The mechanism of cobalamin binding to hog intrinsic factor

Chicken brain Arylsulfatase A (E.C.3.1.6.1) was immobilized by interaction with Concanavalin A. The immobilized enzyme retained its catalytic activity and this enzyme can be reused without appreciable loss of activity. The storage stability of bound and soluble enzymes was comparable and binding of enzyme to Concanavalin A increases its thermal stability. Kinetic studies indicated that bound enzyme shows similar anomalous kinetics as that of free enzyme but slight change was observed in relation to pH optima, Km value and activation energy.     

Effect of Ginkgo biloba extract on rat hepatic microsomal CYP1A activity: role of ginkgolides, bilobalide, and flavonols

The present study investigated the in vitro effect of Ginkgo biloba extracts and some of the individual constituents (ginkgolides, bilobalide, and flavonols such as kaempferol, quercetin, isorhamnetin, and their glycosides) on CYP1A-mediated 7-ethoxyresorufin O-dealkylation in hepatic microsomes isolated from rats induced with beta-naphthoflavone. G. biloba extract competitively inhibited CYP1A activity, with an apparent Ki value of 1.6 +/- 0.4 microg/mL (mean +/- SE). At the concentrations present in the G. biloba extracts, ginkgolides A, B, C, and J and bilobalide did not affect CYP1A activity, whereas kaempferol (IC50 = 0.006 +/- 0.001 microg/mL, mean +/- SE), isorhamnetin (0.007 +/- 0.001 microg/mL), and quercetin (0.050 +/- 0.003 microg/mL) decreased this activity. The monoglycosides (1 and 10 microg/mL) and diglycosides (10 microg/mL) of kaempferol and quercetin but not those of isorhamnetin also inhibited CYP1A activity. The order of inhibitory potency was kaempferol approximately equal to isorhamnetin > quercetin, and for each of these flavonols the order of potency was aglycone > monoglycoside > diglycoside. In summary, G. biloba extract competitively inhibited rat hepatic microsomal CYP1A activity, but the effect was not due to ginkgolides A, B, C, or J, bilobalide, kaempferol, quercetin, isorhamnetin, or the respective flavonol monoglycosides or diglycosides.     

Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract

The Ginkgo biloba extract EGb761 was tested for its ability to inhibit the major human cytochrome P450 enzymes (CYPs). The full extract was found to strongly inhibit CYP2C9 (Ki = 14+/- 4 microg/mL), and to a lesser extent, CYP1A2 (Ki = 106 +/- 24 microg/mL), CYP2E1 (Ki = 127 +/- 42 microg/mL), and CYP3A4 (Ki = 155 +/- 43 microg/mL). The terpenoidic and flavonoidic fractions of the extract were tested separately against the same P450s to identify the source of inhibition by EGb761. The terpenoidic fraction inhibited only CYP2C9 (Ki = 15 +/-6 microg/mL) whereas the flavonoidic fraction of EGb761 showed high inhibition of CYP2C9, CYP1A2, CYP2E1, and CYP3A4 (Ki's between 4.9 and 55 microg/mL). The flavonoidic fraction was further fractionated using extraction and chromatography. Inhibition studies indicated that the majority of these fractions inhibited P450s at a significant level (IC50 < 40 microg/mL).     

Combinatorial modification of natural products: synthesis and in vitro analysis of derivatives of thiazole peptide antibiotic GE2270 A: A-ring modifications

Thiazole peptide GE2270 A (1) possesses potent antimicrobial activity against many gram-positive pathogens, including methicillin resistant Staphylococcus aureus (S. aureus, MRSA; MIC(90)=0.06 microg/mL) and vancomycin resistant Enterococcus spp. (VRE; MIC(90)=0.03 microg/mL); however its poor aqueous solubility has prohibited its development for the clinical treatment of infections. An integrated combinatorial and medicinal chemistry program was employed to identify derivatives of 1 that retain activity but possess greatly enhanced aqueous solubility.     

