Phloem Mobility of Xenobiotics: II. Bioassay Testing of the Unified Mathematical Model

Two bioassays were used to test phloem mobility of selected xenobiotic compounds: (a) excised bean leaf assay; (b) rooted bean leaf assay. Compounds assayed were N-alkylpyridiniums with systematic variation in octanol-water partition coefficients (log K_ow), substituted benzoic acids with about the same log K_ow value but variable acidities. Results of the assays strongly conform, quantitatively, to the predictions of the unified mathematical model. Results also indicate that the membrane permeability value of a compound, which depends directly on log K_ow value, is the overriding factor in determining phloem mobility. When the weak acid functionality of a compound confers increased phloem mobility, it does so principally by making the log K_ow value, and consequently the membrane permeability of the compound more optimal.     

Influence of Phenolic Acids on Ion Uptake: IV. Depolarization of Membrane Potentials

The membrane potentials of aged, excised barley (Hordeum vulgare L.) root cells were rapidly depolarized by the addition of salicylic acid (o-hydroxybenzoic acid) to the buffered medium bathing root segments. Initial values for membrane potentials were restored very slowly (within 100 minutes) by replacing the phenolic solution by phenolic-free buffer. Several other naturally occurring benzoic and cinnamic acids depolarized cell membrane potentials. The cinnamic acids consistently caused a greater depolarization than the correspondingly substituted benzoic acids. A strong positive correlation was found between the depolarization values (ΔE) for the benzoic acids and their lipid solubilities. This study supports the hypothesis that the inhibition of ion uptake brought about by naturally occurring phenolic acids is caused by a generalized increase in membrane permeability to inorganic ions.     

Quantitative Analysis of the Modes of Growth Inhibition by Weak Organic Acids in Saccharomyces cerevisiae

Weak organic acids are naturally occurring compounds that are commercially used as preservatives in the food and beverage industries. They extend the shelf life of food products by inhibiting microbial growth. There are a number of theories that explain the antifungal properties of these weak acids, but the exact mechanism is still unknown. We set out to quantitatively determine the contributions of various mechanisms of antifungal activity of these weak acids, as well as the mechanisms that yeast uses to counteract their effects. We analyzed the effects of four weak organic acids differing in lipophilicity (sorbic, benzoic, propionic, and acetic acids) on growth and intracellular pH (pH_i) in Saccharomyces cerevisiae. Although lipophilicity of the acids correlated with the rate of acidification of the cytosol, our data confirmed that not initial acidification, but rather the cell''s ability to restore pH_i, was a determinant for growth inhibition. This pH_i recovery in turn depended on the nature of the organic anion. We identified long-term acidification as the major cause of growth inhibition under acetic acid stress. Restoration of pH_i, and consequently growth rate, in the presence of this weak acid required the full activity of the plasma membrane ATPase Pma1p. Surprisingly, the proposed anion export pump Pdr12p was shown to play an important role in the ability of yeast cells to restore the pH_i upon lipophilic (sorbic and benzoic) acid stress, probably through a charge interaction of anion and proton transport.     

Microbial Transformation of Esters of Chlorinated Carboxylic Acids

Two groups of compounds were selected for microbial transformation studies. In the first group were carboxylic acid esters having a fixed aromatic moiety and an increasing length of the alkyl component. Ethyl esters of chlorine-substituted carboxylic acids were in the second group. Microorganisms from environmental waters and a pure culture of Pseudomonas putida U were used. The bacterial populations were monitored by plate counts, and disappearance of the parent compound was followed by gas-liquid chromatography as a function of time. The products of microbial hydrolysis were the respective carboxylic acids. Octanol-water partition coefficients (K_ow) for the compounds were measured. These values spanned three orders of magnitude, whereas microbial transformation rate constants (k_b) varied only 50-fold. The microbial rate constants of the carboxylic acid esters with a fixed aromatic moiety increased with an increasing length of alkyl substituents. The regression coefficient for the linear relationships between log k_b and log K_ow was high for group 1 compounds, indicating that these parameters correlated well. The regression coefficient for the linear relationships for group 2 compounds, however, was low, indicating that these parameters correlated poorly.     

