Cytotoxic Activity of Dammarane Triterpenoids from Birch Leaves

Cytotoxic activity of dammarane triterpenoids isolated from birch leaves was studied. These substances differ from the native ginseng genin (20(S)-protopanaxadiol) by the number, location, or configuration of OH-groups. Using fertilized egg cells of sea urchin Strongylocentrotus intermedius we demonstrate that the orientation of C-3 OH-group has no effect on cytotoxic activity of triterpenoids as well as a higher activity of a triterpenoid with 3,12-OH as compared to a C-3 ketone but lower activity as compared to a triterpenoid with 3,17-OH. Depending on the number of OH-groups the cytotoxic activity of triterpenoids decreases in the row: tetraol > pentaol > triol. Dammar-24-ene-3,12,17,20(S)-tetraol (compound IV) is cytotoxic for the Ehrlich ascite carcinoma cells and this effect is additive to cytotoxic activity of anthracycline antibiotic carminomycin in vitro. Compound IV changes the permeability and microviscosity of the tumor cell membranes.     

δ13C values of grass species collected in the northern Sahara desert

Summary ¹³C values were measured for 45 Poaceae species collected in the northern Sahara desert, at the foot of the Saharan Atlas. The results indicate a clear relationship between carbon isotope discrimination and phytogeographical distribution of the grasses. Mediterranean species predominantly had ¹³C values indicating the C₃ pathway of photosynthesis. By contrast, nearly all species belonging to the Saharo-Arabian and /or Sudanian group showed a C₄ like carbon isotope composition. Leaf material of two species, Lygeum spartum and Stipa tenacissima, had ¹³C values in the region of-20, i.e. intermediate between the mean ¹³C values of C₃ and C₄ plants. However, additional speciments of both these grasses obtained from a different source (herbarium of the Hebrew University, Jerusalem) yielded a C₃ like carbon isotope composition.     

Comparative Study of Effects of Various Sterols and Triterpenoids on Permeability of Model Lipid Membranes

Using permeability to labeled glucose as a criterion of stability for liposomal membranes, a comparative study on stabilizing properties of different sterols and triterpenes in phospholipid bilayer has been carried out as well as on structural peculiarities of sterols responsible for membranolytic properties of cucumarioside G₁ from the cucumaria Eupentacta fraudatris. Stabilizing action of the studied sterols and triterpenoides incorporated in the bilayer decreases in the following order: cholesterol sulfate > cholesterol > ⁵-sterols > -sitosterol > ergosterols > ⁷-sterols > epicholesterol > pregnane > androstane > coprosterol > 14-methylcholest-9(11)-en-3-ol > 4, 14-dimethylcholest-9(11)-en-3-ol > holothurinogenin A₁ > glucoside of cholesterol > -xylosidase of ⁷-sterols > betulin > protopanaxatriol > phosphatidylcholine liposomes without sterol > protopanaxadiol > oleanolic acid. Sterol-dependent membranolytic cucumarioside G₁ practically loses its ability to increase permeability of phospholipid membranes containing sterols obtained from this holothuria as well as coprosterol, epicholestrol, sulfated and glycosylated forms of sterols. The obtained results confirm the sterol hypothesis of the mechanism of membranotropic action of holothuria glycosides and of resistance to them of holothuria cell membranes.     

A theoretical investigation of cytotoxic activity of celastroid triterpenoids

Quantum chemical calculations at the B3LYP/6-31G* level of theory have been carried out on 20 celastroid triterpenoids to obtain a set of molecular electronic properties and to correlate these with cytotoxic activities. The cytotoxic activities of these compounds can be roughly correlated with electronic effects related to nucleophilic addition to C(6) of the compounds: The energies of the frontier molecular orbitals (E _HOMO and E _LUMO), the HOMO-LUMO energy gap, the dipole moment, the charge on C(6), and the electrophilicity on C(6). Figure LUMO of Pristimerin.     

Use of 4-Thiouridine and 4-Thiothymidine in Studies on Pyrimidine Nucleoside Phosphorylases

The new substrates 4-thiouridine and 4-thiothymidine were proposed for spectrophotometric measurement of the activity of uridine (UP) and thymidine (TP) phosphorylases. At pH 7.5, 4-thiouridine has an absorbance maximum at 330 nm, and the difference in extinction coefficient () between 4-thiouridine and 4-thiouracil is 3000 ^–1cm^–1. 4-Thiouridine proved to be a good substrate for UP: the Michaelis ( ) and catalytic (k _cat) constants were estimated respectively at 130 M and 49 s^–1 at 25°C. Even a greater (5000 M^–1cm^–1 at 336 nm) was observed for the 4-thiothymidine/4-thiothymine pair.     