S-Euglobals: biomimetic synthesis, antileishmanial, antimalarial, and antimicrobial activities

Several new euglobal analogues (named as S-euglobals) were synthesized from phloroglucinol via a biomimetic three-component reaction involving Knoevenagel condensation followed by [4+2]-Diels-Alder cycloaddition with monoterpene. Newly synthesized euglobal analogues involve monoterpenes that have not yet been encountered in natural euglobals. S-Euglobals along with previously synthesized robustadial A and B were evaluated for in vitro antileishmanial, antimalarial, antimicrobial, and cytotoxic activities. Out of 16, nine analogues were found to exhibit antileishmanial activity against Leishmania donovani promastigotes. Analogue 7 was the most potent with IC(50) of 2.4 microg/mL and IC(90) of 8 microg/mL, followed by analogues 8 and 11 (IC(50) 5.5 and 9.5 microg/mL). Antileishmanial activity of robustadial A (5) and B (6) was moderate with IC(50) of 20 and 16 microg/mL, respectively. Robustadial A and B and S-euglobal 8 exhibited weak antimalarial activity against Plasmodium falciparum (IC(50) of 2.7-4.76 microg/mL). Few of the euglobal analogues showed antibacterial activity against methicillin-resistant Staphylococcus aureus. Amongst these, analogue 11 was the most potent with IC(50) of 1.0 microg/mL and MIC of 5.0 microg/mL. Most of the compounds were not cytotoxic up to 25 microg/mL in a panel of cell lines consisting of both cancer (SK-MEL, KB, BT-549, and SK-OV-3) as well as non-cancer kidney (Vero and LLC-PK11) cells.     

Effects of the dietary phytoestrogen biochanin A on cell growth in the mammary carcinoma cell line MCF-7

Studies of the dietary phytoestrogen biochanin A on cell proliferation of the cultured estrogen responsive cells human breast carcinoma MCF-7 showed that biochanin A exhibits biphasic regulation on MCF-7 cells. At concentrations of less than 10 microg/mL, cells respond to biochanin A by increasing cell growth and de novo DNA synthesis. The addition of biochanin A at concentrations of greater than 30 microg/mL significantly inhibited cell growth and DNA synthesis in a dose-dependent fashion, resulting in an IC(50) value of 40 microg/mL. The reversibility of these inhibitory effects by biochanin A appears also to be concentration dependent. Cells previously treated with high concentrations (>60 microg/mL) of biochanin A did not regain normal growth after treatment ceased. Biochanin A was cytostatic at low concentrations (<40 microg/mL) and cytotoxic at higher concentrations. Upon exposure to 100 microg/mL of biochanin A, cell morphology was severely altered, cell volume decreased, and condensation of cell components was clearly noticeable. In addition, biochanin A damaged cell membranes by increasing membrane permeability. These results suggest possible molecular and cellular mechanisms of the action of dietary phytoestrogens on estrogen target cells.     

In vitro activity of linezolid and eperezolid, two novel oxazolidinone antimicrobial agents, against anaerobic bacteria

Linezolid (formerly U-100766) and eperezolid (formerly U-100592) are novel oxazolidinone antimicrobial agents that are active against multi-drug-resistant staphylococci, streptococci, enterococci, corynebacteria, and mycobacteria. Preliminary studies also demonstrated that the compounds inhibited some test strains of anaerobic bacteria. Therefore, we extended the in vitro evaluation of these agents to include a total of 54 different anaerobic species. Minimal inhibitory concentration (MIC) values were determined using a standard agar dilution method for 143 anaerobic bacterial isolates. Eperezolid and linezolid demonstrated potent activity against the anaerobic Gram-positive organisms with most MIC values in the range of 0.25-4 microg/mL. Viridans streptococci demonstrated MICs of 1-2 microg/mL; Peptostreptococcus species and Propionibacterium species were inhibited by 相似文献   

