The hypolipidemic natural product guggulsterone acts as an antagonist of the bile acid receptor

Ayurveda, the ancient Indian system of health care and medicine, has a well-organized materia medica in which plants form a dominant part. A key illustration of the exploitation of this knowledge toward the development of a modern drug is the isolation and characterization of two antihyperlipidemic compounds, Z-, and E-guggulsterone from the tree Commiphora mukul, the exudate of which has been traditionally used for mitigating lipid disorders. Here, we demonstrate that Z-guggulsterone and an analog, 80-574 currently in clinical trials, act as antagonists of the bile acid receptor (BAR), a member of the intracellular receptor superfamily. These compounds antagonize the activity of BAR in vitro, and in cell culture systems on promoters and endogenous target genes. In biochemical assays, they are able to displace coactivator peptides from the receptor in a dose-dependent manner. The mechanism by which they act as BAR antagonists is likely through their inability to recruit coactivator proteins, failure to release corepressor proteins from unliganded receptor, and ability to compete with BAR agonists to block coactivator recruitment. Our data suggest these compounds may mediate at least some of their effects via the BAR.     

A selective defect in arachidonic acid release from macrophage membranes in high potassium media

Murine peritoneal macrophages cultured in minimal essential medium (alpha-MEM; 118 mM Na+, 5 mM K+) released arachidonic acid (20:4) from phospholipids on encountering a phagocytic stimulus of unopsonized zymosan. In high concentrations of extracellular K+ (118 mM), 3H release from cells prelabeled with [3H]20:4 was inhibited 80% with minimal reduction (18%) in phagocytosis. The inhibitory effect of K+ on 20:4 release was fully reversed on returning cells to medium containing Na+ (118 mM). Preingestion of zymosan particles by macrophages maintained in high K+ medium resulted in cells being "primed" for 20:4 release, which was only effected (without the further addition of particles) by changing the medium to one containing Na+. In contrast, 20:4 release from cells stimulated with the calcium ionophore A23187 was unimpaired by the elevated K+ medium, suggesting no direct effect of high K+ on the phospholipase. Macrophages stimulated with zymosan in alpha-MEM metabolized the released 20:4 to prostacyclin, prostaglandin E2 (PGE2), and leukotriene C (LTC). The smaller quantity of released 20:4 in high K+ medium was recovered as 6-Keto-PGF1 alpha, the breakdown product of prostacyclin, and PGE2. No LTC was synthesized. In high K+, resting (no zymosan) macrophages synthesized hydroxyeicosatetraenoic acids from exogeneously supplied 20:4 in proportions similar to cells maintained in alpha-MEM. These findings and the similarity of products (including LTC) produced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 stimulated cells in alpha-MEM and high K+ medium indicated that the cyclooxygenase and lipoxygenase pathway enzymes were not directly inhibited by high extracellular K+. We conclude that high concentrations of extracellular K+ uncouple phagocytosis of unopsonized zymosan from the induction of the phospholipase responsible for the 20:4 cascade and suggest that the lesion is at the level of signal transduction between the receptor-ligand complex and the phospholipase.     

Low conductance states of a single ion channel are not ‘closed’

We have used a polymer-exclusion method to estimate the sizes of the high and low-conductance states of Staphylococcus aureus -toxin channels across planar lipid bilayers. Despite a >10-fold difference in conductance between high and low-conductance states, the size differs by <2-fold. We conclude that factors other than the dimensions have a strong influence on the conductance of -toxin channels. We also show that the high conductance state is destabilized by the presence of high molecular weight polymers outside the channel, compatible with the removal of channel water as the high conductance state shrinks to the low conductance state.We are grateful to Drs. D.T. Edmonds, A.A. Lev and V.A. Parsegian for fruitful discussion and to the Cell Surface Research Fund, the Science and Engineering Research Council, The Wellcome Trust, UNESCO (Molecular and Cellular Biology Network) and the National Academy of Sciences/National Research Council for financial support.     

Cytotoxic and fluorescent assays for thymocyte subpopulations differing in surface thy-1 level.

