Neoflavonoids and the cinnamylphenol kuhlmannistyrene from Machaerium kuhlmannii and M. Nictitans

The trunkwood of Machaerium kuhlmannii contains methyl palmitate, 3-O-acetyloleanolic acid and sitosterol; the benzene derivatives 2,3-dimethoxyphenol, 2,6-dimethoxyphenol, 2-hydroxy-3-methoxyphenol, 2,3-dimethoxybenzaldehyde and methyl 3-(2-hydroxy-4-methoxyphenyl)-propionate; the isoflavonoids formononetin and (6aS,11aS)-medicarpin; the neoflavonoids (R)-3,4-dimethoxydalbergione, (R)-3,4-dimethoxydalbergiquinol, kuhlmanniquinol [(R)-3-(4-hydroxyphenyl)-3-(5-hydroxy-2,3,4-trimethoxyphenyl)-propene], dalbergin, kuhlmannin (6-hydroxy-7,8-dimethoxy-4-phenylcoumarin) and kuhlmannene (6-hydroxy-7,8-dimethoxy-4-phenylchrom-3-ene), as well as the cinnamylphenol kuhlmannistyrene [Z-1-(5-hydroxy-2,3,4-trimethoxybenzyl)-2-(2-hydroxyphenyl)-ethylene]. Five of these compounds, in addition to (R)-4′-hydroxy-3,4-dimethoxydalbergione, were also isolated from a trunkwood extract of M. nictitans. Structural assignments were confirmed by chemical interconversion and by the synthesis of (±)-kuhlmanniquinol.     

Light-dependent rubidium transport in intact Halobacterium halobium cells

The uptake of rubidium in intact Halobacterium halobium cells was followed, and found to be light-dependent. The exchange process is slow, the steady-state rate of ⁸⁶Rb⁺/Rb⁺ exchange being given by k = 6.3 · 10^?4 min^?1. Starved cells exhibited a faster rate than unstarved cells. The influx of ⁸⁶Rb⁺ was almost completely blocked in the presence of proton conductors (CCCP, FCCP, and SF 6847), and was sensitive to the presence of the permeant cation TPMP⁺. Valinomycin very slightly increased the rate of uptake, while 1 · 10^?6 M nigericin showed significant inhibition. On the other hand, release of ⁸⁶Rb⁺ was not light-dependent, although still affected by uncouplers, TPMP⁺, and nigericin. These experimental observations may be explained in terms of a passive flux driven by an electrical potential difference, and influenced by positive isotope interaction within the membrane. In carefully matched influx-efflux studies, the extent of the positive isotope interaction was measured. Using the formal treatment of Kedem and Essig, the ratio (exchange resistance)/(resistance to net flow) for ⁸⁶Rb⁺ was found to be 1.7.     

Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors

Using artificial electron donors and acceptors, it is shown here that the major HCO₃^? effect in the Hill reaction is after the “primary” electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acceptor Q^? to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q^?, show no significant bicarbonate stimulation of electron flow. However, a 6–7-fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.     

Alkylation of cytosine and 5-hydroxymethyl-cytosine by methyl methanesulphonate and N-methyl-N-nitrosourea: its relevance to mutagenesis.

The reaction of cytosine and 5-hydroxymethyl-cytosine (OHMeCyt) with a variety of monofunctional alkylating agents has been investigated to evaluate further the possible role of cytosine alkylation in mutagenesis and the possibility that the immunity of T-even phages to mutation by methyl methanesulphonate (MMS) was due to the unreactivity of OHMeCyt towards this agent. Both cytosine and OHMeCyt reacted equally well with the methylating agents MMS and N-methyl-N-nitrosourea (MNU) affording 6% and less than 1% respectively of the 3-substituted derivative. No product was isolated following subjection of the bases to reaction with ethyl methane-sulphonate (EMS), N-ethyl-N-nitrosourea (ENU) or iso-propyl methane-sulphonate (iPMS).     

Investigation of human erythrocyte ghost membranes with intramolecular excimer probes

