Acetylenic 2-phenylethylamides and new isobutylamides from Acmella oleracea (L.) R. K. Jansen,a Brazilian spice with larvicidal activity on Aedes aegypti

Ethanol extract obtained from dried leaves of Acmella oleracea afforded after a liquid/liquid partition procedure a larvicidal hexane fraction (LC₅₀ = 145.6 ppm) and a non larvicidal dichloromethane one. From the inactive fraction, three amides were identified, two new structures, named deca-6,9-dihydroxy-(2E,7E)-dienoic acid isobutylamide (1), deca-8,9-dihydroxy-(2E,6Z)-dienoic acid isobutylamide (2) and the known nona-2,3-dihydroxy-6,8-diynoic acid 2-phenylethylamide (3). Bioassay-guided chromatographic fractionation of the hexane partition led to the identification of an amide mixture, nona-(2Z)-en-6,8-diynoic acid 2-phenylethylamide (4) and deca-(2Z)-en-6,8-diynoic acid 2-phenylethlylamide (5). This mixture was active against Aedes aegypti larvae at LC₅₀ = 7.6 ppm. Low toxicity of crude extracts and derived fractions on Artemia salina nauplies showed the possibility of using them to control the A. aegypti mosquito larvae. This is the first report on larvicidal activity of acetylenic 2-phenylethylamides and their identification in A. oleracea leaves.     

Microbial transformation of the triterpene nigranoic acid in Trichoderma sp.

Two new metabolites were obtained by microbial transformation of the triterpene nigranoic acid (3,4-secocyloarta-4 (28), 24 (Z)-diene-3,26-dioic acid), (1) in the culture of Trichoderma sp. JY-1, a fungus obtained from the branches of Kadsura angustifolia. Their structures were established as 15α, 16α-dihydroxy-3,4-secocyloarta-4 (28), 17 (20), 17 (E), 24 (E)-triene-3,26-dioic acid (2) and 16α, 20α-dihydroxy-18 (13 → 17β) abeo-3,4-secocyloarta-4 (28), 12 (13), 24 (Z)-triene-3,26-dioic acid (3) by analysis of NMR and MS data and by analogy with the data for the substrate nigranoic acid (1). Compound 2 was found to possess an unusual 17(20), 17 (E)-ene structure while compound 3 featured an unprecedented 18(13 → 17β)-abeo-secocyloarta skeleton. Additionally, compounds 1–3 showed weak anti-HIV activity with EC₅₀ values of 10.5, 8.8 and 7.6 μg/mL, therapeutic index values (CC₅₀/EC₅₀) of 8.48, 9.12 and 10.1, respectively.     

Benzo[d]isothiazole 1,1-dioxide derivatives as dual functional inhibitors of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1

A series of 6-nitro-3-(m-tolylamino) benzo[d]isothiazole 1,1-dioxide analogues were synthesized and evaluated for their inhibition activity against 5-lipoxygenase (5-LOX) and microsomal prostaglandin E₂ synthase (mPGES-1). These compounds can inhibit both enzymes with IC₅₀ values ranging from 0.15 to 23.6 μM. One of the most potential compounds, 3g, inhibits 5-LOX and mPGES-1 with IC₅₀ values of 0.6 μM, 2.1 μM, respectively.     

Synthesis and biochemical evaluation of a range of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole-based compounds as potent inhibitors of 17α-hydroxylase/17,20-lyase (P45017α) derived from rat testicular microsomes

We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of (4-substituted phenyl)sulfonate derivatives of 4-hydroxybenzyl imidazole against the two components of 17α-hydroxylase/17,20-lyase (P450_17α), namely, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results show the compounds to be highly potent inhibitors with limited selectivity towards the lyase component [e.g., toluene-4-sulfonic acid 4-imidazol-1-ylmethyl-phenyl ester (4) possessed an IC₅₀ value of 40 nM against 17α-OHase and 30 nM against lyase].     