Effects of soybean lipoxygenase on Na+/K+-ATPase activity in vitro

Oxidized metabolites of polyunsaturated fatty acids produced by lipoxygenase are among the endogenous regulators of Na+/K+-ATPase. The direct effect of lipoxygenase on Na+/K+-ATPase activity was assessed in vitro using soybean lipoxygenase. Treatment of 4.2 microg/mL Na+/K+-ATPase (from dog kidneys) with 4.2 microg/mL of soybean lipoxygenase caused 20+/-2% inhibition of ATPase activity. A 10-fold increase in lipoxygenase concentration (41.6 microg/mL) led to 30+/-0.3% inhibition. In the presence of 12 microg/mL phenidone (a lipoxygenase inhibitor) and 15.4 microg/mL glutathione (a tripeptide containing a cysteine residue) inhibition of Na+/K+-ATPase activity was blocked and an increase in ATPase activity was observed. The presence of lipoxygenase enhanced the inhibition of Na+/K+-ATPase activity caused by 20 ng/mL ouabain (31+/-2 vs. 19+/-2) but had little or no effect with higher concentrations of ouabain. These findings suggest that lipoxygenase may regulate Na+/K+-ATPase by acting directly on the enzyme.     

Synthesis and antituberculosis activity of the derivatives of glycoside steviolbioside from the plant Stevia rebaudiana and diterpenoid isosteviol containing hydrazone, hydrazide and pyridinoyl moieties

Conjugates of antitubercular drug Isoniazid (hydrazide of isonicotinic acid), nicotinic and alpha-picolinic acid hydrazides and glycoside steviolbioside from the plant Stevia rebaudiana as well as the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. Besides, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties connected by dihydrazide linker were also obtained. Both initial compounds and their synthetic derivatives inhibit the growth of Mycobacterium tuberculosis (H37Rv in vitro). The minimum concentration at which the growth of M. tuberculosis was inhibited by 100% (MIC) for stevioside and steviolbioside equals 7.5 and 3.8 microg/mL, respectively. MIC values for conjugates of the hydrazides of pyridine carbonic acids and steviolbioside as well as isosteviol are in the ranges 5-10 and 10-20 microg/mL, respectively. Maximum inhibitory effect against M. tuberculosis showed the conjugates of isosteviol and adipic acid dihydrazide (MIC values ranged from 1.7 to 3.1 microg/mL). Antitubercular activity of the compounds studied is higher than the activity of antitubercular drug Pyrizanamide (MIC = 12.5-20 microg/mL) but lower than the activity of antitubercular drug Isoniazid (MIC = 0.02-0.04 microg/mL).     

Optically active antifungal azoles: synthesis and antifungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol

A series of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (11a-n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazole-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (12a-n) has been synthesized. The antifungal activity of compounds was evaluated by in vitro agar diffusion and broth dilution assay. Compounds 11d and its positional isomer 12d having 3-trifluoromethyl substitution on the phenyl ring of piperazine demonstrated significant antifungal activity against variety of fungal cultures (Candida spp. C. neoformans and Aspergillus spp.). The compound 12d showed MIC value of 0.12 microg/mL for C. albicans, C. albicans V-01-191A-261 (resistant strain); 0.25 microg/mL for C. tropicalis, C. parapsilosis ATCC 22019 and C. krusei and MIC value of 0.5 microg/mL for C. glabrata, C. krusei ATCC 6258, which is comparable to itraconazole and better than fluconazole. Further, compound 11d showed significant activity (MIC; 0.25-0.5 microg/mL) against Candida spp. and strong anticryptococcal activity (MIC; 0.25 microg/mL) against C. neoformans.     