Comparison of Ni-sensitive and Ni-resistant strains of Nostoc muscorum

A wild-type Ni-sensitive (Ni^s) strain of Nostoc muscorum ISU spontaneously yielded mutants resistant to inhibition by 40 M Ni with a frequency of about 10^-7. A Ni-resistant (Ni^r) mutant was deficient in the activities of urease and uptake hydrogenase. Cellular Ni uptake in the Ni^s strain was dependent on concentration (40 to 120 M) and time (0 to 30 min) (V_max=0.51 nmol/g protein.min; K_m=92 M). The Ni bioconcentration factor for such cells ranged between 0.95×10³ and 1.89×10³. Ni uptake in spheroplast preparations from Ni^s cells followed almost the same trend as intact cells except that the bioconcentration factor was slightly less [(0.82 to 1.39)×10³]. In contrast, Ni uptake in the Ni^r intact cells was not concentration dependent and also the uptake was saturated, even at 40 M, within 10 min. Spheroplasts from the Ni^r strain showed a Ni bioconcentration factor of 1.19×10³ compared with 4.41×10³ for intact cells. The invariably lower Ni uptake by spheroplasts was attributed to altered membrane transport properties.R.K. Asthana, A.L. Singh and S.P. Singh are with the Algal Research Laboratory, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221 005, India.     

Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor

When growth-phase cell suspension cultures of Scutellaria baicalensis were treated with 50 g of yeast elicitor preparation ml^–1, both oleanolic acid and ursolic acid transiently increased in the culture medium rather than in the cells. The maximal triterpenoid concentration was 13.7 mg l^–1 media approx. 35 h after treatment, whereas the maximum concentration was 2.1 mg l^–1 media after about 20 h following treatment with methyl jasmonate. Elicitor treatment also doubled phospholipase A₂ activity (25 pmol mg^–1 min) and the simultaneous treatment of aristolochic acid, a phospholipase A₂ inhibitor, inhibited triterpenoids accumulation as well as phospholipase A₂ activity.     

Water permeability of bilayer lipid membranes: Sterol-lipid interaction

Summary Bilayer lipid membranes were generated in an aqueous medium from synthetic, egg or plant phosphatidyl choline (PC) or from plant monogalactosyl diglyceride (MG). The water permeability of the black membranes was determined by measuring the net volume flux produced by a NaCl gradient. The osmotic permeability coefficient,P _os, was markedly affected by the number of double bonds in the fatty acid conjugates of the lipids: the greater the degree of unsaturation, the higher the value ofP _os. The temperature dependence ofP _os of the lipid membranes was studied over a range of 29 to 40°C. The experimental activation energy,E _a , estimated from the linear plots of log (P _os)versus 1/T, was significantly higher for MG membranes (17 kcal/mole) than for the various PC membranes (11 to 13 kcal/mole), probably owing to hydrogen bonding between MG and water molecules. In comparison with PC membranes, the membranes generated from PC and cholesterol (11 molar ratio) had lowerP _os but similarE _a values. Likewise, either stigmasterol or -sitosterol decreasedP _os of MG membranes, whileE _a was not affected by the sterols. MG-cholesterol membranes were specifically characterized by a unique value ofE _a (–36 kcal/mole) thus indicating temperature dependent structural changes.     

Bacillus subtilis bacteriophages SP beta c1 is a deletion mutant of SP beta

Summary The restriction fragment patterns of two mutant forms of the temperate Bacillus subtilis bacteriophage SP have been examined. The DNA of a heat-inducible mutant, SPc2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SPc⁺ DNA. The DNA of a clear-plaque mutant, SPc1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SPc1 nor SPc2 DNA is cleaved by the endonuclease HaeIII.     