Response surface methodology: Synthesis of short chain fructooligosaccharides with a fructosyltransferase from Aspergillus aculeatus

A transferase was isolated, purified and characterised from Aspergillus aculeatus. The enzyme exhibited a pH and temperature optima of 6.0 and 60 °C, respectively and under such conditions remained stable with no decrease in activity after 5 h. The enzyme was purified 7.1 fold with a yield of 22.3% and specific activity of 486.1 U mg^?1 after dialysis, concentration with polyethyleneglycol (30%) and DEAE-Sephacel chromatography. It was monomeric with a molecular mass of 85 kDa and K_m and V_max values of 272.3 mM and 166.7 μmol min^?1 ml^?1. The influence of pH, temperature, reaction time, and enzyme and sucrose concentration on the formation of short-chain fructooligosaccharides (FOS) was examined by statistical response surface methodology (RSM). The enzyme showed both transfructosylation and hydrolytic activity with the transfructosylation ratio increasing to 88% at a sucrose concentration of 600 mg ml^?1. Sucrose concentration (400 mg ml^?1) temperature (60 °C), and pH (5.6) favoured the synthesis of high levels of GF₃ and GF₄. Incubation time had a critical effect on the yield of FOS as the major products were GF₂ after 4 h and GF₄ after 8 h. A prolonged incubation of 16 h resulted in the conversion of GF₄ into GF₂ as a result of self hydrolase activity.     

The ameliorating effects of 2,3-dihydroxy-4-methoxyacetophenone on scopolamine-induced memory impairment in mice and its neuroprotective activity

We isolated 2,3-dihydroxy-4-methoxyacetophenone, a neuroprotective compound from Cynenchum paniculatum in our previous study.The present study was conducted to investigate the possible neuroprotective effect of 2,3-dihydroxy-4-methoxyacetophenone that has been previously isolated from Cynenchum paniculatum on hippocampal neuronal cell line, HT22 cells and its possible cognitive-enhancing effect on scopolamine-induced amnesia in mice.Neuroprotective effect against glutamate-induced neurotoxicity in HT22 cells was evaluated by MTT assay. Also, cognitive enhancing effect against scopolamine (1 mg/kg, ip) induced learning and memory deficit was measured by Morris water maze test. Oral administered of 2,3-dihydroxy-4-methoxyacetophenone (1, 10, 20, 40 and 50 mg/kg) to amnesic mice induced by scopolamine. In Morris water maze test, 2,3-dihydroxy-4-methoxyacetophenone (50 mg/kg) improved the impairment of spatial memory induced by scopolamine. 2,3-Dihydroxy-4-methoxyacetophenone protect HT22 cells on glutamate induced cell-death in a dose-dependent manner (EC₅₀ value: 10.94 μM). Furthermore, 2,3-dihydroxy-4-methoxyacetophenone was found to inhibit [Ca²⁺] accumulation in HT22 cells and had antioxidantive activity. The results showed that 2,3-dihydroxy-4-methoxyacetophenone exert neuroprotective and cognitive-enhancing activities through its antioxidant activity. We suggest that 2,3-dihydroxy-4-methoxyacetophenone improves cognitive function and may be helpful for the treatment of Alzheimer’s disease.     

Continuous degradation of maltose by enzyme entrapment technology using calcium alginate beads as a matrix

Maltase from Bacillus licheniformis KIBGE-IB4 was immobilized within calcium alginate beads using entrapment technique. Immobilized maltase showed maximum immobilization yield with 4% sodium alginate and 0.2 M calcium chloride within 90.0 min of curing time. Entrapment increases the enzyme–substrate reaction time and temperature from 5.0 to 10.0 min and 45 °C to 50 °C, respectively as compared to its free counterpart. However, pH optima remained same for maltose hydrolysis. Diffusional limitation of substrate (maltose) caused a declined in V_max of immobilized enzyme from 8411.0 to 4919.0 U ml^?1 min^?1 whereas, K_m apparently increased from 1.71 to 3.17 mM ml^?1. Immobilization also increased the stability of free maltase against a broad temperature range and enzyme retained 45% and 32% activity at 55 °C and 60 °C, respectively after 90.0 min. Immobilized enzyme also exhibited recycling efficiency more than six cycles and retained 17% of its initial activity even after 6th cycles. Immobilized enzyme showed relatively better storage stability at 4 °C and 30 °C after 60.0 days as compared to free enzyme.     