Galactofuranose-rich heteropolysaccharide from Trebouxia sp., photobiont of the lichen Ramalina gracilis and its effect on macrophage activation

A structural study of the carbohydrates from Trebouxia sp., the algal symbiont of the lichen Ramalina gracilis demonstrated a galactofuranan-rich heteropolysaccharide, which was predominated by (1-->5)-linked galactofuranosyl units with side-chains in position 6 on approximately 11.0% of the units. The side-chains have very complex branched structures. This polysaccharide showed cell eliciting activity on peritoneal macrophages in vitro at all concentrations tested (1-150 microg/mL), and at 150 microg/mL an increase of 60% of macrophage activation in comparison to the control group was observed. A potential role of these carbohydrates in lichen recognition process is also discussed.     

Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves

Ayurvedic medicines prepared in India consist of Withania somnifera roots as one of the main ingredients. It is consumed as a dietary supplement around the world. The leaves of W. somnifera were used in the treatment of tumors and inflammation in several Asian countries. We have isolated twelve withanolides such as withaferin A (1), sitoindoside IX (2), 4-(1-hydroxy-2, 2-dimethylcyclpropanone)-2, 3-dihydrowithaferin A (3), 2, 3-dihydrowithaferin A (4), 24, 25-dihydro-27-desoxywithaferin A (5), physagulin D (1-->6)-beta-D-glucopyranosyl- (1-->4)-beta-D-glucopyranoside (6), 27-O-beta-D-glucopyranosylphysagulin D (7), physagulin D (8), withanoside IV (9), and 27-O-beta-D-glucopyranosylviscosalactone B (10), 4, 16-dihydroxy-5beta, 6beta-epoxyphysagulin D (11), viscosalactone B (12) from the leaves of this species. Compounds 1-12 and diacetylwithaferin A (13) were tested for their antiproliferative activity on NCI-H460 (Lung), HCT-116 (Colon), SF-268 (Central Nervous System; CNS and MCF-7 (Breast) human tumor cell lines. The inhibitory concentration to afford 50% cell viability (IC50) for these compounds was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Withaferin A and its derivatives exhibited inhibitory concentrations (50%) ranging from 0.24 +/- 0.01 to 11.6 +/- 1.9 microg/mL. Viscosalactone B (12) showed the 50% inhibition at concentrations ranging from 0.32 +/- 0.05 to 0.47 +/- 0.15 microg/mL whereas its 27-O-glucoside derivative (10) exhibited IC50 between 7.9 +/- 2.9 and 17.3 +/- 3.9 microg/ml. However, Physagulin D type withanolides showed either weak or no activity at 30 microg/mL. Therefore, incorporation of withanolides in the diet may prevent or decrease the growth of tumors in human.     

Carboxylic acid and phosphate ester derivatives of fluconazole: synthesis and antifungal activities

Two classes of fluconazole derivatives, (a) carboxylic acid esters and (b) fatty alcohol and carbohydrate phosphate esters, were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger. All carboxylic acid ester derivatives of fluconazole (1a-l), such as O-2-bromooctanoylfluconazole (1g, MIC=111 microg/mL) and O-11-bromoundecanoylfluconazole (1j, MIC=198 microg/mL), exhibited higher antifungal activity than fluconazole (MIC > or = 4444 microg/mL) against C. albicans ATCC 14053 in SDB medium. Several fatty alcohol phosphate triester derivatives of fluconazole, such as 2a, 2b, 2f, 2g, and 2h, exhibited enhanced antifungal activities against C. albicans and/or A. niger compared to fluconazole in SDB medium. For example, 2-cyanoethyl-omega-undecylenyl fluconazole phosphate (2b) with MIC value of 122 microg/mL had at least 36 times greater antifungal activity than fluconazole against C. albicans in SDB medium. Methyl-undecanyl fluconazole phosphate (2f) with a MIC value of 190 microg/mL was at least 3-fold more potent than fluconazole against A. niger ATCC 16404. All compounds had higher estimated lipophilicity and dermal permeability than those for fluconazole. These results demonstrate the potential of these antifungal agents for further development as sustained-release topical antifungal chemotherapeutic agents.     