A number of analytical techniques for distinguishing and separating the "high theta" "low theta" subpopulations of mouse thymocytes have been compared. A differential cytotoxic assay was compared to a quantitative immunofluorescent assay on individual cells using flow cytofluorometry and cell sorting. Conventional anti-Thy-1 antisera were compared with a monoclonal IgM anti-Thy-1. The monoclonal reagent greatly improved both types of assay, eliminated a number of artifacts and allowed either procedure to be used to give a clear distinction, based on Thy-1 level, between the two subpopulations. The distribution of Thy-1 on thymocytes is bimodal, rather than continuous. These separate "high theta" and "low theta" categories each includes a population a population of dividing cells.     

Patterns of nucleotide substitution in pseudogenes and functional genes

Summary The pattern of point mutations is inferred from nucleotide substitutions in pseudogenes. The pattern obtained suggests that transition mutations occur somewhat more frequently than transversion mutations and that mutations result more often in A or T than in G or C. Our results are discussed with respect to the predictions from Topal and Fresco's model for the molecular basis of point (substitution) mutations (Nature 263:285–289, 1976). The pattern of nucleotide substitution at the first and second positions of codons in functional genes is quite similar to that in pseudogenes, but the relative frequency of the transition CT in the sense strand is drastically reduced and those of the transversions CG and GC are doubled. The differences between the two patterns can be explained by the observation that in the protein evolution amino acid substitutions occur mainly between amino acids with similar biochemical properties (Grantham, Science 185:862–864, 1974). Our results for the patterns of nucleotide substitutions in pseudogenes and in functional genes lead to the prediction that both the coding and non-coding regions of protein coding genes should have high frequencies of A and T. Available data show that the non-coding regions are indeed high in A and T but the coding regions are low in T, though high in A.     

Techniques for universal evaluation of astringency of green tea infusion by the use of a taste sensor system

A practical method for universal evaluation of the astringency of green tea infusion by a taste sensor system was established. The use of EGCg aqueous solution as a standard enabled analysis with high accuracy and reproducibility. The sensor output was converted into taste-intensity on the basis of Weber's and Weber-Fechner laws, which was named the "EIT(ast)" value ("EIT" and "ast" are abbreviations for "Estimated Intensity of Taste" and "astringency" respectively). It was clarified that green tea infusion is to be classified into eight grades on the EIT(ast) scale. Furthermore, the high correlation of the EIT(ast) value with the human gustatory sense and the high stability of the taste sensor were proved.     

Formation of ketone bodies by resting lymphocytes

1. Both beta-hydroxy-beta-methylglutaryl-coenzyme A synthase and lyase activities are present in rat mesenteric lymphocytes: all of the synthase and almost all (80%) of the lyase were present in the mitochondrial compartment of the cell. 2. A high rate of acetoacetate formation was observed in mesenteric lymphocytes incubated in vitro for 60 min in the absence of added substrate; addition of pyruvate or glutamine increased the "endogenous" rate of acetoacetate formation by about 30%. 3. The rates of ketone body formation are similar to maximal rates observed for rat liver. 4. It is suggested that the high rate of endogenous acetoacetate production occurs from long chain fatty acids: this suggestion is consistent with the reported high "endogenous" rate of O2 consumption by lymphocytes. 5. Of the pyruvate metabolized via pyruvate dehydrogenase in lymphocytes, ca 50-70% could be accounted for as acetoacetate, acetate, 3-hydroxybutyrate and citrate: the fate of the remainder is not known. 6. There was a high rate of endogenous acetoacetate formation by isolated mitochondria from these cells. 7. The rate was doubled by addition of pyruvate or butyrate; it was trebled by addition of propionate, ADP or carbonyl cyanide trichloro-methoxyphenylhydrazone; but it was decreased by addition of antimycin A or glutamine. 8. It is suggested that the high rates of acetoacetate formation in these cells acts as a dynamic "buffer" system for the acetyl coenzyme A (CoA) concentration: that is, acetyl CoA is always available for fatty acid synthesis, cholesterologenesis, chain extension of fatty acids or acetylation of proteins (e.g. for covalent control of their activity) which will be demanded at different stages of the cell cycle. 9. This is another example of branch-point sensitivity in control in cells with the potential for rapid cell division.     