Synthesis and evaluation of tricyclic derivatives containing a non-aromatic amide as inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1)

Highly potent poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors, including 9-hydroxy-1,2-dihydro-4H-thiopyrano[3,4-c]quinolin-5(6H)-one derivatives with a non-aromatic A-ring, were synthesized. Among the derivatives, 12a showed low nanomolar enzyme and cellular activity (IC₅₀ = 42 nM, ED₅₀ = 220 nM) with good water solubility. Further, 12a exhibited microsomal stability in vitro and brain permeability in vivo.     

The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium,Anoxybacillus beppuensis TSSC-1

Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55 °C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000 U/mg specific activity and 19.5 fold purification. The molecular weight, K_m and V_max were 43 kD, 0.5 mg ml^?1 and 3571.42 μmol ml^?1 m^?1, respectively. The enzymatic catalysis of soluble starch was optimum at 80 °C and pH 7. The thermodynamic parameters, K_d, t_1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The K_d and t_1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.     

Unusual hepatic mitochondrial arginase in an Indian air-breathing teleost,Heteropneustes fossilis: Purification and characterization

A functional urea cycle with both cytosolic (ARG I) and mitochondrial (ARG II) arginase activity is present in the liver of an ureogenic air-breathing teleost, Heteropneustes fossilis. Antibodies against mammalian ARG II showed no cross-reactivity with the H. fossilis ARG II. ARG II was purified to homogeneity from H. fossilis liver. Purified ARG II showed a native molecular mass of 96 kDa. SDS–PAGE showed a major band at 48 kDa. The native enzyme, therefore, appears to be a homodimer. The pI value of the enzyme was 7.5. The purified enzyme showed maximum activity at pH 10.5 and 55 °C. The K_m of purified ARG II for l-arginine was 5.25 ± 1.12 mM. l-Ornithine and N^ω-hydroxy-l-arginine showed mixed inhibition with K_i values 2.16 ± 0.08 and 0.02 ± 0.004 mM respectively. Mn^+ 2 and Co^+ 2 were effective activators of arginase activity. Antibody raised against purified H. fossilis ARG II did not cross-react with fish ARG I, and mammalian ARG I and ARG II. Western blot with the antibodies against purified H. fossilis hepatic ARG II showed cross reactivity with a 96 kDa band on native PAGE and a 48 kDa band on SDS–PAGE. The molecular, immunological and kinetic properties suggest uniqueness of the hepatic mitochondrial ARG II in H. fossilis.     

Thioether-stapled macrocyclic inhibitors of the EH domain of EHD1

Recycling of receptors from the endosomal recycling compartment to the plasma membrane is a critical cellular process, and recycling is particularly important for maintaining invasiveness in solid tumors. In this work, we continue our efforts to inhibit EHD1, a critical adaptor protein involved in receptor recycling. We applied a diversity-oriented macrocyclization approach to produce cyclic peptides with varied conformations, but that each contain a motif that binds to the EH domain of EHD1. Screening these uncovered several new inhibitors for EHD1’s EH domain, the most potent of which bound with a K_d of 3.1 μM. Several of the most potent inhibitors were tested in a cellular assay that measures extent of vesicle recycling. Inhibiting EHD1 could potentially slow cancer invasiveness and metastasis, and these cyclic peptides represent the most potent inhibitors of EHD1 to date.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Synthesis and pharmacological characterization of benzenesulfonamides as dual species inhibitors of human and murine mPGES-1

The microsomal prostaglandin E₂ synthase 1 (mPGES-1) became a desirable target in recent years for the research of new anti-inflammatory drugs. Even though many potent inhibitors of human mPGES-1, tested in vitro assay systems, have been synthesized, they all failed in preclinical trials in rodent models of inflammation, due to the lack of activity on rodent enzyme. Within this work we want to present a new class of mPGES-1 inhibitors derived from a benzenesulfonamide scaffold with inhibitory potency on human and murine mPGES-1. Starting point with an IC₅₀ of 13.8 μM on human mPGES-1 was compound 1 (4-{benzyl[(4-methoxyphenyl)methyl]sulfamoyl}benzoic acid; FR4), which was discovered by a virtual screening approach. Optimization during a structure–activity relationship (SAR) process leads to compound 28 (4-[(cyclohexylmethyl)[(4-phenylphenyl)methyl]sulfamoyl]benzoic acid) with an improved IC₅₀ of 0.8 μM on human mPGES-1. For the most promising compounds a broad pharmacological characterization has been carried out to estimate their anti-inflammatory potential.     