Permeability of Red Cell Membranes to Small Hydrophilic and Lipophilic Solutes

The permeability coefficients of a series of amides, ureas, and diols have been measured on red cells of man and dog using the minimum volume method of Sha'afi et al. When the molecules are grouped according to their ether-water partition coefficients, k_ether, the behavior of the hydrophilic molecules, with k_ether less than water, is different from that of the lipophilic molecules, characterized by k_ether greater than water. The rate of permeation of the hydrophilic molecules through an aqueous pathway is determined by the molar volume, a parameter in which the geometrical measure of molecular volume is modified by hydrogen-bonding ability. This indicates the importance of chemical interactions within the aqueous path. The permeation of the lipophilic molecules is determined in the first instance by k_ether, taken as a measure of the ease with which the molecule can escape from its aqueous environment. Within the membrane, lipophilic permeability is modified both by steric factors and by the formation of hydrogen bonds with membrane components. These data allow one to infer that lipid-soluble molecules travel through an organized structure within the lipid membrane and come into contact with polar moieties.     

Influence of Phenolic Acids upon Ion Uptake: III. INHIBITION OF POTASSIUM ABSOBPTION

The influence of 12 naturally occurring phenolic acids on theuptake of potassium by barley (Hordeum vulgare L. cv. Karlsberg)was examined using ⁸⁶Rb-labelled potassium solutions. Withoutexception, all compounds tested, including both cinnamic acidand benzoic acid derivatives, caused a significant reductionof uptake. In addition, the inhibitory capacities of 15 variously substitutedbenzoic acids were determined over a range of different concentrationsof inhibitors. For each of these compounds an inhibition constant(K_i), defined as the concentration required to produce a 50per cent reduction of uptake, was determined. The K_i valueswere found to be strongly correlated with the octanol-waterpartition coefficients of the compounds under examination. Itis clear from this study and previous work upon phosphate uptakethat phenolic acids exert a generalized inhibitory effect uponactive ion-uptake which is almost certainly mediated throughreversible alterations in membrane permeability.     

Effect of weak inorganic acids and lower carboxylic acids on the conductivity of bilayer lipid membranes

The ability of weak inorganic acids (H₂S, HCN) and lower carboxylic acids to interact with bilayer lipid membranes, change their conductivity, and act as protonophores has been investigated. The mechanism of changes in membrane conductivity was studied. Factors influencing the interaction of acids with model lipid membranes were determined. Maximum changes in conductivity were observed at pH values equal to the dissociation constants of weak acids and correlated with the octanol-water partition coefficients.     

Structural features required for inhibition of cyanide-insensitive electron transfer by propyl gallate

A series of 19 structural analogs of propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester) were tested for their ability to inhibit the cyanide-insensitive, electron transfer pathway in isolated mung bean mitochondria. The results indicate that the trihydroxy substituent, not the ester, of propyl gallate is the structural feature of the molecule required to produce inhibition. Further, only one OH group, if it is located para to the ester moiety, will bring about specific inhibition. Of the compounds which contained the appropriate hydroxyl group, the lower the pK_α of the hydroxyl group, the lower the observed inhibition constant (K_i′) for blocking the alternative pathway. Even though the observed K_i′ values varied over two orders of magnitude for the compounds tested, the calculated pH-independent, intrinsic inhibition constants (K_i) were markedly similar for all inhibitory compounds. The results indicate that a simple phenolate anion is the minimum structural feature required to observe specific inhibition of the alternative pathway and the more easily the anion can be formed, the better the observed inhibition. Similarities between the above compounds and the structural features associated with hydroxamic acids were also noted.     

A rapid assay for the binding of actinomycin to DNA.

The theory of countercurrent distribution (CCD) was reviewed and extended. The separation function for the fundamental distribution of CCD was presented in the form n = t²(αk₁+β)²/αk₁(β?1)² where n is the number of transfers, t the abscissa of the standard normal distribution, α = v_m/v₈ the phase ratio, β = k₁/k₂≥ 1 the separation factor, and k₁ the partition coefficient of the more radidly moving component; n was found to be minimal on the condition αk₁ = β. The separation function for the single withdrawal of CCD was obtained in the form N = u + 1 = t²{(αk₁ + 1)^1/2 + [β(αk₁ + β)]^1/2}²/(β ? 1)²+ 1, where N is the number of partition units. From this equation it appears that N is minimal when αk₁ = 0. Compared with the former separation functions presented in the literature, these separation functions have the advantage of giving directly the relationships among the phase ratio, the absolute partition coefficient, the separation factor, the resolution degree, and the number of transfers or partition units required. In addition, the dependencies of the elution volumes and the widths of the elution curves on α, β, and the partition coefficients were considered mathematically by means of differential calculus. The elution volumes were found to have minima at certain αk₁ values. The standard deviations, on the contrary, did not have minima in respect to αk₁. The theory presented can be used for selecting proper operating conditions while separating chemical compounds.     

