Deubiquitinases as Anticancer Targets of Gold Complexes

Metal-based anticancer agents with non-DNA targets are anticipated for overcoming cisplatin resistance problems. Gold complexes are generally known to undergo ligand exchange or redox reactions with thiols, and hence, are potentially useful agents that could target thiol-containing enzymes, but not DNA. Recent studies have shown that deubiquitinases (DUBs), key enzymes regulating proteasome-related protein homeostasis, are potential anticancer targets of both gold(I) and gold(III) complexes. In this review, the current status of gold complexes as DUB inhibitors is discussed. In particular, auranofin and cyclometalated gold(III) complexes containing dithiocarbamate ligands (e.g., [(Au^III(C N)(DEDT)]⁺, HC N=2-phenylpyridine, DEDT=diethyldithiocarbamate) are highlighted as examples of DUB inhibitors. The mechanisms of their anticancer action, together with in vitro and/or in vivo antitumor potencies, are also explored.     

Caulobacter segnis Dioxygenase CsO2: A Practical Biocatalyst for Stilbenoid Ozonolysis

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5–3 h) with high conversions (95–99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50–mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.     

Enhancement of oxidation resistance at 1000–1400 °C of low carbon Al2O3–C refractories with pre-synthesized SiCnw/Al2O3

The oxidation resistance of low carbon Al₂O₃–C refractories with the addition of SiC_nw/Al₂O₃ composite powders and the enhancement mechanisms were investigated. The oxidation resistance was evaluated by oxidation index (O.I.) and oxidation rate constant (k). The enhancement mechanisms of SiC_nw/Al₂O₃ on oxidation resistance were analyzed based on the phases and microstructures. The results showed that the SiC_nw/Al₂O₃ can improve the oxidation resistance of Al₂O₃–C refractories, the O.I. and k of A6 (6 wt% SiC_nw/Al₂O₃ addition) were 26.0% and 34.5% lower than those of reference sample A0, respectively. The oxidation resistance of refractories was improved in a range of 1000–1400 °C due to the introduction of SiC_nw/Al₂O₃. The enhancement mechanisms can be explained that SiC_nw is more susceptible to be oxidized due to its high specific surface area, which expanded the action temperature range of other antioxidants and itself. The mullite and dense protective layer generated during oxidation is also beneficial to impede the diffusion of O₂.     

Biosynthesis of Hygrocins,Antitumor Naphthoquinone Ansamycins Produced by Streptomyces sp. LZ35

Hygrocins are naphthoquinone ansamycins with significant antitumor activities. Here, we report the identification and characterization of the hygrocin biosynthetic gene cluster (hgc) in Streptomyces sp. LZ35. A biosynthetic pathway is proposed based on bioinformatics analysis of the hgc genes and intermediates accumulated in selected gene disruption mutants. One of the steps during the biosynthesis of hygrocins is a Baeyer–Villiger oxidation between C5 and C6, catalyzed by luciferase‐like monooxygenase homologue Hgc3. Hgc3 represents the founding member of a previously uncharacterized family of enzymes acting as Baeyer–Villiger monooxygenases.     

Physiological, Nutritional, and Biochemical Bases of Corn Resistance to Foliage-Feeding Fall Armyworm