Human erythrocyte ghost membranes have been investigated using two intramolecular excimer probes, di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether. Values for the viscosity of the direct probe environment in the ghost membranes range from 76 cP at 37°C to 570 cP at 5°C, as reported for di(1-pyrenyl)propane, with liquid paraffin as the reference solvent. For the activation energy of the excimer formation process, determined here mainly by the viscosity of the medium, a value of 37 kJ/mol is obtained. The other probe molecule reports a higher local viscosity, 133 cP at 37°C, as well as a higher activation energy of excimer formation, 54 kJ/mol. Neither thermotropic phase transitions nor temperature hysteresis effects are observed within the temperature range (0 to 40°C) studied. From the vibrational structure of the fluorescence spectrum of di(1-pyrenylmethyl) ether, a polarity of the probe environment close to that of hexanol ($\text{? = 13.3}$) results for the erythrocyte ghost membranes. The polarity measured in egg phosphatidylcholine membranes and in multibilayers of dimyristoylphosphatidylcholine is slightly larger, comparable to that of butanol ($\text{? = 17.5}$), whereas a polarity comparable to that of methanol ($\text{? = 32.7}$) is observed for aqueous micellar solutions of sodium dodecyl sulphate. Further, from the wavelength shifts in the absorption spectrum of di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether, the polarizability of the probe surroundings can be determined, leading to a surprisingly high value for the apparent refractive index. This is attributed to a high local density of the direct environment of the probe, for which a location between the membrane/water interface and the unpolar bilayer mid-plane is deduced.     

Ca2+ and calmodulin antagonists inhibit the proton gradients associated with non-cyclic and cyclic photophosphorylation in spinach chloroplasts

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.     

Synthesis of charged amphipathic nitroxide lipid spin labels and an example of their application in membrane studies

The synthesis of a series of amphipathic nitroxide lipid spin labels is reported. Thus, 12-proxylhexadecanol has been converted into the versatile fatty acid spin label 14-proxylstearic acid. This substance was used to prepare 14-proxylstearyltrimethylammonium methanesulfonate, a positively charged label, and 14-proxylstearylmethyl phosphate sodium salt, a negatively charged label. Also prepared in the doxyl series were quaternary ammonium salts derived from 16-doxyl- and 7-doxylstearic acid. The positively charged and negatively charged proxyl labels were used in a preliminary experiment to investigate the role of charge in their interaction with reconstituted cytochrome oxidase. The average binding affinity of the negatively charged label is approximately 2-fold higher than that of the positively charged label at pH 7.4. At pH 5.5 the average relative affinity for negatively charged label is about 3.5-fold higher than that of positively charged label, suggesting that the ionizable group(s) on the protein can interact with the lipid headgroup.     

Effects of chilling on tomato fruit texture

The effects of chilling on tomato ( Lycopersicon esculentum Mill cv. Caruso) texture were investigated using fruit stored at 22°C (nonchilled) or 5°C (chilled) for 28 days. or at 5°C for 15 days before transfer to 22°C to facilitate ripening during and additional 13 days (prechilled). Prechilled fruit exhibited symptoms of slight chilling injury, i.e. development of mealiness, accelerated softening relative to that of nonchilled fruit and nonuniform surface colour development. The firmness of all fruit decreased during ripening and chilled storage when measured by flat plate compression and puncture, especially during the early stages of ripening of nonchilled and prechilled fruit. The compression firmness of pericarp tissue similarly decreased during ripening of nonchilled and prechilled fruit, but was maintained during chilling. Total moisture content (ca 94%) of tissue, uronide content (32-35% w/w) and extracted β-galactosidase activity did not differ significantly ( P > 0.05) among fruit during ripening and chilled storage. The degree of uronide methyl esterification in ethanol-insoluble solids prepared from pericarp tissue (EIS) was relatively low for all fruit. i.e. <40%. EIS from which greater levels of pectinesterase were extracted (i.e. nonchilled>chilled>prechilled) exhibited decreased levels of uronide methyl esterification. Markedly elevated levels of β-glucosidase activity were extracted from prechilled EIS. Total polygalacturonase activity (mainly as PGI) and autolysis of enzyme-extracted EIS were inversely correlated ( P ≤ 0.05) only with the loss of nonchilled fruit and tissue firmness and prechilled fruit firmness. Results suggest a possible role for β-glucosidase in textural changes of prechilled fruit and tissue (e.g. loss of firmness, development of mealiness) and also implicate loss of skin strength in the softening of whole fruit during chilling.     

Enantioselective hydrolysis of racemic methyl jasmonate using microorganisms and isolated enzymes

A variety of microorganisms were used to hydrolyze racemic methyl jasmonate [I] with varying degrees of enantioselectivity. The fungi tested included species from the genera Aspergillus, Penicillium, and Talaromyces. All fungi tested showed a preference for the [1S,2S(Z)]-(+)-isomer. The yeasts Saccharomyces cerevisiae and Candida albicans showed no activity. A number of bacterial genera were also tested. No activity could be shown for members of the genera Bacillus, Pseudomonas, Escherichia, Nocardia, and Thermoactinomyces. Hydrolytic activity was found in the genera Streptomyces and Mycobacterium. S. henetus showed the same enantioselectivity as the fungi, while M. phlei hydrolyzed the [1R,2R(Z)]-(−)-isomer preferentially. A number of isolated enzymes were also screened for activity. Varying degrees of hydrolytic activity and enantioselectivity were found.     