Calcium-dependent protein kinase gene expression in response to physical and chemical stimuli in mungbean (Vigna radiata)

Protein kinases are important in eukaryotic signal transduction pathways. In this study we designed degenerate oligonucleotides corresponding to two conserved regions of protein kinases and using the polymerase chain reaction (PCR) have amplified a 141 bp fragment of DNA from mungbeans (Vigna radiata Rwilcz cv. Berken). Sequence analysis of the PCR products indicates that they encode several putative protein kinases with respect to their identity with other known plant protein kinases. Using one of the six fragments (CPK3-8), we isolated a 2022 bp cDNA (VrCDPK-1) from a Vigna radiata gt11 library. VrCDPK-1 has a 96 bp 5-untranslated region and a 465 bp 3-untranslated region and an open reading frame of 1461 bp. VrCDPK-1 contains all of the conserved regions commonly found in calcium dependent protein kinases (CDPK). VrCDPK-1 shares 24 to 89% sequence identity with previously reported sequences for plant CDPKs at the protein level. southern analysis revealed the presence of several copies of the CDPK gene. VrCDPK-1 expression was stimulated when mungbean cuttings were treated with CaCl₂, while treatment with MgCl₂ had no effect. We are reporting for the first time a CDPK gene in mungbean which is inducible by mechanical strain. Cuttings treated with indole-3-acetic acid (IAA) or subjected to salt stress showed an increase in VrCDPK-1 expression. There was a dramatic stimulation in VrCDPK-1 expression 6 h after cuttings were treated with cycloheximide.     

Simulation of chaotic EEG patterns with a dynamic model of the olfactory system

The main parts of the central olfactory system are the bulb (OB), anterior nucleus (AON), and prepyriform cortex (PC). Each part consists of a mass of excitatory or inhibitory neurons that is modelled in its noninteractive state by a 2nd order ordinary differential equation (ODE) having a static nonlinearity. The model is called a KO_e or a KO_t set respectively; it is evaluated in the open loop state under deep anesthesia. Interactions in waking states are represented by coupled KO sets, respectivelyKI _e (mutual excitation) andKI _i (mutual inhibition). The coupledKI _e andKI _i sets form aKII set, which suffices to represent the dynamics of theOB, AON, andPC separately. The coupling of these three structures by both excitatory and inhibitory feedback loops forms aKIII set. The solutions to this high-dimensional system ofODEs suffice to simulate the chaotic patterns of the EEG, including the normal low-level background activity, the high-level relatively coherent bursts of oscillation that accompany reception of input to the bulb, and a degenerate state of an epileptic seizure determined by a toroidal chaotic attractor. An example is given of the Ruelle-Takens-Newhouse route to chaos in the olfactory system. Due to the simplicity and generality of the elements of the model and their interconnections, the model can serve as the starting point for other neural systems that generate deterministic chaotic activity.Supported by a grant MH06686 from the National Institute of Mental Health     

Composition of soluble cuticular lipids and water permeability of cuticular membranes from Citrus leaves

Water permeability and composition of soluble cuticular lipids of isolated cuticular membranes from leaves of Citrus aurantium L. were investigated for 3 successive years. The average water permeability coefficient determined using 169 cuticular membranes was 1.09·10^–7 cm s^–1 with a standard deviation of 0.78·10^–7 cm s^–1. There were no significant differences in water permeability between years. Cuticular membranes are characterized by a great variability in water permeability both within and between years. Both water permeability of individual membranes and variability between membranes are shown to be determined by soluble cuticular lipids contained within the cuticular membranes. The soluble cuticular lipids of Citrus leaves are composed of fatty acids, primary alcohols, esters, and hydrocarbons. They occur in amounts of 9.84 g cm^–2, which represents approx. 3% of the total mass of isolated cuticular membranes. The specific weight of cuticular membranes (365.4 g cm^–1) and total amount of soluble cuticular lipids did not vary significantly between years. Significant differences were observed for the amounts and composition of the constituent classes of lipids. Six homologues comprise 86% of the fatty acids (C₁₆; C₁₈; C₁₉; C₂₁; C₂₄; C₂₆), 83% of the primary alcohols (C₂₄; C₂₆; C₂₈; C₃₀; C₃₂; C₃₄) and 88% of the esters (C₃₆; C₃₈; C₄₀; C₄₁; C₄₂; C₄₄). Eleven major homologues amount only to 62% of the total hydrocarbons (C₁₆; C₁₇; C₁₈; C₂₀; C₂₆; C₂₇; C₂₉; C₃₀; C₃₁; C₃₂; C₃₃). Variability in the composition of soluble cuticular lipids between years was much smaller than variability of water permeability and, therefore, no relation between composition of soluble cuticular lipids and water permeability could be found. It is suggested that this may be due to the fact that the lipid composition observed represents the averages of 20 to 30 membranes analyzed so that differences between individual membranes may have been leveled out.Abbreviations CM cuticular membranes - MX polymer matrix - P_d permeability coefficient for diffusion of water - SCL soluble cuticular lipids - MES morpholinoethane sulphonic acid     