Three corn (Zea mays) germplasm lines [i.e., Ab24E (susceptible control), Mp708 (resistant control), and a locally selected partial inbred line FAW7050 (resistant)] were examined for Spodoptera frugiperda (J.E. Smith; Lepidoptera: Noctuidae) resistance. Nutritional [i.e., total protein content, amino acids, glucose, total nonstructural carbohydrates (TNC), protein to TNC (P/C) ratios] and biochemical (i.e., peroxidase and lipoxygenase 3) properties in the seedlings of these corn lines were examined to categorize resistance mechanisms to S. frugiperda. Physiological changes in photosynthetic rates also were examined in an attempt to explain nutritional and biochemical dynamics among corn germplasm lines and between insect-infested and noninfested corn plants within a germplasm line. Results indicated that S. frugiperda larvae survived better and developed faster in susceptible Ab24E than in resistant FAW7050 or Mp708. The three germplasm lines differed in resistance mechanisms to S. frugiperda, and the observed patterns of resistance were probably collective results of the P/C ratio and defensive proteins. That is, the susceptibility of Ab24E to S. frugiperda was due to a high P/C ratio and a low level of induced defensive compounds in response to insect herbivory, while the resistance of FAW7050 resulted from elevated defensive proteins following insect herbivory, low P/C ratio, and elevated defensive proteins in Mp708 contributed to its resistance to S. frugiperda. The elevated protein amounts in resistant Mp708 and FAW7050 following S. frugiperda injury were likely due to greater conversion of photosynthates to defensive proteins following the greater photosynthetic rates in these entries. Greater photosynthetic capacity in Mp708 and FAW7050 also led to higher amino acid and glucose contents in these two lines. Neither amino acid nor lipoxygenase 3 played a critical role in corn resistance to S. frugiperda. However, high inducibility of peroxidase may be an indicator of S. frugiperda susceptibility as observed elsewhere.     

Chemical Synthesis and Gas Chromatographic Behaviour of γ‐Stearidonic (18:4n‐6) Acid

γ‐Stearidonic acid, 18:4n‐6, a potential product of β‐oxidation of arachidonic acid (20:4n‐6), was only recently positively identified in a living organism—a thermophilic cyanobacterium Tolypothrix sp., albeit at low levels, whilst some indirect evidence suggests its wider presence, e.g. in a unicellular marine alga. We have prepared 18:4n‐6 using an iodolactonisation chain‐shortening approach from 22:5n‐6 and obtained its ¹H‐, ¹³C‐, COSY‐ and HSQC NMR spectra, with 18:5n‐3 spectra also recorded for a comparison. The GC and GC‐MS behaviour of its methyl ester was also studied. Like another Δ3 polyunsaturated acid, octadecapentaenoic (18:5n‐3), 18:4n‐6 rapidly yields 2‐trans isomer upon formation of dimethyloxazoline derivative. On a polar ionic liquid phase (SLB‐IL100, 200 °C) the methyl ester could be mistaken for 18:3n‐3, while on methylsilicone phase (BP1, 210 °C) it eluted ahead of 18:3n‐6 and 18:4n‐3, suggesting that when present it may be easily misidentified during GC analysis of fatty acids.     

Cell membrane localization of sterols with conventional and unusual side chains in two marine demonsponges

Subcellular fractionation by differential centrifugation was performed on two previously unstudied marine sponges that predominantly contain either conventional (Reniera sp.) or unconventional (Pseudaxinyssa sp.) sterols. Direct evidence for the presence of unconventional sterols with C₂₄ alkylated side chains in the cellular membranes ofPseudaxinyssa sp. is provided, but the presence of unconventional sterols in sponge membranes is shown not to be a universal feature of the Porifera. Possible structural and functional roles of unconventional lipid molecules in sponge cell membranes are discussed. Part 5 of “Sterols in Marine Invertebrates”. Fort Part 58 in this series, see Cho, J.-H., and Djerassi, C. (1987)J. Org. Chem. 52, 4517–4521.     

