglandin release, particularly of PGE₂ and its metabolite. It is suggested that PG's may play a role in the local effects of luminal hyperosomolarity on digestive tract functions.     

Seasonal variation in the secondary chemistry of foliar and reproductive tissues of Delphinium nuttallianum

Plant secondary compounds are critical in affecting interactions between plants and their herbivores. The norditerpene alkaloids are secondary compounds in Delphinium (larkspur) species which are divided into two classes: the N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type) and non MSAL-type, and are known to be toxic to herbivorous insects and livestock. Alkaloid concentrations were measured in a whole plant context in vegetative and reproductive tissues in Delphinium nuttallianum at different stages of plant maturity at two locations to explore how plant maturity affected alkaloid concentrations within a growing season. Alkaloid concentrations differed between vegetative and reproductive tissues, with vegetative tissues having significantly lower alkaloid concentrations than reproductive tissues. However, no systematic differences in alkaloid concentrations were observed at different plant maturity stages across the growing season. Based on the data we suggest that alkaloid allocation in different plant parts of D. nuttallianum is influenced by life history of the plant, consistent with plant defense theory. At one location, as pods mature the qualitative alkaloid composition changed through structural diversification of the alkaloids present. The ecological significance of this structural diversification awaits further exploration.     

Acetylenes from the callus of Panax ginseng

Two new polyacetylenes were isolated from dried callus of Panax ginseng. The structures of the polyacetylenes were confirmed as heptadeca-3-oxo-4,6-diyne-9,10-diol and its dihydro derivative by their IR, ¹H NMR, ¹³C NMR and mass spectra, and some chemical reactions. The new acetylenes exhibited growth inhibition against Yoshida sarcoma cells in tissue culture.