Polysaccharides from Astragalus membranaceus promote phagocytosis and superoxide anion (O2-) production by coelomocytes from sea cucumber Apostichopus japonicus in vitro

The potential immunostimulatory effects of Astralagus membranaceus polysaccharides (APS) on sea cucumber, Apostichopus japonicus (Selenka), were investigated in vitro. Phagocytosis and superoxide anion (O(2)(-)) production by phagocytic amoebocytes (PA) from A. japonicus coelomic fluid were measured during incubation at 18 degrees C, 22 degrees C, or 25 degrees C with APS at 0, 10, 20, or 40 microg mL(-1) (n=3). Phagocytic activity against yeast cells was quantified by direct visualization, and O(2)(-) production by nitroblue tetrazolium (NBT) reduction assay. Compared with controls, including APS at 20 microg mL(-1) significantly increased (P<0.05) the percentage of phagocytic capacity (PC) and phagocytic index (PI) at 18 degrees C and 22 degrees C, but no significant enhancement was observed at 25 degrees C. In contrast, the coelmocytes of A. japonicus can have an obvious generation of O(2)(-) after the stimulation. The concentration of 20 microg mL(-1) APS resulted in a significant increase in nitroblue tetrazolium (NBT) positive cells (P<0.05) at different temperature and even 10 microg mL(-1) APS could increase O(2)(-) generation significantly at 18 degrees C and 22 degrees C. Both phagocytosing and O(2)(-) production increased with the increase of APS concentration from 0 to 20 microg mL(-1) at different temperature, and when APS at 40 microg mL(-1), they were decreased. It suggested that immunocytes activity in A. japonicus decreased with the temperature increasing from 18 degrees C to 25 degrees C, and APS could be an effective immunostimulant to enhance phagocytic activity and O(2)(-) production.     

Effect of growth hormone therapy on the proinflammatory cytokine profile in growth hormone-deficient children

The aim of the present study was to establish whether growth hormone (GH) treatment in vivo affects pro-inflammatory cytokine production by resting or in vitro, activated, cultured, peripheral blood mononuclear cells (PBMC) from children with complete growth hormone deficiency (GHD). We evaluated 11, pre-pubertal children (6 males and 5 females) with GHD, aged between 6 and 14 years, and 9, age- and sex-matched healthy subjects were studied as controls (CTRLs). Freshly isolated PBMC were cultured for 4 or 24 h in X-VIVO medium in the presence or absence of 0.01 microg/mL lipopolysaccharide for the determination of TNF-alpha and IL-6 production; alternatively, cells were incubated 24 h in X-VIVO medium with or without 25 microg/mL Concanavalin A for IFN-gamma production. Cytokines were measured in the cell supernatants by enzyme-linked immunosorbent assay kits. The results of the present study provide evidence that spontaneous and/or mitogen-induced, in vitro PBMC production of pro-inflammatory cytokines is lower in GHD children than in healthy, age-matched individuals (p<0.05 by the Mann-Whitney U-test). After 3 months of GH therapy, cytokine production was significantly (p<0.05 by the Wilcoxon test) increased, but was still lower than in healthy controls. It is reasonable to speculate that severe GH deficiency can cause alterations in the pro-inflammatory cytokine-induced immune response in humans, and that GH treatment can ameliorate this important immunological function.     

Synthesis, absolute stereochemistry and molecular design of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201

The absolute stereochemistry of the new antifungal and antibacterial antibiotic produced by Streptomyces sp.201 has been established by achieving the total synthesis of the product. A series of analogues have also been synthesized by changing the side chain and their bioactivity assessed against different microbial strains. Among them, 1e (R = C8H17) was found to be the most potent with MIC of 8 microg/mL against Mycobacterium tuberculosis, 12 microg/mL against Escherichia coli and 16 microg/mL against Bacillus subtilis 6 microg/mL against Proteus vulgaris. This was followed by 1b (R = C5H11) with MIC of 10-20 microg/mL range and 1d (R = C7H15) with MIC of 14-24 g/mL, whereas 1a (R = C4H9) and 1f (R = C18H35) were found to be completely inactive. Besides, 1c (R = C6H13) showed certain extent of antibacterial activity in the range of 24-50 microg/mL. Mycobacterium tuberculosis was very sensitive to 1e (R = C8H17) with MIC of 8 microg/mL. Antifungal activity of analogues 1d (R = C7H15) and 1e, (R = C8H17) against Fusarium oxysporum and Rhizoctonia solani were found promising with MFCs in the 15-18 microg/mL range.     