Semiconductor-electrochemical aspects of bacterial leaching. Part 2. Survey of rate-controlling sulphide properties

Sixteen metal sulphides with widely differing solid state, chemical and electrochemical properties have been investigated with respect to their suitability as an energy source for Thiobacillus ferrooxidans. The most critical rate-determining parameter for bacterial oxidation (in the absence of any electron acceptors, e.g. Fe³⁺) was the solubility product of the sulphide. Deviations from the systematic dependence are, however, observed when a large concentration of the holes are present in the semiconducting sulphide (p-type conduction), or when holes are generated through electron extraction from the sulphide surface (e.g. by Fe³⁺). The experimental results are consistent with a mechanism in which Th. ferrooxidans utilises H⁺ and Fe³⁺ as catalytic agents which break surface bonds by chemical and electrochemical mechanisms, respectively. They are subsequently recycled while the bacterium oxidises the generated surface products, — SH^δ and S_o, etc., to sulphate. On the basis of the derived mechanism it should be possible to estimate and predict the suitability of sulphides for bacterial oxidation and to analyse the influence of crystalline quality and impurities, as well as the composition of the solution, on the rate of oxidation.     

Heavy Metal Induced Antibiotic Resistance in Bacterium LSJC7

Co-contamination of antibiotics and heavy metals prevails in the environment, and may play an important role in disseminating bacterial antibiotic resistance, but the selective effects of heavy metals on bacterial antibiotic resistance is largely unclear. To investigate this, the effects of heavy metals on antibiotic resistance were studied in a genome-sequenced bacterium, LSJC7. The results showed that the presence of arsenate, copper, and zinc were implicated in fortifying the resistance of LSJC7 towards tetracycline. The concentrations of heavy metals required to induce antibiotic resistance, i.e., the minimum heavy metal concentrations (MHCs), were far below (up to 64-fold) the minimum inhibition concentrations (MIC) of LSJC7. This finding indicates that the relatively low heavy metal levels in polluted environments and in treated humans and animals might be sufficient to induce bacterial antibiotic resistance. In addition, heavy metal induced antibiotic resistance was also observed for a combination of arsenate and chloramphenicol in LSJC7, and copper/zinc and tetracycline in antibiotic susceptible strain Escherichia coli DH5α. Overall, this study implies that heavy metal induced antibiotic resistance might be ubiquitous among various microbial species and suggests that it might play a role in the emergence and spread of antibiotic resistance in metal and antibiotic co-contaminated environments.     

Enhanced maize (Zea mays L.) pericarp browning: Associations with insect resistance and involvement of oxidizing enzymes

The kernel pericarp of a maize (Zea mays L.) inbred, Mp313E, that browns rapidly at milk stage when damaged and that is resistant toAspergillus flavus Link and the dusky sap beetleCarpophilus lugubris Murray compared to a susceptible inbred, SC212M, was examined for differing oxidizing enzymes (peroxidases) and their substrate specificity. Additional pericarp enzymes were constitutively produced by the Mp313E inbred compared to the SC212M inbred, as indicated by gel electrophoresis and isoelectric focusing. These enzymes oxidized relevent pericarp substrates such as ferulic acid. Similar results were seen with two varieties of maize containing theCh mutant gene, which brown upon senescence in that enhanced oxidation of ferulic acid was seen in homogenates of browned pericarp compared to that which was cold-shocked and did not brown. Corn powder that was browned by mixing oxidizing enzymes with corn pericarp components ferulic acid and coumaric acid were typically less preferred/more toxic to caterpillars such asHelicoverpa zea (Boddie) and sap beetles such asC. lugubris. Thus, enhanced browning of maize pericarp can promote resistance to insects and is at least partly influenced by the presence of oxidizing enzymes. This mechanism may also promote resistance to maize pathogens, including those that produce mycotoxins.     

Regioselective Acetylation of C21 Hydroxysteroids by the Bacterial Chloramphenicol Acetyltransferase I

Chloramphenicol acetyltransferase I (CATI) detoxifies the antibiotic chloramphenicol and confers a corresponding resistance to bacteria. In this study we identified this enzyme as a steroid acetyltransferase and designed a new and efficient Escherichia‐coli‐based biocatalyst for the regioselective acetylation of C21 hydroxy groups in steroids of pharmaceutical interest. The cells carried a recombinant catI gene controlled by a constitutive promoter. The capacity of the whole‐cell system to modify different hydroxysteroids was investigated, and NMR spectroscopy revealed that all substrates were selectively transformed into the corresponding 21‐acetoxy derivatives. The biotransformation was optimized, and the reaction mechanism is discussed on the basis of a computationally modeled substrate docking into the crystal structure of CATI.     