An algebraic model for the kinetics of covalent enzyme inhibition at low substrate concentrations

This article describes an integrated rate equation for the time course of covalent enzyme inhibition under the conditions where the substrate concentration is significantly lower than the corresponding Michaelis constant, for example, in the Omnia assays of epidermal growth factor receptor (EGFR) kinase. The newly described method is applicable to experimental conditions where the enzyme concentration is significantly lower than the dissociation constant of the initially formed reversible enzyme–inhibitor complex (no “tight binding”). A detailed comparison with the traditionally used rate equation for covalent inhibition is presented. The two methods produce approximately identical values of the first-order inactivation rate constant (k_inact). However, the inhibition constant (K_i), and therefore also the second-order inactivation rate constant k_inact/K_i, is underestimated by the traditional method by up to an order of magnitude.     

Relationship between 1-aminocyclopropanecarboxylate malonyltransferase and D-amino acid malonyltransferase

An enzyme preparation isolated from mungbean hypocotyls catalyses the malonyl-CoA-dependent N-malonylation of 1-aminocyclopropane-1-carboxylic acid (ACC), D-phenylalanine (Phe), D-methionine and 2-aminoisobutyric acid with K_m values of 0.15, 0.8, 3.4 and 5.1 mM, respectively L-enantiomers of Phe and methionine were, however, not malonylated by the enzyme preparation. When ACC was tested on D-Phe malonyltransferase activity, or when D-Phe was tested on ACC malonyltransferase activity, these compounds exhibited competitive inhibition kinetics with K_i values similar to their respective K_m values. Such a relationship suggests that malonylations of ACC and D-amino acids are catalysed by the same enzyme. This view was further supported by the observations that the ratio ACC-D-Phe malonyltransferase activities remained constant throughout various fractionation steps and both enzyme activities were inhibited similarly by various sulphydryl reagents and 1-aminocycloalkane-1-carboxylic acids.     

Novel PDE5 inhibitors derived from rutaecarpine for the treatment of Alzheimer’s disease

A series of novel rutaecarpine derivatives were synthesized and subjected to pharmacological evaluation as PDE5 inhibitors. The structure–activity relationships were discussed and their binding conformation and simultaneous interaction mode were further clarified by the molecular docking studies. Among the 25 analogues, compound 8i exhibited most potent PDE5 inhibition with IC₅₀ values about 0.086 μM. Moreover, it also produced good effects against scopolamine-induced cognitive impairment in vivo. These results might bring significant instruction for further development of potential PDE5 inhibitors derived from rutaecarpine as a good candidate drug for the treatment of Alzheimer’s disease.     

Phloem mobility of xenobiotics: tabular review of physicochemical properties governing the output of the Kleier model

The Kleier model of phloem-mobility of xenobiotics combines the intermediate permeability hypothesis with the acid trap mechanism for weak acids. The output of the model is dependent on the lipophilicity of a compound, for which octanol/water partition coefficients (log K_ow) have been used as a measure. The membrane permeability of xenobiotics is predicted from these partition coefficients, and the nature of the sieve tube membranes has been modelled using regressions derived from Nitella or potato permeability data. A wide range of log K_ow values for herbicides, fungicides, insecticides and experimental compounds (400) have been tabulated along with the model output for various membrane parameters. The application of the model is in broad agreement with literature and experimental observations on many of the known phloem mobile herbicides and predicts low phloem mobility for the fungicides and insecticides considered here, again in agreement with the literature. The behaviour of herbicides representative of the main chemical families and modes of action are reviewed, along with examples of the few phloem-mobile fungicides and insecticides identified.Abbreviations K_ow octanol-water partition coefficient - pK_a –log₁₀ acid dissociation constant - C_f Concentration factor - P membrane permeability     