Breast cancer aromatase expression evaluated by the novel antibody 677: Correlations to intra-tumor estrogen levels and hormone receptor status

Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E₂; estrone, E₁; estrone sulfate, E₁S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E₂ levels comparing 677+ versus 677? tumors, although a non-significant trend towards higher tumor E₁ and E₁S levels was observed in 677+ breast cancers. In contrast, tumor levels of E₂ were significantly higher in ER+ tumors compared to ER? tumors (P < 0.001) and to benign breast tissue from the same breast (P < 0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E₂ (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E₁ (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E₁S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677? tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E₂ were also found to be significantly higher among 677+ compared to 677? tumors: 873.2 fmol/g (95% CI 395.9–1925.6) versus 217.9 fmol/g (95% CI 88.8–534.9) (P = 0.015).In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis.     

α-l-rhamnosidase selective for rutin to isoquercitrin transformation from Penicillium griseoroseum MTCC-9224

An α-l-rhamnosidase secreting fungal strain has been isolated from the decaying goose berry (Emblica officinalis) fruit peel. The fungal strain has been identified as Penicillium greoroseum MTCC-9224. The α-l-rhamnosidase of this fungal strain has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and an anion exchange chromatography on DEAE-cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 97 kDa in SDS-PAGE analysis. The native-PAGE analysis also gave a single protein band confirming the purity of the enzyme. Using p-nitrophenyl-α-l-rhamnopyranoside as the substrate, K_m and k_cat values of the enzyme were 0.65 mM and 43.65 s⁻¹, respectively. The pH and temperature optima of the enzyme were 6.5 and 57 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 27.9 kJ/mol. The purified α-l-rhamnosidase hydrolyzed rutin to isoquercitrin and l-rhamnose but has no effect on naringin and hesperidin.     

Purification and characterization of α-l-rhamnosidase from Penicillium corylopholum MTCC-2011

An extracellular α-l-rhamnosidase has been purified to electrophoretic homogeneity from the culture filtrate of Penicillium corylopholum MTCC-2011 using a simple procedure consisting of concentration by ultrafiltration and cation exchange column chromatography on carboxymethyl cellulose. The sodium dodesyl sulphate polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass of 67.0 kDa. The native – polyacrylamide gel electrophoresis analysis also gave a single protein band confirming the purity of the enzyme and also showing that the enzyme is a monomer in the native state. The K_m and k_cat values of the enzyme were 0.42 mM and 35.7 s^?1, respectively, using p-nitrophenyl α-l-rhamnopyranoside as the substrate. The pH and temperature optima of the enzyme were 6.5 and 57.0 °C, respectively. The purified enzyme preparation successfully hydrolyzed naringin and rutin to prunin and quercetin glucoside, respectively. Thus it can be used for the preparation of these pharmaceutically important compounds.     

Class II α-mannosidase from Aspergillus fischeri: Energetics of catalysis and inhibition

Energetics of the catalysis of Class II α-mannosidase (E.C.3.2.1.24) from Aspergillus fischeri was studied. The enzyme showed K_cat/K_m for Man (α1-3) Man, Man (α1-2) Man and Man (α1-6) Man as 7488, 5376 and 3690 M^?1 min^?1, respectively. The activation energy, E_a was 15.14, 47.43 and 71.21 kJ/mol for α1-3, α1-2 and α1-6 linked mannobioses, respectively, reflecting the energy barrier in the hydrolysis of latter two substrates. The enzyme showed K_cat/K_m as 3.56 × 10⁵ and 4.61 × 10⁵ M^?1 min^?1 and E_a as 38.7 and 8.92 kJ/mol, towards pNPαMan and 4-MeUmbαMan, respectively. Binding of Swainsonine to the enzyme is stronger than that of 1-deoxymannojirimycin.     