Free radical scavenging capacity as related to antioxidant activity and ginsenoside composition of Asian and North American ginseng extracts

Different Panax species derived from Asia (Panax ginseng C.A. Meyer) and North America (Panax quinquefolium L.) were extracted by methanol and evaluated for relative ginsenoside composition and antioxidant activities. Ginseng root contained a greater proportion of total ginsenoside compared to ginseng hair analyzed by high-performance liquid chromatography. North American ginseng root was characterized with undetectable ginsenoside Rf and greater Rb₁/Rb₂ than Asian ginseng root. Panax quinquefolium exhibited a relatively higher (P<0.05) affinity to scavenge free radical than panax ginseng using the 2,2-azobis (3-ethylbenzothine-6-thine-6-surfonic acid) radical model. In a bilayer lamella suspension oxidation model induced by peroxyl radicals, ginseng samples exhibited notable antioxidant activity. Specifically, however, the P. quinquefolium extracts delayed lipid peroxidation longer (P<0.05) than the P. ginseng extracts. Ginseng extracts from both Panax species protected human low-density lipoprotein against cupric ion-mediated oxidation. Similar protection was observed against peroxyl radical-induced supercoiled DNA breakage. A pure ginsenoside standard (e.g., Rb₁) produced similar results. The antioxidant activities of different ginseng species and specific plant parts include free radical scavenging and may be related to ginsenoside Rb₁/Rb₂ content.     

Isolation of Marine Paracoccus sp. Ss63 from the Sponge Sarcotragus sp. and Characterization of its Quorum-Sensing Chemical-Signaling Molecules by LC-MS/MS Analysis

Gram-negative bacteria often use N-acyl homoserine lactones (AHLs) as quorum-sensing (QS) signal molecules. The present study aimed to identify and characterize the production of AHLs by Paracoccus sp. Ss63, which was isolated from the Mediterranean sponge Sarcotragus sp. High performance liquid chromatography – high resolution tandem mass spectrometry (LC-HRMS/MS) provided evidence for the production, by Paracoccus sp. Ss63, of twelve saturated long-chain AHLs (3-OH-C10-HSL, 3-OH-C12-HSL, C10-HSL, 3-OH-C14-HSL, C12-HSL, 3-O-C16-HSL, 3-OH-C16-HSL, C14-HSL, 3-OH-C18-HSL, C16-HSL, 3-O-C18-HSL, and C18-HSL), along with four putative unsaturated long-chain AHLs (C10 : 1-HSL, 3-OH-C18 : 1-HSL, C16 : 1-HSL, and C18 : 1-HSL). The distribution of Paracoccus sp. Ss63 in different sponge species, as well as in seawater and marine sediment samples, suggests a mixed lifestyle for this bacterium, i.e., free-living and host-associated. The high pH of seawater is likely to inactivate the AHL signal molecules, and limit the possibility of AHLs to accumulate to the concentration required for QS-based gene regulation. We hypothesize that Paracoccus sp. Ss63 utilizes quorum sensing to sense and respond to the different environments it experiences, with an active QS only when found inside enclosed niches (host-associated and at lower pH).     