Gallic acid oxidation by turnip peroxidase

Both ellagic and gallic acids non competitively inhibited guaiacol oxidation by turnip peroxidase. The K_i values were 3 and 26 μm for ellagic and gallic acid respectively. Enzymatic oxidation of gallic acid by the isolated major turnip peroxidase was characterized with respect to spectral behaviour, affinity constant and pH effect. The K_m for H₂O₂ and gallic acid are 2.5 and 8.0 mM for turnip peroxidase. The pH optimum for gallic acid oxidation is about 6.5 and the rate constant k₄ decreased with the increase of pH in presence of both guaiacol and Gallic acid. When the gallic acid oxidation products were subjected to chromatographic analysis, it was found to be converted mainly to ellagic and an unknown quinone.     

Kinetic study on the inhibition of xanthine oxidase by acylated derivatives of flavonoids synthesised enzymatically

Studies have reported that flavonoids inhibit xanthine oxidase (XO) activity; however, poor solubility and stability in lipophilic media limit their bioavailability and applications. This study evaluated the kinetic parameters of XO inhibition and partition coefficients of flavonoid esters biosynthesised from hesperidin, naringin, and rutin via enzymatic acylation with hexanoic, octanoic, decanoic, lauric, and oleic acids catalysed by Candida antarctica lipase B (CALB). Quantitative determination by ultra-high performance liquid chromatography–mass spectrometry (UHPLC–MS) showed higher conversion yields (%) for naringin and rutin esters using acyl donors with 8C and 10C. Rutin decanoate had higher partition coefficients (0.95), and naringin octanoate and naringin decanoate showed greater inhibitory effects on XO (IC₅₀ of 110.35 and 117.51?μM, respectively). Kinetic analysis showed significant differences (p?K_m values, whereas the values for V_max were the same, implying the competitive nature of XO inhibition.     

Adenosine metabolism: Modification by S-adenosylhomocysteine and 5′-methylthioadenosine

To elucidate potential toxic properties of S-adenosylhomocysteine and 5′-methylthioadenosine, we have examined the inhibitory properties of these compounds upon enzymes involved with adenosine metabolism. S-Adenosylhomocysteine, but not S-adenosylmethionine, was a noncompetitive inhibitor of adenosine kinase with K_i values ranging from 100 to 400 μm. Methylthioadenosine competitively inhibited adenosine kinase with variable adenosine below 1 μm with a K_i of 120 μm, increased adenosine kinase activity when the adenosine concentration exceeded 2 μm, and did not appear to be a substrate for adenosine kinase. Methylthioadenosine inactivated S-adenosylhomocysteine hydrolase from erythrocytes, B-lymphoblasts, and T-lymphoblasts with K_i values ranging from 65 to 117 μm and “k₂” from 0.30 to 0.55 min^?1. Adenosine deaminase was not inhibited by 5′-methylthioadenosine up to 1000 μm. To clarify how 5′-methylthioadenosine might accumulate, 5′-methylthioadenosine phosphorylase was evaluated. This enzyme was not blocked by up to 500 μm adenosine, deoxyadenosine, S-adenosylhomocysteine, or S-adenosylmethionine and was not decreased in erythrocytes from patients with adenosine deaminase deficiency, purine nucleoside phosphorylase deficiency, or hypogammaglobulinemia. These observations suggest that the inhibitory properties of 5′-methylthioadenosine upon adenosine kinase and S-adenosylhomocysteine hydrolase may contribute to the toxicity of the exogenously added compound. The toxicity resulting from S-adenosylhomocysteine accumulation intracellularly may be related to adenosine kinase inhibition in addition to disruption of transmethylation reactions.     