Carotenoid pigments of Stigmatella aurantiaca (Myxobacterales)

Summary In this first article on the carotenoids of Myxobacterales we report on the minor carotenoids of Stigmatella aurantiaca: phytoene, phytofluene, lycopene, -carotene, 4-keto--carotene, 1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy-torulene, and 1,2,1,2-tetrahydro-1,1-dihydroxy-lycopene. These pigments account for about 10% of total carotenoids.     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.     

Influence of Triterpene Glycosides of the Holothurian Cucumaria japonica on Root Growth in Cucumis sativus L. Seedlings

We have shown that triterpene glycosides of the Far Eastern edible holothurian Cucumaria japonica (monosulfated subfractions A ₂ and A ₄, disulfated subfraction A ₆, trisulfated subfraction A ₇, and the total fraction of monosulfated glycosides including subfractions A ₀, A ₁, A ₂, and A ₄) inhibited growth of the main root of Cucumis sativus seedlings. Cucumariosides were arranged as follows according to their inhibitory activity: subfraction A ₂ (ED₅₀ = 53.1 ± 3.6 g/ml) > total fraction of monosulfated glycosides (ED₅₀ = 127.4 ± 5.8 g/ml) > subfraction A ₄ (ED₅₀ = 346.5 ± 15.2 g/ml) > subfraction A ₆ (ED₅₀ = 375.7 ± 17.2 g/ml) > subfraction A ₇ (ED₅₀ = 539.4 ± 11.5 g/ml). The average length of the main root decreased with the increase of glycoside concentrations as a result of a growing amount of seedlings with low germination capacity and the absence of seedlings with high germination capacity.     

Alnion forests in Lower Saxony (FRG), their ecological requirements,classification and position within Carici elongatae-Alnetum of Northern Central Europe

In the diluvial plain of Lower Saxony ( = Niedersachsen) two types of alder fen woods are found: alder swamp woods (alder carrs) with a more changing water table and inundations in winter, and alder spring woods without hibernal inundation and a more or less constant water table. While all alder swamp woods are Alnion forests, the alder spring woods can be devided into Alnion and Alno-Ulmion forests.All Alnion forests in the diluvial plain of Lower Saxony belong to the association Carici elongatae-Alnetum Schwick. 1933. This association can be devided into three subassociations, C. el.-Al. betuletosum Bodeux 1955, C. el.-Al. typicum Meijer Drees 1936 and C. el.-Al. cardaminetosum Meijer Drees 1936.Low pH-values and high C/N-ratios of the soil are good indicators to distinguish the Carici elongatae-Alnetum betuletosum from the other two subassociations. Humus contents has a tendency to decrease in the sequence C. el.-Al. betuletosum > C. el.-Al. typicum > C. el.-Al cardaminetosum, but is no reliable factor for classification of a single plot. Differences in annual groundwater level variations cause a floristical differentiation only within alderwoods of meso- to eutrophic sites. Here a nearly constant water table or at least no hibernal inundation leads to a Carici elongatae-Alnetum cardaminetosum, while sites which are inundated in winter carry the Carici elongatae-Alnetum typicum. On oligotrophic sites both, swamp and spring woods belong to the Carici elongatae-Alnetum betuletosum.The subdivision of the Carici elongatae-Alnetum for the whole lowlands of northern Central Europe is generally the same as for Lower Saxony. Source reference: This paper was presented as a poster with the title Alnion Forests in Lower Saxony (FRG)-Syntaxonomical position and ecological requirements on the IAVS-Symposium Forests of the World, Upsala 20.–27.8.1989.     