Dissolved and suspended mercury. species in the Wabigoon River (Ontario,Canada): seasonal and regional variations

Seasonal and regional variations in the speciation, sediment-water partitioning, and dynamics of mercury (Hg) were studied at selected sites along the Hg-polluted Wabigoon River, and at unpolluted headwater and tributary sites, during April–September, 1979. ‘Dissolved’ and ‘particulate’ forms of Hg in the water were separated by continuous-flow centrifugation in the field. The Hg and other pollutants such as wood chips and salt had been discharged from a chlor-alkali plant and paper mill at Dryden, Ontario. Concentrations and loadings of particulate methyl mercury (CH₃Hg⁺) and total particulate Hg (and loadings of total ‘dissolved’ Hg) were greatest during the spring flood (April-May) owing to accelerated resuspension and transport of sediments. Concentrations of ‘dissolved’ CH₃Hg⁺, however, were highest in the summer (July–September), probably reflecting stimulation of microbial methylating activity by elevated temperatures, together with factors such as reduced levels of metal-scavenging particulates and minimal dilution by runoff. Total dissolved Hg concentrations were relatively high in September at polluted sites only, possibly because of desorption from sediments due to elevated concentrations of Cl⁻ ions. Loadings of dissolved CH₃Hg⁺ tended to be high in the summer but were generally depressed (suggesting sorption by suspended particles) during the major spring-flood episode in May. During July–August dissolved CH₃Hg⁺ was a function of total dissolved Hg, suggesting rapid biomethylation of desorbed inorganic Hg; but in general dissolved and suspended CH₃Hg⁺ levels depended on environmental variables and were unrelated to total Hg concentrations. In the summer only, total dissolved Hg was a function of dissolved Cl⁻. Hg species in particulates were associated with sulfides, hydrated Fe and Mn oxides, organic matter (notably high molecular weight humic and humic-Fe components), and selenium (Se); but CH₃Hg⁺ and total Hg differed in their specific preferences for binding agents, implying that binding sites discriminate between CH₃Hg⁺ and Hg²⁺ ions. CH₃Hg⁺ was associated with sulfide and (in the spring only) with Fe oxides, whereas total Hg was associated with organic matter and Se and with DTPA- and NaOH-extractable Fe in the spring but with Mn oxide and NaOH-extractable organics in the summer. Sulfides were most abundant in May, indicating that they were eroded from bottom sediments, but Fe and Mn oxides were most abundant in the summer, probably owing to activities of filamentous iron bacteria and other micro-organisms. Particulate Hg was 98–100% nonextractable by mild solvents such as Ca acetate, CaCl₂, dilute acetic acid, and (at polluted sites only) DTPA solutions, suggesting that the particulate Hg mobilized in the spring may not be readily available to organisms; association with Se and high molecular weight humic matter also supports this hypothesis. Hg probably becomes more bio-available in the summer, as suggested by the upsurge in dissolved CH₃Hg⁺ and total dissolved Hg levels, and by increases in the solubility of particulate Hg in acetic acid, DTPA, H₂O₂, and NaOH solutions, as well as an increase in the relative importance of lower molecular weight fractions of NaOH-extractable Hg (in September). Regional variations in Hg speciation and partitioning reflected a gradient in sediment composition from wood chips near Dryden to silt-clay mud further downstream. Hg in silt-clay mud relatively far (> 35 km) downstream from the source of pollution or in unpolluted areas appeared to be more readily solubilized by Cl⁻ ions or chelators such as DTPA, more readily methylated (as indicated by downstream increases in dissolved CH₃Hg⁺ levels and CH₃Hg⁺/total Hg ratios), and was to a greater degree organically bound (H₂O₂-extractable), and thus was probably more bio-available, than Hg in wood-chip deposits. Possible explanations include weaker binding of Hg by the mud, the more finely divided state of the mud, and improved microbial growth at lower concentrations of toxic pollutants. Owing to enrichment in sulfides and Fe oxides, resuspended wood-chip sediments were especially efficient scavengers of CH₃Hg⁺. The results indicate that in any pollution abatement plan aimed at lowering the Hg levels in the biota of lakes fed by the Wabigoon River, immobilization, removal, or detoxification of dissolved as well as particulate forms of Hg in the river would probably have to be considered. Possibly, Hg species could be ‘scrubbed’ from the river water by increasing the suspended load and by sedimentation and treatment with Hg-binding agents in special receiving basins.