A potential role of alkaloid extracts from Salsola species (Chenopodiaceae) in the treatment of Alzheimer's disease

From the aerial parts of Salsola oppositofolia, S. soda and S. tragus an alkaloid extract was obtained and tested to evaluate antioxidant and anti-cholinesterase activities. The in vitro study of the antioxidant activity by the DPPH method revealed a significant activity of Salsola alkaloid extracts with IC(50) values ranging from 16.30 microg/mL for S. oppositifolia to 26.17 microg/mL for S. tragus. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities were evaluated. S. tragus alkaloid extract exerted the highest inhibitory activity against AChE (IC(50) of 30.2 microg/mL) and BChE (IC(50) of 26.5 microg/mL). Interestingly, S. soda and S. oppositifolia exhibited a selective inhibitory activity against BChE with IC(50) values of 34.3 microg/mL and 32.7 microg/mL, respectively. Tetrahydroisoquinoline alkaloids were identified and quantified by GC/MS analysis.     

Antioxidant activity of carotenoid lutein in vitro and in vivo

Carotenoid lutein was evaluated for its antioxidant potential both in vitro and in vivo. Lutein was found to scavenge superoxide radicals, hydroxyl radicals and inhibited in vitro lipid peroxidation. Concentrations needed for 50% inhibition (IC50) were 21, 1.75 and 2.2 microg/mL respectively. It scavenged 2,2-diphenyl-1-picryl hydrazyl (IC50 35 microg/mL) and nitric oxide radicals (IC50 3.8 microg/mL) while 2,2-azobis-3-ethylbenzthiozoline-6-sulfonic acid radicals were inhibited at higher concentration. Ferric reducing power (50%) of lutein was found to be equal 0.3 micromols/mL of FeSO4.7H2O. Its oral administration inhibited superoxide generation in macrophages in vivo by 34.18, 64.32 and 70.22% at doses of 50, 100 and 250 mg/kg body weight. The oral administration of lutein in mice for 1 month significantly increased the activity of catalase, superoxide dismutase, glutathione reductase and glutathione in blood and liver while the activity of glutathione peroxidase and glutathione-S-transferase were found to be increased in the liver tissue. Implication of these results in terms of its role in reducing degenerative diseases is discussed.     

Neoplaether, a new cytotoxic and antifungal endophyte metabolite from Neoplaconema napellum IFB-E016

A new diphenyl ether, named neoplaether, together with five known compounds monomethylsulochrin, physcion, helvolic acid, ergosterol and ergosterol peroxide, was isolated from the culture of Neoplaconema napellum IFB-E016, an endophytic fungus residing in the healthy leaves of Hopea hainanensis. The structure of neoplaether was elucidated by a correlative interpretation of its infrared spectrometry, mass spectroscopy and nuclear magnetic resonance spectra, and confirmed by its single-crystal X-ray diffraction analysis. Neoplaether exhibited significant cytotoxic activity against the human nasopharyngeal epidermoid tumor KB cell line, with an IC(50) value of 13.0 microg mL(-1), comparable to that of 5-fluorouracil (2.5 microg mL(-1)) co-assayed as a positive reference. In addition, it showed antifungal activity against Candida albicans, with a minimal inhibitory concentration value of 6.2 microg mL(-1) (amphotericin as a positive control had a value of 1.5 microg mL(-1)).