Cytotoxicity and inhibition of DNA topoisomerase I of polyhydroxylated triterpenoids and triterpenoid glycosides

Cytotoxicity and inhibition on human DNA topoisomerase I (TOP1) and II (TOP2) of 74 plant-originated triterpenoids and triterpenoid glycosides were investigated. The cytotoxic compounds are primarily polyhydroxylated oleananes (GI₅₀ of A549: 1.0–10.19 μM). Sixteen cytotoxic aesculiosides isolated from Aesculus pavia inhibited TOP1 catalytic activity by interacting directly with the free enzyme and preventing the formation of the DNA–TOP1 complex. Interestingly, hydrolysis of six active aesculiosides (1, 4, 6, 8, 10, and 23) lost their TOP1 activities but enhanced their cytotoxicities. None of the test compounds showed any activity against TOP2. Structure–activity relationship (SAR) investigations indicated that cytotoxic oleananes required at least one angeloyl moiety at either C-21 or C-22 but the sugar moiety at C-3 may decrease their cytotoxicities. An angeloyl or tigeloyl group at C-21 is required for oleananes to bind the free TOP1 enzyme although the type and length of acyl moiety at C-22 also affects their activity. However, sugar moiety at C-3 is necessary for their TOP1 activities.     

Tetracyclines,verapamil and nifedipine induce callose deposition at specific cell sites in Riella helicophylla

The Ca²⁺ indicator 7-chlorotetracycline has been shown to bind to a pore complex on both outer surfaces of all non-meristematic cells in the unistratose thallus of Riella (chlorotetracycline-binding surface region=CSR; Grotha, 1983, Planta 158, 473–481). Prolonged treatment of the thallus with 7-chlorotetracycline, 5-hydroxytetracycline, verapamil and desmethoxyverapamil induces the deposition of callose at the same region. The influence of various treatments on verapamil-induced CSR-callose was measured in situ by microfluorometry of aniline-blue-stained material. Callose deposition is maximal at 10^-4M verapamil or 5·10^-5M desmethoxyverapamil with 2·10^-4M Ca²⁺ or Mg²⁺ in the medium. The reaction is completely inhibited at pH 5.5 and is optimal between pH 6.5 and 7.5. The production of CSR-callose is absolutely light-dependent with callose being first visible after 30 min of light. La³⁺, ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid and amiprophosmethyl, antagonists of Ca²⁺ functions, and 2-deoxy-D-glucose suppress the verapamil induction of CSR-callose. Furthermore the ionophores A 23187, valinomycin and monensin effectively block the reaction. The deposition of CSR-callose is diminished at increasing external osmolarity and is abolished at osmotic values that stimulate plasmolysis-callose. Wounding causes the formation of wound-callose but inhibits the induction of CSR-callose in cells of the wound edge. Nifedipine increases or prolongs callose synthesis in cell plates. The Ca²⁺-channel blocker diltiazem is completely ineffective. It is suggested as a working hypothesis that verapamil-induced CSR-callose synthesis is caused by a local change in membrane permeability, possibly as a consequence of the opening of Ca²⁺ channels being involved in Golgi-vesicle mediated exocytosis (A. Kramer and H. Lehmann, 1986, Ber. Dtsch. Bot. Ges. 99, 111–121).Abbreviations APM amiprophosmethyl - APW artificial pond water - CSR chlorotetracycline-binding surface region - CTC 7-chlorotetracycline - DDG 2-deoxy-D-glucose - EGTA ethylone glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - OTC 5-hydroxytetracycline - Pipes 1,4-piperazinediethane sulfonic acid Dedicated to Professor Luise Stange on the occasion of her 60^th birthday     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.     

Effects of ovine prolactin on iodide metabolism and thyroid activity in the eel

Summary In the eel, ovine prolactin (oPrl) treatment (0.018 IU/day·g body weight), for 8 to 13 days modifies neither iodide absorption from the water nor excretion, extrathyroidal metabolism and plasma level of iodide.Thyroid activity, evaluated by epithelial cell height, radioiodine uptake and absolute iodide uptake is approximately twice that of controls. However, the amounts of total iodine, thyroxine (T₄) and triiodothyronine (T₃) in thyroid are unaltered by oPrl. Therefore, the decrease of plasma T₄ and the increase of plasma T₃, previously observed in oPrl-treated eels, do not result from a preferential thyroidal secretion of T₃, but only from a stimulation of peripheral conversion of T₄ to T₃. Furthermore, the increased thyroid activity probably originates from a decreased feedback inhibition following the fall of circulating T₄ induced by oPrl.Abbreviations oPrl ovine prolactin - T ₄ Thyroxine - T ₃ 3.5.3 triiodothyronine - TRH thyrotropin releasing hormone - TSH thyroid stimulating hormone - PBI protein bound iodine