THE POWER OF THE " A" -" NOT A" METHOD

The " A" - " Not A" method is a rating method with two categories. It is often treated as a discrimination method. Unlike forced choice procedures, the Thurstonian model for this method involves a choice criterion. In statistical tests, it is treated as a comparison of two proportions. In this paper, the power for hypothesis tests involving the monadic and replicated monadic " A" - " Not A" method is discussed. The power functions and the sample sizes needed for 80% power are given based on Thurstone's δ. Designs with equal and unequal allocations for A and A (Not A) samples are considered. The power of the method is also compared with that of four forced choice methods under the assumption that the perceptual variance is identical among methods. The comparison shows that, in general, the power for the five methods ranks from high to low: the 3-AFC, 2-AFC, " A" - " Not A", triangular and duo-trio. The comparison also shows that, based on the same number of panelists and/or the same sample size for the A and A⁻ samples for the methods, if the panelists are not too discrepant and the choice criterion in the " A" - " Not A" method is not too strict or too lax, the power of the " A" - " Not A" method is very close to that of the 2-AFC method.     

Effect of high concentrations of sucrose on the enzymatic activity of alpha-chymotrypsin

The effect of the low-molecular-mass natural reagents in high concentrations is important for investigating enzymatic reactions in near "in vivo" conditions and for optimisation of biotechnology processes. A model system, including p-nitrophenyl acetate as substrate and alpha-chymotrypsin as proteolytic enzyme, has been used to study the effect of high concentrations of sucrose, both influencing the viscosity of the reaction medium and acting as a nucleophilic effector (activator) on the enzymatic reaction. A kinetic scheme at high concentrations of nucleophilic effectors (sucrose) has been proposed.     

Genotypic Variation for a Quantitative Character Maintained under Stabilizing Selection without Mutations: Epistasis

A model of the gene action on a quantitative character is suggested. The model takes into account epistasis by combining multiplicative with the traditional additive approximation of the action of loci. It is demonstrated on the basis of this model that a high level of genotypic variation can be maintained in a population for a quantitative character under stabilizing selection in the absence of mutations, if there is epistasis. It is also shown that a large amount of additive variation as well as high heritability can be "hidden" in such a population and "released" if stabilizing selection is relaxed.     

Voltage dependence of calcium current deactivation in the somatic membrane of mouse sensory neurons

Voltage dependence of the deactivation kinetics of calcium inward currents was investigated in the somatic membrane of murine spinal ganglia neurons. It was found that deactivation of high threshold calcium current has a slower component (=0.80–0.85 msec at a repolarizing potential of –80 mV) as well as the principal transient exponential component (130 sec at the same potential repolarizing level). A dissimilar relationship exists between amplitudes of the transient and slower exponential components, describing deactivation of high threshold calcium current and degree of activation of the depolarizing shift in membrane potential; the former dependence is expressed by a sigmoid and the latter by a V-shaped curve. The slower component of deactivation of high threshold current was inhibited substantially by perfusing the cell with a Tris-PO₄-containing solution. Low-threshold calcium tail current undergoes slower deactivation (=1.1–1.2 msec) at a repolarizing potential of –160 mV. A relationship between the time constant of low threshold current deactivation and the type of penetrating cation used was observed. A kinetic model of calcium current deactivation is suggested, taking account of the three different types of calcium channels, (one low and two high threshold) present in the somatic membrane.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 185–193, March–April, 1988.     

Metabolic interaction between purine nucleotide cycle and oxypurine cycle during skeletal muscle contraction of different intensities: a biochemical reappraisal

Background

A substrate cycle is a metabolic transformation in which a substrate A is phosphorylated to A?P at the expense of ATP (or another “high energy” compound), and A?P is converted back to A by a nucleotidase or a phosphatase. Many biochemists resisted the idea of such an ATP waste. Why a non-phosphorylated metabolite should be converted into a phosphorylated form, and converted back to its non-phosphorylated form through a “futile cycle”?

Aim of review

In this Review we aim at presenting our present knowledge on the biochemical features underlying the interrelation between the muscle purine nucleotide cycle and the oxypurine cycle, and on the metabolic responses of the two cycles to increasing intensities of muscle contraction.