Covalent Linkage of an R-ω-Transaminase to a d-Amino Acid Oxidase through Protein Splicing to Enhance Enzymatic Catalysis of Transamination

R-ω-Transaminases (RTAs) catalyse the conversion of R-configured amines [e.g., (R)-1-phenylethylamine] into the corresponding ketones (e.g., acetophenone), by transferring an amino group from an amino donor [e.g., (R)-1-phenylethylamine] onto an amino acceptor (e.g., pyruvate), resulting in a co-product (e.g., d -alanine). d -Alanine can be deaminated back to pyruvate by d -amino acid oxidase (DAAOs). Here, through in vivo subunit splicing, the N terminus of an RTA subunit (RTA^S) was specifically ligated to the C terminus of a DAAO subunit (DAAO^S) through native peptide bonds (RTA&DAAO). RTA^S is in close proximity to DAAO^S, at a molecular-scale distance. Thus the transfer of pyruvate and d -alanine between RTA and DAAO can be directional and efficient. Pyruvate→d -alanine→pyruvate cycles are efficiently formed, thus promoting the forward transamination reaction. In a different, in vitro noncovalent approach, based on coiled-coil association, the RTA^S N terminus was specifically associated with the DAAO^S C terminus (RTA#DAAO). In addition, the two mixed individual enzymes (RTA+DAAO) were also studied. RTA&DAAO has a shorter distance between the paired subunits (RTA^S–DAAO^S) than RTA#DAAO, and the number of the paired subunits is higher than in the case of RTA#DAAO, whereas RTA+DAAO cannot form the paired subunits. RTA&DAAO exhibited a transamination catalysis efficiency higher than that of RTA#DAAO and much higher than that of RTA+DAAO.     

Cell membrane localization of long chain C24−C30 fatty acids in two marine demosponges

Subcellular fractionation by differential centrifugation was performed on two previously unstudied marine sponges (Reniera sp. andPseudaxinyssa sp.) that represent both major subclasses of the Demospongiae. Long chain fatty acids (LCFA) with 24–30 carbon units were found as major constituents of cell membrane isolates of both sponges. Most LCFA were polyunsaturated and were constituents of the phospholipids, which are typical membrane lipids, and in particular the amino-phospholipids. The LCFA composition of phospholipids from whole sponge tissue was shown to provide a reliable indication of the LCFA composition of cell membrane phospholipids in the sponges studied. An unusual triply branched C₁₆ isoprenoid fatty acid, 4,8,12-trimethyltridecanoic acid, also was identified as a cell membrane acid in the spongePseudaxinyssa sp. Part 16 of “Phospholipid Studies of Marine Organisms.” For Part 15 in this series see Dasgupta, A., Ayanoglu, E., Tomer, K.B., and Djerassi, C. (1987)Chem. Phys. Lipids 43, 101–111.     

Effect of Enzymatic Hydrolysis on the Antioxidant Activity of Red and Green Seaweeds and Characterization of the Active Extracts

In the present study, the effect of enzymatic hydrolysis on the antioxidant activity of three red seaweeds (Chondria cornuta, Chondria dasyphylla, and Murrayella periclados) and two green seaweeds (Cladophora sp. and Ulva lactuca) from the Kuwait coast were evaluated. The seaweeds were hydrolyzed with five carbohydrases and three proteases, and the resulting extracts were tested for antioxidant activity. The total phenolic content and yield of the extracts varied greatly depending upon the species and enzyme used for hydrolysis. Of the 40 enzymatic extracts screened for antioxidant activity, the Viscozyme and Alcalase extracts of M. periclados, Neutrase and Ultraflo extracts of Cladophora sp., and Neutrase extracts of C. cornuta had high antioxidant activity compared to other enzymatic extracts in various in vitro assays. Fractionation of the extracts revealed that the radical scavenging and reducing power of the extracts were mainly due to phenolic fractions. In contrast, the iron-chelating ability was mainly due to protein fractions. The level of prevention of lipid oxidation in the liposome model system varied for different fractions of extracts and did not correlate with total phenolic content and other antioxidant properties. The results of the study show that, by hydrolyzing seaweeds with specific enzymes, customized seaweed extracts with specific bioactivity could be obtained.     