Stimulation of gallbladder fluid and electrolyte absorption by butyrate

Summary Gallbladder fluid and electrolyte transport was investigatedin vitro. In guinea pig gallbladder, equimolar substitution of acetate, propionate, butyrate or valerate for HCO₃ was increasingly effective in stimulating fluid absorption. The stimulatory potency of these compounds was a function of their chloroform water partition coefficients. The stimulatory effects of the isomers isobutyrate and isovalerate were less than predicted from their partition coefficients. Acidification of the gallbladder lumen, however, was strictly dependent on the partition coefficients for all of the above fatty acids. Unidirectional²²Na fluxes were measured in rabbit and guinea pig gallbladders under short-circuit conditions. In the presence of butyrate stimulation of net Na flux was due entirely to an increase in the mucosal-to-serosal Na flux. Stimulation by butyrate was abolished by its omission from the mucosal bathing solution. The transepithelial electrical potential difference in both rabbit and guinea pig gallbladder became more lumen positive following mucosal but not serosal addition of butyrate. Net¹⁴C-butyrate fluxes were too small to account for stimulation of Na absorption in either species. Butyrate stimulation of Na absorption by guinea pig gallbladder was abolished by increasing the bathing pH from 7.4 to 8.1. Tris buffer (25mm) partially inhibited butyrate-dependent gallbladder fluid absorption by rabbit and guinea pig at pH 6.4 and 7.0, respectively, and completely at pH 8.4. These results reveal a marked similarity between butyrate and HCO₃ stimulation of gallbladder NaCl and fluid absorption. The results are best explained by a double ion-exchange model, in which butyrate (HCO₃) in the mucosal solution acts to maintain the intracellular supply of H⁺ and butyrate (HCO₃) for countertransport of Na and Cl, respectively.     

Species Differences in the Selective Inhibition of Monoamine Oxidase (1-methyl-2-phenylethyl)hydrazine and its Potentiation by Cyanide

The potentiating effects of cyanide on the inhibition of rat liver mitochondrial monoamine oxidase-A & B and of ox liver mitochondrial MAO-B by pheniprazine [(1-methyl-2-phenylethyl)hydrazine] has been studied. Pheniprazine was shown to behave as a mechanism-based MAO inhibitor. For rat liver MAO-B, the initial non-covalent step was characterized by dissociation constant (K _i) of 2450 nM and the first-order rate constant (k ₊₂) for the covalent adduct formation was 0.16 min⁻¹. As a reversible inhibitor it was selective towards rat liver MAO-A (K _i = 420 nM) but the rate of irreversible inhibition of that enzyme was considerably slower (k ₊₂ = 0.06 min⁻¹). MAO-B from ox liver more closely resembled MAO-A from the rat in sensitivity to reversible inhibition by pheniprazine (K _i = 450 nm) but it was closer to rat liver MAO-B in rate of irreversible inhibition (k ₊₂ = 0.29 min⁻¹). The K _i values were significantly decreased in the presence of KCN but there was little effect on the k ₊₂ values. However, sensitivities of the different enzymes to KCN varied widely and considerably higher concentrations of KCN were required for this effect to be apparent with the rat liver mitochondrial MAO-A than with MAO-B from rat and ox liver. The kinetic behaviour of cyanide activation was consistent with partial (non-essential) competitive activation in all cases. Special issue dedicated to Dr. Moussa Youdim.     

Ivermectin and curcumin cause plasma membrane rigidity in Leishmania amazonensis due to oxidative stress

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to study the mechanisms of action of ivermectin and curcumin against Leishmania (L.) amazonensis promastigotes. EPR spectra showed that treatment of the parasites with both compounds results in plasma membrane rigidity due to oxidative processes. With the IC₅₀ and EPR measurements for assays using different parasite concentrations, estimations could be made for the membrane-water partition coefficient (K_M/W), and the concentration of the compound in the membrane (c_m50) and in the aqueous phase (c_w50), which inhibits cell growth by 50%. The K_M/W values indicated that ivermectin has a greater affinity than curcumin for the parasite membrane. Therefore, the activity of ivermectin was higher for experiments with low cell concentrations, but for concentrations greater than 1.5 × 10⁸ parasites/mL the compounds did not show significantly different results. The c_m50 values indicated that the concentration of compound in the membrane leading to growth inhibition or membrane alteration is approximately 1 M for both ivermectin and curcumin. This high membrane concentration suggests that many ivermectin molecules per chlorine channel are needed to cause an increase in chlorine ion influx.     

Synthesis and antiglycation potentials of bergenin derivatives

Bergenin (1), major bioactive compound isolated from methanolic extract of Mallotus philippinensis, displayed moderate AGE inhibition activity (IC₅₀ = 186.73 μM). A series of derivatives of bergenin (3a-k) containing variety of aromatic acids were synthesized under mild conditions by modification of sugar part. Selective esterification of hydroxyl groups on the sugar part enhanced antiglycation potential of bergenin. Compounds 3j and 3k exhibited potent antiglycation activity with the IC₅₀ values of 60.75 and 12.28 μM, respectively.