Key scientific concepts of review

Nowadays it is widely accepted that the substrate cycles regulate many vital functions depending on the expense of large amounts of ATP, including skeletal muscle contraction, so that the expense of some extra ATP and “high energy” compounds, such as GTP and PRPP via substrate cycles, is not surprising. The Review emphasizes the strict metabolic interrelationship between the purine nucleotide cycle and the oxipurine cycle.

     

Regioselective C-H hydroxylation of omeprazole sulfide by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> CYP102A1 to produce a human metabolite

Objectives

To find a simple enzymatic strategy for the efficient synthesis of the expensive 5′-hydroxyomeprazole sulfide, a recently identified minor human metabolite, from omeprazole sulfide, which is an inexpensive substrate.

Results

The practical synthetic strategy for the 5′-OH omeprazole sulfide was accomplished with a set of highly active CYP102A1 mutants, which were obtained by blue colony screening from CYP102A1 libraries with a high conversion yield. The mutant and even the wild-type enzyme of CYP102A1 catalyzed the high regioselective (98 %) C-H hydroxylation of omeprazole sulfide to 5′-OH omeprazole sulfide with a high conversion yield (85–90 %).

Conclusions

A highly efficient synthesis of 5′-OH omeprazole sulfide was developed using CYP102A1 from Bacillus megaterium as a biocatalyst.

     

Light-induced reactions of ubiquinone in photosynthetic bacterium, Chromatium D. II. Effects of inhibitors and other experimental conditions

Light-induced reactions of ubiquinone in starved cells of ChromatiumD, were investigated under various experimental conditions. The "low amplitude" photoreduction observed on illuminationwithout addition of substrate under aerobic conditions and the"light-off spikes" observed under anaerobic conditions wereshown to be related to the cyclic flow of electrons in the photosyntheticsystem. Both of these were inhibited by o-phenanthroline, HOQNOand piericidin A. The "high amplitude" photoreduction of ubiquinoneobserved in the presence of thiosulfate under aerobic conditionswas inhibited by PCMB, PMA and KCN. The "high amplitude" photooxidationof ubiquinone in the presence of malate under anaerobic conditionswas inhibited by HOQNO, piericidin A, rotenone and PMA. In the presence of KCN and succinate, similar "high amplitude"photooxidation was obtained even under aerobic conditions. Underaerobic conditions, the addition of malate did not affect the"high amplitude" photoreduction due to thiosulfate. On the contrary,under anaerobic conditions, the "high amplitude" photooxidationwith malate was almost completely abolished by the additionof thiosulfate. This apparent counteraction of the oxidationand reduction of ubiquinone was inferred to represent an optimumstate of its efficient turn-over in the noncyclic electron transportchain of photosynthesis inChromatium. ¹This article is the second of a series of studies previouslyreported under the same title in Vol. 8 of this journal (Reference14). (Received August 23, 1968; )     

昆虫抗药性的治理策略:一个数学模型的提出

本文用计算机模型分析及评价了昆虫抗药性的三种治理策略:顺序轮用,“高杀死”策略及棋盘式用药.假设昆虫具有一定的生物学特性:如抗性为单基因,单抗性,抗性基因为半显性,两性生殖,昆虫有扩散习性,感性系在不施药情况下有微弱的适应优势等.分析了用不同剂量造成的不同的死亡率,不同的反选择作用,不同的基因稀释作用等情况对三个策略的影响.结果显示出,在这些情况下,用3—5种杀虫剂隔代顺序轮用加上微弱的反选择作用是最为有效的.“高杀死”策略加上一定的稀释作用也极为有效.棋盘式用药在特殊情况下才有用.     