In situ FT-IR Spectroscopic Studies on the Mechanism of the Catalytic Oxidation of Carbon Monoxide over Supported Cobalt Catalysts

Three types of supported cobalt catalysts (5% as metal Co loading on SiO₂, Al₂O₃ and TiO₂) were prepared by incipient wetness impregnation with aqueous Co(NO₃)₂·6H₂O solution. Then, all catalysts were calcined in air at 400 °C (assigned as 5Co/Si C400, 5Co/Al C400 and 5Co/Ti C400). Their catalytic activities towards the CO oxidation were studied in a continuous flow micro-reactor. Adsorption of carbon monoxide (CO) and the co-adsorption of CO/O₂ over cobalt oxide were further tested under in situ FT-IR. The results showed that both 5Co/Si C400 and 5Co/Al C400 had higher activity than 5Co/Ti C400. The T₅₀ (50% conversion) for both 5Co/Si C400 and 5Co/Al C400 was reached at temperatures as low as ambient temperature. According to the in situ FT-IR analysis, the variation in oxidation of CO was interpreted with different mechanisms, i.e., the reaction between adsorbed CO and lattice oxygen of cobalt oxide, and part of CO₂ formation via carbonates on 5Co/Si C400; both types of carbonates are formed on 5Co/Al C400 to promote the CO oxidation; while both strong adsorption of CO on TiO₂ and CO₂ on cobalt oxide for 5Co/Ti C400 leads to affect the activity.     

Kinetics of vanillin oxidation

Vanillin can be produced by oxidation of kraft lignin, with air, in alkaline medium. The optimal conditions for vanillin production strongly depend on pH and temperature. This paper addresses the effect of temperature and pH on vanillin degradation by oxidation. Experiments were carried out in a wide range of vanillin concentration, oxygen partial pressure, temperature and pH. Simple models are proposed to explain the observed rate of vanillin consumption under conditions of high alkalinity (pH>12) and lower alkalinity (pH<12). At pH>12,the reaction rate of vanillin oxidation is first order in dissolved oxygen concentration [O₂] and in vanillin concentration [C], i.e., (?r_C) ∝[O₂][C]; at pH<12, the rate is zero order in oxygen concentration and second order in vanillin concentration, i.e., (?r_C)∝f(pH)[C]².     

A Genetic Circuit System Based on Quorum Sensing Signaling for Directed Evolution of Quorum‐Quenching Enzymes

Quorum sensing is a cell–cell communication mechanism that is involved in the regulation of biological functions such as luminescence, virulence, and biofilm formation. Quorum‐quenching enzymes, which interrupt quorum‐sensing signaling through degradation of quorum‐sensing molecules, have emerged as a new approach to controlling and preventing bacterial virulence and pathogenesis. In an effort to develop quorum‐quenching enzymes with improved catalytic activities, a genetic circuit system based on acylhomoserine‐lactone (AHL)‐mediated quorum‐sensing signaling was constructed. The genetic circuit system was composed of lux‐R, lux‐I promoter, β‐lactamase, and β‐lactamase inhibitor, and designed to confer antibiotic resistance on host cells expressing an AHL‐degrading enzyme, thereby enabling rapid screening of quorum‐quenching enzymes. To demonstrate the utility of the genetic circuit system, we attempted the directed evolution of the AHL hydrolase from Bacillus sp. The genetic circuit system was shown to be effective in screening of quorum‐quenching enzymes with high catalytic efficiency. From these results it is expected that the genetic circuit system can be widely used for the isolation and directed evolution of quorum‐quenching enzymes with greater potential.     

Catalytic Oxidation of C(sp3)?H Bonds Induced by a Radical Cation Salt: Construction of 1,4‐Dihydropyridines Using a Fragment‐Reassembly Strategy

A fragment‐reassembly strategy was applied to the construction of 1,4‐dihydropyridines and phosphorus‐substituted 1,4‐dihydropyridines under catalytic radical cation salt‐induced C(sp)³ H functionalization of glycine derivatives. Mechanistic studies show that domino C(sp)³ H bond oxidation and C N bond cleavage reactions are involved.