Synthesis and aromatase inhibitory activity of some new 16E-arylidenosteroids

A new series of 16E-arylidene androstene derivatives has been synthesized and evaluated for aromatase inhibitory activity. The impact of various aryl substituents at 16 position of the steroid skeleton on aromatase inhibitory activity has been observed. The 16E-arylidenosteroids 6, 10 and 11 exhibited significant inhibition of the aromatase enzyme. 16-(4-Pyridylmethylene)-4-androstene-3,17-dione (6, IC₅₀: 5.2 μM) and 16-(benzo-[1,3]dioxol-5-ylmethylene)androsta-1,4-diene-3,17-dione (11, IC₅₀: 6.4 μM) were found to be approximately five times more potent in comparison to aminoglutethimide.     

Characterization,purification, and temperature/pressure stability of polyphenol oxidase extracted from plums (Prunus domestica)

In this study, polyphenol oxidase (PPO) was extracted from Prunus domestica and partially purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and ion exchange chromatography. The final purification step revealed a 32.81-fold purification, and the molecular mass was estimated to be 65 kDa by SDS-PAGE. The purified PPO showed enzymatic activity mainly toward five substrates, namely catechol, catechin, 4-methyl catechol, chlorogenic acid, and L-3,4-dihydroxyphenylalanine, whereas it showed no activity toward caffeic acid, ferulic acid, p-coumaric acid, p-cresol, and l-tyrosine. The optimum pH and temperature values were 6.0 and 25 °C, respectively. The enzyme showed high stability in the pH range of 5.0–7.0 and in the temperature range of 25–65 °C. The most effective inhibitors of this enzyme were found to be ascorbic acid and l-cysteine. The thermal inactivation followed a first-order kinetic model, with activation energy of E_a 150.46 ± 1.29 kJ/mol. PPO extracted from plum showed stability at high pressure, with enzyme activation at 500 MPa.     

Reproductive biology and endocrine regulation of final oocyte maturation of captive white bass

The ovarian development, and plasma levels of gonadotropin II (GtH II) and sex-steroid hormones at the end of vitellogenesis were examined in captive white bass Morone chrysops. The changes in plasma hormone levels and oocyte morphology associated with gonadotropinreleasing hormone agonist (GnRHa)-induced final oocyte maturation (FOM) were studied. Although plasma 17β-oestradiol (E₂) and oocyte diameter increased, there were no changes in GtH II, testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) or 17,20β,21-dihydroxy-4-pregnen-3-one (17,20β,21-P) in non-hormone-treated females, and no FOM was observed. Treatment with a sustained-release GnRHa delivery system (GnRHa implant) induced two FOM cycles separated by about 24 h, with the release of approximately equal numbers of eggs in each spawn. Plasma GtH II levels were elevated significantly throughout FOM, reaching a maximum of 9·07 ± 1·55 ng ml^?1 in ovulated fish. Both plasma E₂ and T increased soon after the GnRHa treatment, but E₂ declined in fish undergoing germinal vesicle (GV) migration. Plasma T increased further during FOM (7·55 ± 2·87 ng ml^?1), but declined precipitously at ovulation. A surge in plasma 17,20β-P and 17,20β,21-P (4·11 ± 0·97 ng ml^?1 and 3·10 ± 0·77 ng ml^?1, respectively) was observed in females undergoing GV breakdown (GVBD). Based on the involvement of different sex-steroid hormones, FOM was separated into two stages. Early FOM included lipid-droplet coalescence and GV migration, and was associated with elevations in plasma GtH II and T. Late FOM included GVBD and yolk-globule coalescence, and was associated with elevations in plasma GtH II, 17,20β-P and 17,20β,21-P. The results of this study point to the absence of a surge in plasma GtH II as the missing link in the reproductive axis responsible for the failure of captive white bass to undergo FOM at the end of vitellogenesis. Sustained elevation of plasma GtH II via treatment with a GnRHa implant induced two consecutive spawns with an overall egg production two- to eightfold higher than previously obtained from captive broodstocks, and similar to annual egg production Values reported for wild fish.