Correlation between conformation distortion of the DNA sugar-phosphate backbone and high optical activity of its compact form

Different physico-chemical methods (CD, ORD, small-angle X-ray diffraction, etc) were used for investigating the properties of the DNA compact particles formed in PEG-containing water-salt solutions. It has been shown that small-angle reflection, characteristic of the DNA compact particles, changes from 36.8 A (CPEG = 140 mg/ml) to 25 A (CPEG = 300 mg/ml). The maximal optical activity (the intense negative CD-band and optical rotation [alpha] = 60 000 degrees) are inherent properties of the DNA compact particles formed at CPEG 120--180 mg/ml. The high optical activity points to the twist of DNA chromophores through the DNA molecule resulting in a long-rang pitch (P approximately 2000A).Such macroscopic superhelical structure (diameter 40--30 A) is due to conformational distortion of the DNA double-helix with alternating "left" and "right" orientation of chromophoes. Disappearance of conformation distortion is accompanied by disappearance of the high optical activity of the DNA compact particles and results in a small-angle reflection of 25 A. Taking into account the reasons of formation of the optically-active DNA compact particles conditions are suggested to conserve high optical activity at CPEG equal to 400 mg/ml.     

Dragma hippomanicum X: Astragali (Leguminosae) nevadenses novi criticive,singulo peruviano adjecto

One new species,Astragalus tiehmii, and two new varieties,A. convallarius var.margaretae andA. oophorus var.lavinii, are described from Nevada; the relationships of each are discussed and the first is illustrated. Two new combinations,A. eurylobus (Barneby) Barneby andA. serenoi var.shockleyi (M. E. Jones) Barneby are proposed and defended. A new species,A. bryogenes, is described from high Andean Peru (Junín).     

Periplasmic protein-protein contacts in the inner membrane protein Wzc form a tetrameric complex required for the assembly of Escherichia coli group 1 capsules

The K antigenic capsular polysaccharide forms a structural layer, the capsule, on the surfaces of Escherichia coli cells. The capsule provides an important protective covering that helps protect encapsulated bacteria from host immune defenses. The assembly and translocation of the capsule requires proteins in the inner and outer membranes. The inner membrane protein Wzc is a tyrosine autokinase that plays an essential role in what is believed to be a coordinated biosynthesis and secretion process. Mutants lacking Wzc can form K antigen oligosaccharides but are unable to polymerize high molecular weight capsular polymers. Wzc homologs have been identified in exopolymer biosynthesis systems in many different Gram-negative and -positive bacteria. Using single particle averaging on cryo-negatively stained samples, we have produced the first three-dimensional structure of this type of membrane protein in its phosphorylated state at approximately 14 A resolution. Perfluoro-octanoate-PAGE analysis of detergent-solubilized oligomeric Wzc and symmetry analysis of the transmission electron microscopy data clearly demonstrated that Wzc forms a tetrameric complex with C4 rotational symmetry. Viewed from the top of the complex, the oligomer is square with a diameter of approximately 100 A and can be divided into four separate densities. From the side, Wzc is approximately 110 A high and has a distinctive appearance similar to an extracted molar tooth. The upper "crown" region is approximately 55 A high and forms a continuous ring of density. Four unconnected "roots" ( approximately 65 A high) emerge from the underside of the crown. We propose that the crown is formed by protein-protein contacts from the four Wzc periplasmic domains, while each root represents an individual cytoplasmic tyrosine autokinase domain.     

A gene affecting liver activities of three glycosidases in the house mouse

A gene locus is described controlling liver activities in the house mouse of three glycosidases, i.e., -galactosidase, -glucuronidase, and N-acetyl--hexosaminidase. An allele conferring low activity is present in the inbred strain LIS/A, and an allele for high activity is present in A/Br Af mice. The three enzyme activities are correlated with each other. The possible linkage between this gene and the Bgs locus on chromosome 9 is discussed.     

Recent advances in anaerobic biological processes for textile printing and dyeing wastewater treatment: a mini-review

Textile printing and dyeing wastewater is usually characterized by high pH, high turbidity, poor bio-degradability, complex composition, and high chrominance, and is discharged in large amounts. It has been regarded as one of the hardest to treat forms of industrial wastewater. Conventional physicochemical technologies can remove these contaminants from water bodies, but at the expense of high energy consumption and high cost. Alternatively, biological processes with limited energy consumption, low cost and high efficiency are considered as promising technologies. Among them, the anaerobic biological processes have been proven to be effective for the treatment of high-concentration textile printing and dyeing wastewater. In this mini-review, recent advances on high-rate anaerobic technologies for such purposes are reviewed. Current limitations of these technologies are summarized, and future research directions are indicated.

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